WO2011018898A1 - Cdc45l as tumor marker and therapeutic target for cancer - Google Patents

Cdc45l as tumor marker and therapeutic target for cancer Download PDF

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WO2011018898A1
WO2011018898A1 PCT/JP2010/005049 JP2010005049W WO2011018898A1 WO 2011018898 A1 WO2011018898 A1 WO 2011018898A1 JP 2010005049 W JP2010005049 W JP 2010005049W WO 2011018898 A1 WO2011018898 A1 WO 2011018898A1
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cdc45l
pif1
gene
cancer
double
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PCT/JP2010/005049
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French (fr)
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Yataro Daigo
Yusuke Nakamura
Takuya Tsunoda
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Oncotherapy Science, Inc.
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Priority to EP10808083A priority Critical patent/EP2464749A1/en
Priority to CN2010800461722A priority patent/CN102575297A/en
Priority to JP2012507486A priority patent/JP2013501501A/en
Publication of WO2011018898A1 publication Critical patent/WO2011018898A1/en

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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to lung cancer, more particularly the diagnosis and treatment thereof.
  • NPL 1 Primary lung cancer is the leading cause of cancer deaths worldwide (NPL 1). Many genetic alterations involved in lung carcinogenesis have been reported, but the precise molecular mechanisms still remain unclear (NPL 2). In the last few decades cytotoxic agents including paclitaxel, docetaxel, gemcitabine, and vinorelbine have emerged to offer multiple therapeutic choices for patients with advanced NSCLC, however, those regimens provide a limited survival benefit compared with cisplatin-based therapies (NPL 3).
  • VEGF vascular endothelial growth factor
  • EGFR i.e., cetuximab/anti-EGFR
  • inhibitors for EGFR tyrosine kinase i.e., gefitinib and erlotinib
  • RNA interference (RNAi) technique was established (NPL 13-37).
  • CDC45 cell division cycle 45-cell (CDC45) gene is an essential gene required for initiation of DNA replication, and together with MCM and CDC6 proteins it forms a complex necessary for the initiation of DNA replication in eukaryotic cells (NPL 38).
  • a human homolog of yeast CDC45 gene termed CDC45L encodes a protein of 566 amino acids and shows 27.6% identity to yeast CDC45 (NPL 39).
  • CDC45L interacts in HeLaS3 cells with the elongating DNA polymerases delta and -epsilon, and with Psf2, which is a component of the GINS complex as well as with MCM5 and -7, subunits of the putative replicative DNA helicase complex (NPL 40).
  • NPL 41 A higher level of CDC45L protein expression in proliferating cell populations of cancer cell lines has been reported (NPL 41).
  • NPL 41 the roles of CDC45L activation in the development and progression of cancer have not been clarified.
  • PIF1 petite integration frequency 1 gene was first identified in Saccharomyces cerevisiae and was classified as a member of SFI 5'-to-3' DNA helicase conserved from yeast to human (NPL 42).
  • the function of PIF1 in yeast is implicated in mtDNA repair, rDNA replication and in the regulation of telomere length (NPL 42, 43).
  • yeast PIF1 is thought to be a region-specific DNA helicase because in vitro it requires a specific DNA structure for the efficient DNA unwinding and unwinds DNA in a 5'-to-3' direction (NPL 43).
  • NPL 1 Jemal A, et al. CA Cancer J Clin. 2008;58:71-96.
  • NPL 2 Sozzi G. European Journal of Cancer 2001;37 Suppl 7:S63-S73.
  • NPL 3 Schiller JH, et al. N Engl J Med 2002;346:92-8.
  • NPL 4 Sandler A, et al. N Engl J Med 2006;355:2542-50.
  • NPL 5 Shepherd FA, et al. N Engl J Med 2005;353:123-32.
  • NPL 6 Thatcher N. Lung Cancer 2007;57 Suppl 2:S18-23.
  • NPL 7 Daigo Y, Nakamura Y.
  • NPL 8 Kikuchi T, et al. Oncogene 2003;22:2192-205.
  • NPL 9 Kakiuchi S, et al. Mol Cancer Res 2003;1:485-99.
  • NPL 10 Kakiuchi S, et al. Hum Mol Genet 2004;13:3029-43.
  • NPL 11 Kikuchi T, et al. Int J Oncol 2006; 28:799-805.
  • NPL 12 Taniwaki M, et al. Int J Oncol 2006;29:567-75.
  • NPL 13 Suzuki C, et al. Cancer Res 2003;63:7038-41.
  • NPL 14 Ishikawa N, et al. Clin Cancer Res 2004;10:8363-70.
  • NPL 15 Kato T, et al. Cancer Res 2005;65:5638-46.
  • NPL 16 Furukawa C, et al. Cancer Res 2005;65:7102-10.
  • NPL 17 Ishikawa N, et al. Cancer Res 2005;65:9176-84.
  • NPL 18 Suzuki C, et al. Cancer Res 2005;65:11314-25.
  • NPL 19 Ishikawa N, et al. Cancer Sci 2006;97:737-45.
  • NPL 20 Takahashi K, et al. Cancer Res 2006;66:9408-19.
  • NPL 21 Hayama S, et al. Cancer Res 2006;66:10339-48.
  • NPL 22 Kato T, et al. Clin Cancer Res 2007;13:434-42.
  • NPL 23 Suzuki C, et al. Mol Cancer Ther 2007;6:542-51.
  • NPL 24 Yamabuki T, et al. Cancer Res 2007;67:2517-25.
  • NPL 25 Hayama S, et al. Cancer Res 2007; 67:4113-22.
  • NPL 26 Taniwaki M, et al. Clin Cancer Res 2007;13:6624-31.
  • NPL 27 Ishikawa N, et al. Cancer Res 2007;67:11601-11.
  • NPL 28 Mano Y, et al. Cancer Sci 2007;98:1902-13.
  • NPL 29 Kato T, et al. Cancer Res 2007; 67:8544-53.
  • NPL 30 Kato T, et al. Clin Cancer Res 2008;14:2363-70.
  • NPL 31 Dunleavy EM, et al. Cell 2009;137:485-97.
  • NPL 32 Hirata D, et al. Clin Cancer Res 2009,15:256-66.
  • NPL 33 Takano A, et al. Cancer Res (2009 in press)
  • NPL 34 Suda T, et al. Cancer Sci 2007;98:1803-8.
  • NPL 35 Mizukami Y, et al.
  • NPL 36 Harao M, et al. Int J Cancer 2008;123:2616-25.
  • NPL 37 Kono K, et al. Cancer Sci (2009 in press)
  • NPL 38 Hopwood B, Dalton S. Proc Natl Acad Sci U S A 1996;93:12309-14.
  • NPL 39 P, et al. J Biol Chem 1998;273:18205-9.
  • NPL 40 Bauerschmidt C, et al. Genes Cells 2007;12:745-58.
  • NPL 41 Pollok S, et al. FEBS J 2007;274:3669-84.
  • NPL 42 Foury F, Dyck EV. EMBO J 1985;4:3525-30.
  • NPL 43 Bessler JB, et al. Trends Cell Biol 2001;11:60-5.
  • the present invention relates to cancer-related gene CDC45L and PIF1, which are commonly up-regulated in tumors, and strategies for the development of molecular targeted drugs for cancer treatment using CDC45L and/or PIF1.
  • the present invention provides a method for diagnosing cancer, e.g., a cancer overe-expressing a CDC45L gene and/or PIF1 gene, e.g., lung cancer, using the expression level of the CDC45L and/or PIF1 gene as an index.
  • the mRNA of CDC45L or PIF1 gene can be detected by appropriate primers or probes or, alternatively, the CDC45L or PIF1 protein can be detected by anti- CDC45L or PIF1 antibody in order to determine the expression level of the gene.
  • the cancer is mediated or promoted by a CDC45L and/or PIF1.
  • the cancer is lung cancer.
  • the cancer is a lung adenocarcinoma (ADC), a lung squamous-cell carcinoma (SCC), and a lung large-cell carcinoma (LCC), or a small-cell lung cancers (SCLC).
  • ADC lung adenocarcinoma
  • SCC lung squamous-cell carcinoma
  • the present invention also provides a method for predicting the progress of a subject with cancer, e.g., lung cancer, using the expression level of the CDC45L as an index.
  • the present invention provides a method for predicting the prognosis of the cancer, e.g., lung cancer, patient using the expression level of the CDC45L and/or PIF1 gene or biological activity of the CDC45L and/or PIF1 protein as an index.
  • the present invention provides a method for screening a candidate compound for treating or preventing cancer, e.g., lung cancer, using the binding to the CDC45L and/or PIF1 polypeptide, the expression level of the CDC45L and/or PIF1 gene, or biological activity of the CDC45L and/or PIF1 polypeptide as an index.
  • the present invention provides a method for screening a candidate compound for treating or preventing cancer, e.g., lung cancer, using the interaction between CDC45L polypeptide and PIF1 polypeptide as an index.
  • the present invention provides double-stranded molecules, e.g., siRNA, against the CDC45L or PIF1 gene , that inhibits the expression of the gene, and vectors encoding the double stranded molecule.
  • double-stranded molecules of the present invention are useful for treating or preventing cancers, e.g., a cancer mediated by a CDC45L and/or PIF1 or resulting from overexpression of a CDC45L and/or PIF1, e.g., lung cancer.
  • the present invention provides methods of treating or preventing cancer in a subject, comprising administering to said subject a pharmaceutically effective amount of a double-stranded molecule against CDC45L gene or PIF1 gene, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits cell proliferation as well as the expression of the gene.
  • the present invention provides compositions for treating or preventing cancer, comprising a double-stranded molecule against a CDC45L or PIF1, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits cell proliferation as well as the expression of the gene, and a pharmaceutically acceptable carrier.
  • Figure 1 depicts the expression of CDC45L in lung tumor tissues and cell lines.
  • A, B Expression of CDC45L in a normal lung tissue and 15 clinical lung cancer tissue samples (5 lung ADC, 5 lung SCC, and 5 SCLC) (A) and normal airway epithelia derived cells (SAEC) and 15 lung-cancer cell lines (B), detected by semi-quantitative RT-PCR analysis.
  • C Expression of CDC45L protein in 6 lung-cancer cell lines and SAEC cells examined by western-blot analysis.
  • D Subcellular localization of endogenous CDC45L protein in LC319 cells. CDC45L was stained strongly in the nucleus and weakly in the cytoplasm.
  • Figure 2 depicts the expression of CDC45L in normal tissues and association of CDC45L overexpression with poorer prognosis for NSCLC patients.
  • A Immunohistochemical analysis of CDC45L protein expression in 5 normal tissues (heart, lung, liver, kidney, and testis) and lung cancers. CDC45L expressed abundantly in testis and lung cancer cells (mainly in nucleus and/or cytoplasm), but its expression was hardly detectable in the remaining four normal tissues.
  • B Examples for positive and negative staining of CDC45L expression in cancer tissues (original magnification X100).
  • Figure 3 depicts the growth suppressive effect of siRNA against CDC45L on lung cancer cells.
  • A Gene knockdown effect on CDC45L expression in A549 (left) and SBC-3 (right) cells by two different siRNAs for CDC45L, si-CDC45L-#1 and si-CDC45L-#2, and two control siRNAs (si-LUC and si-EGFP), analyzed by semi-quantitative RT-PCR.
  • B C, Colony formation (B) and MTT (C) assays of A549 (left) and SBC-3 (right) cells transfected with si-CDC45Ls or control siRNAs. Columns, relative absorbance of triplicate assays; bars, SD.
  • D Flow cytometric analysis of A549 cells transfected with si-CDC45L-#2 or si-LUC (48 hours after the transfection).
  • Figure 4 depicts the interaction of CDC45L with PIF1.A, Expression analysis of CDC45L and PIF1 in a normal lung tissue and 15 clinical lung cancer tissue samples by semi-quantitative RT-PCR analysis.
  • B Interaction of endogenous CDC45L with exogenous PIF1 in lung cancer SBC-3 cells. Immunoprecipitations using cell lysates from SBC-3 cells transfected with FLAG-tagged PIF1-expression plasmids were carried out with anti-FLAG antibody. Immunoprecipitates were subjected to western-blot analysis using anti-CDC45L antibody.
  • IB immunoblotting
  • IP immunoprecipitation.
  • Figure 5 depicts the growth suppressive effect by siRNA for PIF1 on lung cancer cells.
  • A Gene knockdown effect on PIF1 expression in A549 (left) and SBC-3 (right) cells by two kinds of si-PIF1 (si-PIF1-#1 and si-PIF1-#2) and two control siRNAs (si-LUC and si-EGFP), detected by semi-quantitative RT-PCR analysis.
  • B C, Colony formation (B) and MTT (C) assays of A549 (left) and SBC-3 (right) cells transfected with si-PIF1s or control siRNAs. Columns, relative absorbance of triplicate assays; bars, SD.
  • isolated and purified used in relation with a substance indicates that the substance is substantially free from at least one substance that can be included in the natural source.
  • an isolated or purified antibody refers to antibodies that are substantially free of cellular material for example, carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • substantially free of cellular material includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein").
  • heterologous protein also referred to herein as a "contaminating protein”
  • the polypeptide is recombinantly produced, in some embodiments it is also substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation.
  • the polypeptide is produced by chemical synthesis, in some embodiments it is substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation.
  • That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel.
  • proteins including antibodies of the present invention are isolated or purified.
  • nucleic acid molecule for example, a cDNA molecule
  • a cDNA molecule can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • nucleic acid molecules encoding proteins of the present invention are isolated or purified.
  • polypeptide polypeptide
  • peptide protein
  • protein polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, for example, an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine).
  • amino acid analog refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium).
  • modified R group or modified backbones e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium.
  • amino acid mimetic refers to chemical compounds that have different structures but similar functions to general amino acids.
  • Amino acids can be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • nucleic acid and nucleic acid molecule
  • gene refers to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers.
  • the gene, polynucleotide, oligonucleotide, nucleic acid, or nucleic acid molecule can be composed of DNA, RNA or a combination thereof.
  • biological sample refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • body fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen.
  • Biological sample further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof.
  • biological sample refers to a medium, for example, a nutrient broth or gel in which an organism has been propagated, which contains cellular components, for example, proteins or polynucleotides.
  • cancer refers to cancers over-expressing the CDC45L and/or PIF1 gene, such as lung cancer, including adenocarcinoma (ADC), squamous-cell carcinoma (SCC), large-cell carcinoma (LCC), and small-cell lung cancer (SCLC).
  • ADC adenocarcinoma
  • SCC squamous-cell carcinoma
  • LCC large-cell carcinoma
  • SCLC small-cell lung cancer
  • CDC45L gene encompasses the human CDC45L gene as well as those of other animals including non-human primate, mouse, rat, dog, cat, horse, and cow but is not limited thereto, and includes allelic mutants and genes found in other animals as corresponding to the CDC45L gene.
  • the amino acid sequence encoded by the human CDC45L gene is shown as SEQ ID NO: 14 and is also available as GenBank Accession No. NM_003504.3 (NP_003495.1).
  • the polypeptide encoded by the CDC45L gene is referred to as “CDC45L”, and sometimes as “CDC45L polypeptide” or "CDC45L protein”.
  • PIF1 gene encompasses the human PIF1 gene as well as those of other animals including non-human primate, mouse, rat, dog, cat, horse, and cow but is not limited thereto, and includes allelic mutants and genes found in other animals as corresponding to the PIF1 gene.
  • the amino acid sequence encoded by the human PIF1 gene is shown as SEQ ID NO: 16 and is also available as GenBank Accession No. NM_025049.2 (NP_079325.2).
  • the polypeptide encoded by the PIF1 gene is referred to as "PIF1", and sometimes as “PIF1 polypeptide” or "PIF1 protein”.
  • a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to the protein. Namely, any polypeptide that retains at least one biological activity of CDC45L or PIF1 can be used as such a functional equivalent in the present invention.
  • functional equivalents of CDC45L and the functional equivalents of PIF1 retain promoting activity of cell proliferation.
  • the biological activity of CDC45L contains binding activity to PIF1. Therefore, in a preferred embodiment, a functional equivalent of CDC45L can contain a PIF1 binding region.
  • the biological activity of PIF1 contains binding activity to CDC45L. Therefore, in a preferred embodiment, a functional equivalent of PIF1 can contain a CDC45L binding region.
  • Functional equivalents of CDC45L include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the CDC45L protein.
  • functional equivalents of PIF1 include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the PIF1 protein.
  • amino acid side chains examples include hydrophobic amino acids (alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, valine), hydrophilic amino acids (arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, serine, threonine), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (glycine, alanine, valine, leucine, isoleucine, praline); a hydroxyl group containing side-chain (serine, threonine, tyrosine); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (aspartic acid, asparagine, glutamic acid, glutamine); a base containing side-chain (arginine, lysine
  • conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Glycine (G); (2) Aspartic acid (D), Glutamic acid (E); (3) Aspargine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (7) Serine (S), Threonine (T); and (8) Cysteine (C), Methionine (M) (see, e.g., Thomas E. Creighton, Proteins Publisher: New York: W.H. Freeman, c1984).
  • Such conservatively modified polypeptides are included in the CDC45L or PIF1 protein.
  • the present invention is not restricted thereto and the CDC45L or PIF1 protein includes non-conservative modifications so long as they retain any one of the biological activity of the CDC45L or PIF1 protein.
  • the number of amino acids to be mutated in such a modified protein is generally 10 amino acids of less, for example, 6 amino acids of less, for example, 3 amino acids or less.
  • Fusion proteins include fusions of the CDC45L or PIF1 protein and other peptides or proteins, which also can be used in the present invention. Fusion proteins can be made by techniques well known to a person skilled in the art, for example, by linking the DNA encoding the CDC45L or PIF1 gene with a DNA encoding other peptides or proteins, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. There is no restriction as to the peptides or proteins fused to the CDC45L or PIF1 protein so long as the resulting fusion protein retains any one of the objective biological activity of the CDC45L or PIF1 proteins.
  • FLAG Hopp TP, et al., Biotechnology 6: 1204-10 (1988)
  • 6xHis containing six His (histidine) residues 10xHis
  • Influenza agglutinin HA
  • human c-myc fragment VSP-GP fragment
  • p18HIV fragment T7-tag
  • HSV-tag HSV-tag
  • E-tag E-tag
  • modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
  • the proteins used for the present invention include those that are encoded by DNA that hybridize under stringent conditions with a whole or part of the DNA sequence encoding the human CDC45L protein or the human PIF1 protein and are functional equivalent to the human CDC45L protein or the human PIF1 protein.
  • These proteins include mammal homologues corresponding to the proteins derived from human or mouse (for example, proteins encoded by monkey, rat, rabbit or bovine genes).
  • isolating a cDNA highly homologous to the DNA encoding the human CDC45L gene or human PIF1 gene lung cancer tissues or cell lines, or tissues from testis can be used.
  • hybridization refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will differ under different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • Tm thermal melting point
  • stringent conditions are selected to be about 5-10 degrees Centigrade lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
  • Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions can also be achieved with the addition of destabilizing agents for example, formamide.
  • a positive signal is at least two times of background, for example, 10 times of background hybridization.
  • hybridization can be performed by conducting prehybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer.
  • the following washing step can be conducted, for example, in a low stringent condition.
  • a low stringent condition is, for example, 42 degrees C, 2x SSC, 0.1% SDS, for example, 50 degrees C, 2x SSC, 0.1% SDS.
  • high stringent condition is used.
  • a high stringent condition is, for example, washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min.
  • temperature and salt concentration can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.
  • a gene amplification method for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a DNA encoding a protein functional equivalent to the human CDC45L or PIF1 gene, using a primer synthesized based on the sequence information of the DNA (SEQ ID NO: 13 or 15) encoding the human CDC45L or PIF1 protein (SEQ ID NO: 14 or 16), examples of primer sequences are pointed out in Semi-quantitative RT-PCR in [EXAMPLE].
  • PCR polymerase chain reaction
  • Proteins that are functional equivalent to the human CDC45L or PIF1 protein encoded by the DNA isolated through the above hybridization techniques or gene amplification techniques normally have a high homology (also referred to as sequence identity) to the amino acid sequence of the human CDC45L or PIF1 protein.
  • “High homology” typically refers to the degree of identity between two optimally aligned sequences (either polypeptide or polynucleotide sequences).
  • high homology or sequence identity refers to homology of 40% or higher, for example, 60% or higher, for example, 80% or higher, for example, 85%, 90%, 95%, 98%, 99%, or higher.
  • the degree of homology or identity between two polypeptide or polynucleotide sequences can be determined by following the algorithm (Wilbur WJ & Lipman DJ. Proc Natl Acad Sci U S A. 1983 Feb; 80 (3):726-30).
  • BLAST and BLAST 2.0 algorithms are described (Altschul SF, et al., J Mol Biol. 1990 Oct 5; 215 (3):403-10; Nucleic Acids Res. 1997 Sep 1;25(17):3389-402).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/).
  • the algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff S & Henikoff JG. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10915-9).
  • a protein useful in the context of the present invention can have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has any one of the biological activity of the CDC45L protein (SEQ ID NO: 14) or PIF1 protein (SEQ ID NO: 16), it is useful in the present invention.
  • the present invention also encompasses the use of partial peptides of the CDC45L protein and the PIF1 protein.
  • a partial peptide has an amino acid sequence specific to the CDC45L protein or the PIF1 protein and consists of less than about 400 amino acids, usually less than about 200 and often less than about 100 amino acids, and at least about 7 amino acids, for example, about 8 amino acids or more, for example, about 9 amino acids or more.
  • a partial CDC45L peptide used for the screenings of the present invention suitably contains at least a binding domain of CDC45L.
  • a partial CDC45L peptide used for the screenings of the present invention suitably contains PIF1 binding region.
  • Such partial peptides are also encompassed by the phrase "functional equivalent” of the CDC45L protein.
  • a partial PIF1 peptide used for the screenings of the present invention suitably contains at least a binding domain of PIF1.
  • a partial PIF1 peptide used for the screenings of the present invention suitably contains CDC45L binding region.
  • Such partial peptides are also encompassed by the phrase "functional equivalent" of the PIF1 protein.
  • the polypeptide or fragments used for the present method can be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence.
  • conventional peptide synthesis methods that can be adopted for the synthesis include: (1) Peptide Synthesis, Interscience, New York, 1966; (2) The Proteins, Vol. 2, Academic Press, New York, 1976; (3) Peptide Synthesis (in Japanese), Maruzen Co., 1975; (4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985; (5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991; (6) WO99/67288; and (7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.
  • the protein can be obtained adopting any known genetic engineering methods for producing polypeptides (e.g., Morrison DA., et al., J Bacteriol. 1977 Oct;132(1):349-51; Clark-Curtiss JE & Curtiss R 3rd. Methods Enzymol. 1983;101:347-62).
  • a suitable vector comprising a polynucleotide encoding the objective protein in an expressible form (e.g., downstream of a regulatory sequence comprising a promoter) is prepared, transformed into a suitable host cell, and then the host cell is cultured to produce the protein.
  • a gene encoding the CDC45L or PIF1 is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, for example, pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8.
  • a promoter can be used for the expression. Any commonly used promoters can be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic engineering, vol. 3. Academic Press, London, 1982, 83-141), the EF- alpha promoter (Kim DW, et al. Gene. 1990 Jul 16;91(2):217-23), the CAG promoter (Niwa H, et al., Gene. 1991 Dec 15;108(2):193-9), the RSV LTR promoter (Cullen BR. Methods Enzymol. 1987;152:684-704), the SR alpha promoter (Takebe Y, et al., Mol Cell Biol.
  • the introduction of the vector into host cells to express the CDC45L or PIF1 gene can be performed according to any methods, for example, the electroporation method (Chu G, et al., Nucleic Acids Res. 1987 Feb 11;15(3):1311-26), the calcium phosphate method (Chen C & Okayama H. Mol Cell Biol. 1987 Aug;7(8):2745-52), the DEAE dextran method (Lopata MA, et al., Nucleic Acids Res. 1984 Jul 25;12(14):5707-17; Sussman DJ & Milman G. Mol Cell Biol. 1984 Aug;4(8):1641-3), the Lipofectin method (Derijard B, et al., Cell.
  • the proteins can also be produced in vitro by using an in vitro translation system.
  • the phrase “CDC45L gene” encompasses polynucleotides that encode the human CDC45L or any of the functional equivalents of the human CDC45L gene.
  • the phrase “PIF1 gene” encompasses polynucleotides that encode the human PIF1 or any of the functional equivalents of the human PIF1 gene.
  • the CDC45L gene and the PIF1 gene can be obtained from nature as naturally occurring proteins via conventional cloning methods or through chemical synthesis based on the selected nucleotide sequence. Methods for cloning genes using cDNA libraries and such are well known in the art.
  • antibody as used herein is intended to include immunoglobulins and fragments thereof which are specifically reactive to the designated protein or peptide thereof.
  • An antibody can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabels, and antibody fragments.
  • an antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.
  • An “antibody” indicates all classes (e.g. IgA, IgD, IgE, IgG and IgM).
  • the present invention may use antibodies against CDC45L protein or PIF1 protein. These antibodies can be useful for diagnosing lung cancer. Furthermore, the present invention may use antibodies against partial peptides of CDC45L polypeptides or PIF polypeptides.
  • antibodies against the PIF binding region of CDC45L polypeptides or the CDC45L binding region of the PIF1 polypeptides may be preferably used. These antibodies can be useful for inhibiting and/or blocking an interaction, e.g. binding, between CDC45L polypeptides and PIF1 polypeptides and can be useful for treating and/or preventing cancer (over)expressing CDC45L and/or PIF1, e.g. lung cancer. These antibodies will be provided by known methods. For techniques for the production of the antibodies used in the present invention, conventional methods can be used.
  • double-stranded molecules refers to a nucleic acid molecule that inhibits the expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g. double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).
  • siRNA short interfering RNA
  • dsRNA double-stranded ribonucleic acid
  • shRNA small hairpin RNA
  • siD/R-NA short interfering DNA/RNA
  • siRNA refers to a double-stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed.
  • the siRNA includes a ribonucleotide corresponding to a sense nucleic acid sequence of a target gene (also referred to as “sense strand”), a ribonucleotide corresponding to an antisense nucleic acid sequence of a target gene (also referred to as "antisense strand”) or both.
  • the siRNA can be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin.
  • the siRNA can either be a dsRNA or shRNA.
  • dsRNA refers to a construct of two RNA molecules comprising complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule.
  • the double-stranded RNA molecule may also refer to siRNA, or small interfering RNA molecule.
  • the sequence of two strands can comprise not only the "sense” or "antisense” RNAs selected from a protein coding sequence of a target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.
  • shRNA refers to an siRNA having a stem-loop structure, comprising a first and second regions complementary to one another, i.e., sense and antisense strands.
  • the degree of complementarity and orientation of the region is sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and can also be referred to as "intervening single-strand".
  • siD/R-NA refers to a double-stranded molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA.
  • a hybrid indicates a molecule wherein an oligonucleotide composed of DNA and an oligonucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule can contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used.
  • the siD/R-NA includes a sense nucleic acid sequence of a target gene (also referred to as "sense strand"), an antisense nucleic acid sequence of a target gene (also referred to as “antisense strand”) or both.
  • the siD/R-NA can be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin.
  • the siD/R-NA can either be a dsD/R-NA or shD/R-NA.
  • dsD/R-NA refers to a construct of two molecules comprising complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule.
  • the nucleotide sequence of two strands can comprise not only the "sense” or "antisense” polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene.
  • One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).
  • shD/R-NA refers to an siD/R-NA having a stem-loop structure, comprising a first and second regions complementary to one another, i.e., sense and antisense strands.
  • the degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and can also be referred to as "intervening single-strand".
  • Target sequence is a nucleotide sequence within an mRNA or cDNA sequence of a target gene, which will result in suppress of translation of the whole mRNA of the target gene if a double-stranded molecule targeting the sequence is introduced into a cell expressing the target gene.
  • a nucleotide sequence within an mRNA or cDNA sequence of a target gene can be determined to be a target sequence when a double-stranded molecule comprising a sequence corresponding to the target sequence inhibits expression of the target gene in a cell expressing the gene.
  • the double stranded polynucleotide by which suppresses the gene expression may consist of the target sequence and 3' overhang (e.g., uu).
  • the sense strand of the double-stranded cDNA i.e., a sequence that mRNA sequence is converted into DNA sequence
  • a double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule.
  • the phrase "corresponding to” means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule.
  • a target sequence is shown in DNA sequence and a sense strand of a double-stranded molecule has an RNA region
  • base “t”s within the RNA region is replaced with base “u”s.
  • base "u"s within the DNA region is replaced with "t”s.
  • a sequence corresponding to a target sequence is "5'- GCAAACACCUGCTCAAGTC-3'".
  • a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand. For example, when a target sequence is shown in the RNA sequence of SEQ ID NO: 9 and the antisense strand of the double-stranded molecule has the 5' side half region composed of DNA, "a complementary sequence to a target sequence" is 3'- CGUUUGUGGACGAGTTCAG -5'".
  • the sequence corresponding to a target sequence of SEQ ID NO: 9 is the RNA sequence of SEQ ID NO: 9
  • the complementary sequence corresponding to a target sequence of SEQ ID NO: 9 is the RNA sequence of "3'- CGUUUGUGGACGAGUUCAG -5'".
  • a double-stranded molecule may has one or two 3'overhangs having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto.
  • a double-stranded molecule against CDC45L gene which molecule hybridizes to a CDC45L mRNA, inhibits or reduces production of CDC45L protein encoded by the gene by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and thus, inhibiting expression of the CDC45L protein.
  • a double-stranded molecule against PIF1 gene which molecule hybridizes to a PIF1 mRNA, inhibits or reduces production of PIF1 protein encoded by the genes by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and thus, inhibiting expression of the PIF1 protein.
  • the present invention provides isolated double-stranded molecules having the property to inhibit or reduce the expression of CDC45L gene or PIF1 gene in cancer cells when introduced into a cell.
  • the target sequences of double-stranded molecules may be designed by siRNA design algorithm mentioned below.
  • target sequences for CDC45L include, for example, 5'- GCAAACACCUGCUCAAGUC -3' (SEQ ID NO: 9) or 5'- GGACGUGGAUGCUCUGUGU -3' (SEQ ID NO: 10).
  • target sequences for PIF1 include, for example, 5'- GAAAGGCCAGAGCAUCUUC-3' (SEQ ID NO: 11) or 5'- GGCAUGACCCUGGAUUGUG-3' (SEQ ID NO: 12).
  • the present invention also provides a double-stranded molecule whose target sequence comprises or consisting of SEQ ID NO: 9, 10, 11 or 12.
  • the present invention provides the following double-stranded molecules [1] to [18]:
  • An isolated double-stranded molecule which, when introduced into a cell, inhibits in vivo expression of a CDC45L gene or PIF1 gene and cell proliferation, wherein the double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form a double strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:
  • [8] The double-stranded molecule of [7], which has a length of between about 19 and about 25 nucleotides.
  • [9] The double-stranded molecule of any one of [1] to [8], which consists of a single oligonucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
  • [10] The double-stranded molecule of [9], which has a general formula 5'-[A]-[B]-[A']-3', wherein [A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 [B] is an intervening single-strand; and [A'] is an antisense strand comprising a nucleotide sequence corresponding to a sequence complementary to the target sequence selected in [A]. [11] The double-stranded molecule of any one of [1] to [10], which comprises RNA.
  • [12] The double-stranded molecule of any one of [1] to [11], which comprises both DNA and RNA.
  • the double-stranded molecule of [12] which is a hybrid of a DNA polynucleotide and an RNA polynucleotide.
  • the double-stranded molecule of [12] which is a chimera of DNA and RNA.
  • [16] The double-stranded molecule of [15], wherein a 5'-end region of the target sequence in the sense strand, and/or a 3'-end region of the complementary sequence of the target sequence in the antisense strand consists of RNA. [17] The double-stranded molecule of [16], wherein the RNA region consists of 9 to 13 nucleotides; and [18] The double-stranded molecule of any one of [1] to [2], which contains one or two 3' overhang(s).
  • the double-stranded molecule of the present invention will be described in more detail below.
  • Methods for designing double-stranded molecules having the ability to inhibit target gene expression in cells are known. (See, for example, US Pat No. 6,506,559, herein incorporated by reference in its entirety).
  • a computer program for designing siRNAs is available from the Ambion website (on the worldwide web at ambion.com/techlib/misc/siRNA_finder.html).
  • the computer program selects target nucleotide sequences for double-stranded molecules based on the following protocol.
  • Target Sites 1. Beginning with the AUG start codon of the transcript, scan downstream for AA di-nucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. recommend to avoid designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these can be richer in regulatory protein binding sites, and UTR-binding proteins and/or translation initiation complexes can interfere with binding of the siRNA endonuclease complex. 2. Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences.
  • BLAST which can be found on the NCBI server at: on the worldwide web at ncbi.nlm.nih.gov/BLAST/, is used (Altschul SF, et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). 3. Select qualifying target sequences for synthesis. Selecting several target sequences along the length of the gene to evaluate is typical.
  • Preferred target sequences for CDC45L gene designed in “Examples” include 5'- GCAAACACCUGCUCAAGUC -3' (SEQ ID NO: 9) or 5'- GGACGUGGAUGCUCUGUGU -3' (SEQ ID NO: 10).
  • preferred target sequences for PIF1 gene designed in “Examples” include, 5'- GAAAGGCCAGAGCAUCUUC-3' (SEQ ID NO: 11) or 5'- GGCAUGACCCUGGAUUGUG-3' (SEQ ID NO: 12).
  • the present invention provides the double-stranded molecules targeting any one of above-mentioned target sequences that were respectively examined for their ability to inhibit and reduce the growth of cancer cells expressing the target genes.
  • the present invention provides double-stranded molecules targeting a target sequence for CDC45L gene selected from the group consisting of 5'- GCAAACACCUGCUCAAGUC -3' (SEQ ID NO: 9) and 5'- GGACGUGGAUGCUCUGUGU -3' (SEQ ID NO: 10).
  • the present invention provides double-stranded molecules targeting a target sequence for PIF1 gene selected from the group consisting of 5'- GAAAGGCCAGAGCAUCUUC-3' (SEQ ID NO: 11) and 5'- GGCAUGACCCUGGAUUGUG-3' (SEQ ID NO: 12).
  • the double-stranded molecules of the present invention targeting the above-mentioned target sequence of CDC45L or PIF1 gene include isolated polynucleotide(s) that comprises any one of the nucleic acid sequences of target sequences and/or complementary sequences to the target sequences.
  • Examples of a double-stranded molecule targeting CDC45L or PIF1 gene include an oligonucleotide comprising the sequence corresponding to SEQ ID NO: 9, 10, 11 or 12, and complementary sequences thereto.
  • the present invention is not limited to these examples, and minor modifications in the aforementioned nucleic acid sequences are acceptable so long as the modified molecule retains the ability to suppress the expression of CDC45L or PIF1 gene.
  • “minor modification" in a nucleic acid sequence indicates one, two or several substitution, deletion, addition or insertion of nucleic acids to the sequence.
  • a double-stranded molecule is composed of two polynucleotides, one polynucleotide has a sequence corresponding to a target sequence, i.e., sense strand, and another polypeptide has a complementary sequence to the target sequence, i.e., antisense strand.
  • the sense strand polynucleotide and the antisense strand polynucleotide hybridize to each other to form double-stranded molecule.
  • double-stranded molecules include dsRNA and dsD/R-NA.
  • a double-stranded molecule is composed of a polynucleotide that has both a sequence corresponding to a target sequence, i.e., sense strand, and a complementary sequence to the target sequence, i.e., antisense strand.
  • the sense strand and the antisense strand are linked by an intervening strand, and hybridize to each other to form a hairpin loop structure.
  • Examples of such double-stranded molecule include shRNA and shD/R-NA.
  • a double-stranded molecule of the present invention comprises a sense strand polynucleotide having a nucleotide sequence of the target sequence and anti-sense strand polynucleotide having a nucleotide sequence complementary to the target sequence, and both of polynucleotides hybridize to each other to form the double-stranded molecule.
  • a part of the polynucleotide of either or both of the strands may be RNA, and when the target sequence is defined with a DNA sequence, the nucleotide "t" within the target sequence and complementary sequence thereto is replaced with "u".
  • such a double-stranded molecule of the present invention comprises a stem-loop structure, composed of the sense and antisense strands.
  • the sense and antisense strands may be joined by a loop.
  • the present invention also provides the double-stranded molecule comprising a single polynucleotide containing both the sense strand and the antisense strand linked or flanked by an intervening single-strand.
  • double-stranded molecules targeting the CDC45L gene or PIF1 gene may have a sequence selected from among SEQ ID NOs: 9, 10, 11 and 12 as a target sequence.
  • preferable examples of the double-stranded molecule of the present invention include polynucleotides that hybridize to each other at a sequence corresponding to SEQ ID NO: 9, 10, 11 or 12 and a complementary sequence thereto, and a polynucleotide that has a sequence corresponding to SEQ ID NO: 9, 10, 11 or 12 and a complementary sequence thereto.
  • a double-stranded molecule of the present invention can be tested for its ability using the methods utilized in the Examples (see, RNA interference assay in "EXAMPLES").
  • the double-stranded molecules comprising sense strands and antisense strands complementary thereto of various portions of mRNA of CDC45L or PIF1 genes were tested in vitro for their ability to decrease production of CDC45L or PIF1 gene product in cancers cell lines (e.g., using SBC-3 and A549) according to standard methods.
  • CDC45L or PIF1 gene product in cells contacted with the candidate double-stranded molecule compared to cells cultured in the absence of the candidate molecule can be detected by, e.g. RT-PCR using primers for CDC45L or PIF1 gene mRNA mentioned (see,(b)) Semi-quantitative RT-PCR in "EXAMPLES"). Sequences which decrease the production of CDC45L or PIF1 gene product in vitro cell-based assays can then be tested for there inhibitory effects on cell growth. Sequences which inhibit cell growth in vitro cell-based assay can then be tested for their in vivo ability using animals with cancer, e.g. nude mouse xenograft models, to confirm decreased production of CDC45L or PIF1 gene product and decreased cancer cell growth.
  • the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a polynucleotide
  • binding means the physical or chemical interaction between two polynucleotides.
  • the polynucleotide comprises modified nucleotides and/or non-phosphodiester linkages, these polynucleotides can also bind each other as same manner.
  • complementary polynucleotide sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches.
  • the sense strand and antisense strand of the isolated polynucleotide of the present invention can form double-stranded molecule or hairpin loop structure by the hybridization.
  • such duplexes contain no more than 1 mismatch for every 10 matches.
  • such duplexes contain no mismatches.
  • the polynucleotide of the invention are typically less than 500, 200, 100, 75, 50, or 25 nucleotides in length.
  • the isolated polynucleotides of the present invention are useful for forming double-stranded molecules against CDC45L or PIF1 gene or preparing template DNAs encoding the double-stranded molecules.
  • the sense strand of the polynucleotide can be longer than 19 nucleotides, for example, longer than 21 nucleotides, for example, between about 19 and 25 nucleotides.
  • the present invention provides the double-stranded molecules comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence.
  • the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
  • the double-stranded molecule serves as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells.
  • the identified target RNA is cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA.
  • a double-stranded molecule of the invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the CDC45L or PIF1 gene under stringent conditions.
  • target sequence or “target nucleic acid” or “target nucleotide”.
  • nucleotide sequence of the “target sequence” can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.
  • the double-stranded molecules of the invention can contain one or more modified nucleotides and/or non-phosphodiester linkages.
  • Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule.
  • the skilled person will be aware of other types of chemical modification which can be incorporated into the present molecules (WO03/070744; WO2005/045037).
  • modifications can be used to provide improved resistance to degradation or improved uptake.
  • modifications include phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base” nucleotides, 5'-C- methyl nucleotides, and inverted deoxyabasic residue incorporation (US Pat Appl. No. 20060122137).
  • modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule.
  • Modifications include chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2 -fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212).
  • modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976).
  • an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine.
  • an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine.
  • the double-stranded molecule when the double-stranded molecule is a double-stranded molecule with a 3' overhang, the 3'- terminal nucleotide overhanging nucleotides can be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200).
  • deoxyribonucleotides Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200.
  • published documents for example, US Pat Appl. No.20060234970 are available.
  • the present invention is not limited to these examples and any known chemical modifications can be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.
  • the double-stranded molecules of the invention can comprise both DNA and RNA, e.g., dsD/R-NA or shD/R-NA.
  • RNA e.g., dsD/R-NA or shD/R-NA.
  • a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability.
  • RNA i.e., a hybrid type double-stranded molecule made of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule comprising both DNA and RNA on any or both of the single strands (polynucleotides), or the like can be formed for enhancing stability of the double-stranded molecule.
  • the hybrid of a DNA strand and an RNA strand can be either where the sense strand is DNA and the antisense strand is RNA, or the opposite so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA.
  • the chimera type double-stranded molecule may have the structure either where both of the sense and antisense strands are composed of DNA and RNA, or where any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
  • the molecule contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression.
  • an upstream partial region i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands
  • RNA is RNA
  • the upstream partial region means the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand. That is, in some embodiments, a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand consists of RNA.
  • the chimera or hybrid type double-stranded molecule of the present invention comprise following combinations.
  • sense strand 5'-[DNA]-3' 3'-(RNA)-[DNA]-5': antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(RNA)-[DNA]-5': antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(RNA)-5': antisense strand
  • sense strand 5'-(RNA)-[DNA]-3' 3'-(RNA)-5': antisense strand.
  • the upstream partial region can be a domain of about 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules.
  • examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA.
  • the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US Pat Appl. No. 20050004064).
  • the double-stranded molecule can form a hairpin, for example, a short hairpin RNA (shRNA) and short hairpin made of DNA and RNA (shD/R-NA).
  • shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference.
  • the shRNA or shD/R-NA comprises the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence.
  • the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the target sequence of the dsRNA or dsD/R-NA.
  • RISC RNA-induced silencing complex
  • a loop sequence composed of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure.
  • the present invention also provides a double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to [A].
  • the target sequence can be selected from the group consisting of, for example, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
  • the present invention is not limited to these examples, and the target sequence in [A] can be modified sequences from these examples so long as the double-stranded molecule retains the ability to suppress the expression of the targeted CDC45L or PIF1 gene and result in inhibits or reduces the cell expressing these genes.
  • the region [A] hybridizes to [A'] to form a loop comprising the region [B].
  • the intervening single-stranded portion [B], i.e., the loop sequence can be 3 to 23 nucleotides in length.
  • the loop sequence for example, can be selected from group consisting of following sequences (on the worldwide web at ambion.com/techlib/tb/tb_506.html).
  • loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26): CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26; UUCG: Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb 18, 100(4): 1639-44, Epub 2003 Feb 10; and UUCAAGAGA: Dykxhoorn DM et al., Nat Rev Mol Cell Biol 2003 Jun, 4(6): 457-67.
  • the loop sequence can be selected from group consisting of AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA; however, the present invention is not limited thereto: GCAAACACCUGCUCAAGUC-[B]- GACUUGAGCAGGUGUUUGC (for target sequence of SEQ ID NO: 9); GGACGUGGAUGCUCUGUGU-[B]-ACACAGAGCAUCCACGUCC (for target sequence of SEQ ID NO: 10); GAAAGGCCAGAGCAUCUUC-[B]-GAAGAUGCUCUGGCCUUUC (for target sequence of SEQ ID NO: 11);and GGCAUGACCCUGGAUUGUG-[B]- CACAAUCCAGGGUCAUGCC (for target sequence of SEQ ID NO: 12).
  • nucleotides can be added to 3'end of the sense strand and/or the antisense strand, as 3' overhangs.
  • the number of nycleotides to be added is at least 2, generally 2 to 10, for example, 2 to 5.
  • the added nucleotides form single strand at the 3'end of sense strand and/or the antisense strand of the double-stranded molecule.
  • the nucleotides for 3' overhang are preferably "u" or "t", but are not limited to.
  • the method of preparing the double-stranded molecule can use any chemical synthetic method known in the art. According to the chemical synthesis method, sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule. Alternatively, a double stranded molecule or siRNA molecule of the present invention may also be synthesized with in vitro translation. In this embodiment, DNA encoding a nucleotide sequence that comprises the target sequence and antisense thereof is transcribed into the double stranded molecule in vitro.
  • the synthesized single-stranded polynucleotides are mixed in a molar ratio of at least about 3:7, for example, about 4:6, for example, substantially equimolar amount (i.e., a molar ratio of about 5:5).
  • the mixture is heated to a temperature at which double-stranded molecules dissociate and then is gradually cooled down.
  • the annealed double-stranded polynucleotide can be purified by usually employed methods known in the art.
  • Example of purification methods include methods utilizing agarose gel electrophoresis or wherein remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.
  • the regulatory sequences flanking target sequences can be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner.
  • the double-stranded molecules can be transcribed intracellularly by cloning CDC45L or PIF1 gene templates into a vector containing, e.g., a RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
  • snRNA small nuclear RNA
  • the double-stranded molecules may be transcribed intracellularly by cloning its coding sequence into a vector containing a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell (e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) adjacent to the coding sequence.
  • a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter
  • the regulatory sequences flanking the coding sequences of double-stranded molecule may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. Details of vectors which are capable of producing the double-stranded molecules will be described below.
  • Vector Also included in the invention is a vector containing one or more of the double-stranded molecules described herein, and a cell containing the vector.
  • a vector of the present invention encodes a double-stranded molecule of the present invention in an expressible form.
  • the phrase "in an expressible form” indicates that the vector, when introduced into a cell, will express the molecule.
  • the vector includes regulatory elements necessary for expression of the double-stranded molecule.
  • the expression vector encodes the double-stranded molecule of the present invention and is adapted for expression of the double-stranded molecule.
  • Such vectors of the present invention can be used for producing the present double-stranded molecules, or directly as an active ingredient for treating cancer.
  • the present invention provides vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to SEQ ID NOs: 9, 10, 11 or 12, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits expression of said gene.
  • the polynucleotide is an oligonucleotide of between about 19 and 25 nucleotides in length (e.g., contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 13, or 15). More preferably, the combination of polynucleotide comprises a single nucleotide transcript comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
  • the combination of polynucleotide has the general formula 5'-[A]-[B]-[A']-3', wherein [A] is a nucleotide sequence comprising SEQ ID NO: 9, 10, 11 or 12; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotide; and [A'] is a nucleotide sequence complementary to [A].
  • Vectors of the present invention can be produced, for example, by cloning a sequence comprising target sequence into an expression vector so that regulatory sequences are operatively-linked to the sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5).
  • RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • a first promoter e.g., a promoter sequence flanking to the 3' end of the cloned DNA
  • RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA).
  • the sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene.
  • two vectors constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and antisense strands and then forming a double-stranded molecule construct.
  • the cloned sequence can encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector contains both the sense and complementary antisense sequences of the target gene.
  • the vectors of the present invention can also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Pat Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720.
  • DNA-based delivery technologies include "naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., US Pat No. 5,922,687).
  • the vectors of the present invention can be, for example, viral or bacterial vectors.
  • expression vectors include attenuated viral hosts, for example, vaccinia or fowlpox (see, e.g., US Pat No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell.
  • Another example of useable vector includes Bacille Calmette Guerin (BCG).
  • BCG vectors are described in Stover et al., Nature 1991, 351: 456-60.
  • a wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.
  • the present invention provides methods for inhibiting cell growth, i.e., cancerous cell growth of a cell from a cancer resulting from overexpression of a CDC45L and/or PIF1 gene, or that is mediated by a CDC45L and/or PIF1 gene, by inhibiting the expression of the CDC45L and/or PIF1 gene.
  • CDC45L or PIF1 gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention which specifically target the expression of CDC45L or PIF1 gene or the vectors of the present invention that can express any of the double-stranded molecules of the present invention.
  • Such ability of the present double-stranded molecules and vectors to inhibit cell growth of cancerous cells indicates that they can be used for methods for treating cancer, a cancer resulting from overexpression of a CDC45L and/or PIF1 gene, or that is mediated by a CDC45L and/or PIF1 gene.
  • the present invention provides methods to treat patients with a cancer resulting from overexpression of a CDC45L and/or PIF1 gene, or that is mediated by a CDC45L and/or PIF1 gene by administering a double-stranded molecule, i.e., an inhibitory nucleic acid, against a CDC45L or PIF1 gene or a vector expressing the molecule without adverse effect because those genes were hardly detected in normal organs.
  • a double-stranded molecule i.e., an inhibitory nucleic acid
  • the present invention provides the following methods [1] to [23]: [1] A method for inhibiting or reducing a growth of a cell (over)expressing a CDC45L and/or PIF1 gene or a method for treating or preventing cancer (over)expressing CDC45L and/or PIF1 gene, wherein said method comprising the step of administering to a subject at least one double-stranded molecule or vector encoding the double-stranded molecule, wherein said double-stranded molecule, when introduced into a cell, inhibits or reduces in vivo expression of said CDC45L or PIF1 gene.
  • the method of the present invention will be described in more detail below.
  • the growth of cells (over)expressing a CDC45L and/or PIF1 gene is inhibited by contacting the cells with a double-stranded molecule against CDC45L or PIF1 gene, a vector expressing the molecule or a composition comprising the same.
  • the cell may be further contacted with a transfection agent. Suitable transfection agents are known in the art.
  • the phrase "inhibition of cell growth" indicates that the cell proliferates at a lower rate or has decreased viability compared to a cell not exposed to the molecule.
  • Cell growth can be measured by methods known in the art, e.g., using the MTT cell proliferation assay.
  • any kind of cell can be suppressed according to the present method so long as the cell expresses or over-expresses the target gene of the double-stranded molecule of the present invention.
  • Exemplary cells include cancers cells.
  • patients suffering from or at risk of developing disease related to CDC45L and/or PIF1 gene can be treated by administering at least one of the present double-stranded molecules, at least one vector expressing at least one of the molecules or at least one composition comprising at least one of the molecules.
  • patients of cancers can be treated according to the present methods.
  • the type of cancer can be identified by standard methods according to the particular type of tumor to be diagnosed.
  • patients treated by the methods of the present invention are selected by detecting the (over)expression of a CDC45L and/or PIF1 gene in a biopsy from the patient by RT-PCR, hybridization or immunoassay.
  • the biopsy specimen from the subject is confirmed for CDC45L and/or PIF1 gene over-expression by methods known in the art, for example, immunohistochemical analysis, hybridization or RT-PCR (see, Semi-quantitative RT-PCR, Western-blotting or Immunohistochemistry in "EXAMPLES”).
  • each of the molecules can direct to the different target sequence of same gene, or different target sequences of different gene.
  • the method can utilize different double-stranded molecules directing to CDC45L or PIF1 gene transcript.
  • the method can utilize double-stranded molecules directed to one, two or more target sequences selected from same gene.
  • a double-stranded molecule of present invention can be directly introduced into the cells in a form to achieve binding of the molecule with corresponding mRNA transcripts.
  • a DNA encoding the double-stranded molecule can be introduced into cells as a vector.
  • transfection-enhancing agent for example, FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical
  • FuGENE FuGENE (Roche diagnostics)
  • Lipofectamine 2000 Invitrogen
  • Oligofectamine Oligofectamine
  • Nucleofector Nucleofector
  • a treatment is determined efficacious if it leads to clinical benefit for example, reduction in expression of a CDC45L or PIF1 gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject.
  • "efficacious” means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.
  • prevention and prophylaxis can occur “at primary, secondary and tertiary prevention levels.” While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
  • the treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis.
  • Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer.
  • reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis include 10%, 20%, 30% or more reduction, or stable disease.
  • the double-stranded molecule of the invention degrades the target mRNA (gene transcripts) in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, compared to standard cancer therapies, significantly less a double-stranded molecule needs to be delivered at or near the site of cancer to exert therapeutic effect.
  • an effective amount of the double-stranded molecule of the invention to be administered to a given subject, by taking into account factors for example, body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic.
  • an effective amount of the double-stranded molecule of the invention comprises an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, for example, from about 2 nM to about 50 nM, for example, from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered.
  • the present methods can be used to inhibit the growth or metastasis of cancer; for example, a cancer resulting from overexpression of a CDC45L and/or PIF1 gene or that is mediated by a CDC45L and/or PIF1 gene, e.g., lung cancer.
  • a double-stranded molecule directed to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10 for CDC45L, and SEQ ID NO: 11 and SEQ ID NO: 12 for PIF1 find use for the treatment of cancers.
  • the double-stranded molecule of the invention can also be administered to a subject in combination with a pharmaceutical agent different from the double-stranded molecule.
  • the double-stranded molecule of the invention can be administered to a subject in combination with another therapeutic method designed to treat cancer.
  • the double-stranded molecule of the invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents, for example, cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).
  • chemotherapeutic agents for example, cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.
  • the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the double-stranded molecule.
  • Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
  • the delivery reagent is a liposome.
  • Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, for example, retinal or tumor tissue, and can also increase the blood half-life of the double-stranded molecule.
  • Liposomes suitable for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, for example, cholesterol. The selection of lipids is generally guided by consideration of factors for example, the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.
  • the liposomes encapsulating the present double-stranded molecule comprises a ligand molecule that can deliver the liposome to the cancer site.
  • Ligands which bind to receptors prevalent in tumor or vascular endothelial cells for example, monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, find use.
  • the liposomes encapsulating the present double-stranded molecule are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure.
  • a liposome of the invention can comprise both opsonization-inhibition moieties and a ligand.
  • Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane.
  • an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids.
  • These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No.
  • Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
  • Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature.
  • target tissue characterized by such microvasculature defects for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53.
  • the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen.
  • liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.
  • Opsonization inhibiting moieties suitable for modifying liposomes can be water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, for example, from about 2,000 to about 20,000 daltons.
  • Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers for example, polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, for example, ganglioside GM 1 .
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • synthetic polymers for example, polyacrylamide or poly N-viny
  • Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
  • the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
  • the opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
  • natural polysaccharides containing amino acids or carboxylic acids e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan
  • aminated polysaccharides or oligosaccharides linear or branched
  • the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof.
  • Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
  • the opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques.
  • an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane.
  • a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH 3 and a solvent mixture for example, tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.
  • Vectors expressing a double-stranded molecule of the invention are discussed above. Such vectors expressing at least one double-stranded molecule of the invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
  • a suitable delivery reagent including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes.
  • the double-stranded molecule of the invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites.
  • the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
  • Suitable enteral administration routes include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (for example, by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation.
  • injections or infusions of the double-stranded molecule or vector can be given at or near the site of cancer.
  • the double-stranded molecule of the invention can be administered in a single dose or in multiple doses.
  • the infusion can be a single sustained dose or can be delivered by multiple infusions.
  • Injection of the agent can be directly into the tissue or near the site of cancer. Multiple injections of the agent into the tissue at or near the site of cancer can be administered.
  • the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site.
  • the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, for example, from about seven to about ten days.
  • the double-stranded molecule is injected at or near the site of cancer once a day for seven days.
  • a dosage regimen comprises multiple administrations, it is understood that the effective amount of a double-stranded molecule administered to the subject can comprise the total amount of a double-stranded molecule administered over the entire dosage regimen.
  • a cancer overexpressing CDC45L and/or PIF1 can be treated with at least one active ingredient selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof.
  • the cancer includes, but is not limited to, lung cancer. Accordingly, prior to the administration of the double-stranded molecule of the present invention as active ingredient, it is preferable to confirm whether the expression level of CDC45L and/or PIF1 in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ.
  • the present invention provides a method for treating a cancer (over)expressing CDC45L and/or PIF1, which method may include the steps of: i) determining the expression level of CDC45L and/or PIF1 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated; ii) comparing the expression level of CDC45L and/or PIF1 with normal control; and iii) administrating at least one component selected from the group consisting of (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, to a subject with a cancer overexpressing CDC45L and/or PIF1 as compared with normal control.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one component selected from the group consisting of: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, and (c) a vector encoding thereof, for use in administrating to a subject having a cancer overexpressing CDC45L and/or PIF1.
  • the present invention further provides a method for identifying a subject to be treated with: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, or (c) a vector encoding thereof, which method may include the step of determining an expression level of CDC45L and/or PIF1 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, or (c) a vector encoding thereof.
  • a subject to be treated by the present method is preferably a mammal.
  • exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • the expression level of CDC45L and/or PIF1 in cancer cell in cancer cells or tissues obtained from a subject is determined.
  • the expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art.
  • the mRNA of CDC45L or PIF1 may be quantified using probes by hybridization methods (e.g., Northern hybridization).
  • the detection may be carried out on a chip or an array. The use of an array is preferable for detecting the expression level of CDC45L and/or PIF1.
  • Those skilled in the art can prepare such probes utilizing the sequence information of CDC45L or PIF1.
  • the cDNA of CDC45L or PIF1 may be used as the probes.
  • the probes may be labeled with a suitable label, such as dyes, fluorescent substances and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
  • the transcription product of CDC45L or PIF1 may be quantified using primers by amplification-based detection methods (e.g., RT-PCR).
  • primers may be prepared based on the available sequence information of the gene.
  • a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of CDC45L or PIF1.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but not to other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degree Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to their target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degree Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degree Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • the translation product may be detected for the diagnosis of the present invention.
  • the quantity of CDC45L or PIF1 protein SEQ ID NO: 14, or 16
  • Methods for determining the quantity of the protein as the translation product include immunoassay methods that use an antibody specifically recognizing the protein.
  • the antibody may be monoclonal or polyclonal.
  • any fragment or modification e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining may be measured via immunohistochemical analysis using an antibody against the CDC45L or PIF1 protein. Namely, in this measurement, strong staining indicates increased presence/level of the protein and, at the same time, high expression level of CDC45L or PIF1 gene.
  • the expression level of a target gene, i.e., the CDC45L or PIF1 gene, in cancer cells can be determined to be increased if the level increases from the control level (e.g., the level in normal cells) of the target gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
  • the control level may be determined at the same time with the cancer cells by using a sample(s) previously collected and stored from a subject/subjects whose disease state(s) (cancerous or non-cancerous) is/are known.
  • control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of CDC45L and/or PIF1 gene in samples from subjects whose disease states are known.
  • control level can be derived from a database of expression patterns from previously tested cells.
  • the expression level of CDC45L and/or PIF1 gene in a biological sample may be compared to multiple control levels, which are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample.
  • the standard value of the expression levels of CDC45L and/or PIF1 gene in a population with a known disease state.
  • the standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as the standard value.
  • control level determined from a biological sample that is known to be non-cancerous is referred to as a "normal control level”.
  • control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level”.
  • the subject When the expression level of CDC45L and/or PIF1 gene is increased as compared to the normal control level, or is similar/equivalent to the cancerous control level, the subject may be diagnosed with cancer to be treated.
  • compositions comprising at least one of the present double-stranded molecules or the vectors coding for the molecules.
  • the present invention provides the following compositions [1] to [24]: [1] A composition for inhibiting or reducing a growth of cell expressing CDC45L and/or PIF1 gene, or for treating or preventing a cancer expressing a CDC45L and/or PIF1 gene, which comprises at least one double-stranded molecule or vector encoding the double-stranded molecule, wherein said double-stranded molecule, when introduced into a cell, inhibits or reduces in vivo expression of said gene.
  • composition of [1], wherein said double-stranded molecule acts at mRNA which matched a target sequence selected from the group SEQ ID NO: 9 and 10 for CDC45L and SEQ ID NO: 11 and 12 for PIF1.
  • composition of [1], wherein said double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form a double strand, wherein said sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12.
  • composition of any one of [1] to [3], wherein the cancer to be treated is lung cancer; [5] The composition of [4], wherein the lung cancer is small cell lung cancer or non-small cell lung cancer; [6] The composition of any one of [1] to [5], wherein the composition contains plural kinds of the double-stranded molecules; [7] The composition of [6], wherein the plural kinds of the double-stranded molecules target the same gene; [8] The composition of any one of [1] to [7], wherein the double-stranded molecule has a length of less than about 100 nucleotides; [9] The composition of [8], wherein the double-stranded molecule has a length of less than about 75 nucleotides; [10] The composition of [9], wherein the double-stranded molecule has a length of less than about 50 nucleotides; [11] The composition of [10], wherein the double-stranded molecule has a length of less than about 25 nucleotides
  • [14] The composition of [13], wherein said double-stranded molecule has a general formula 5'-[A]-[B]-[A']-3', wherein [A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12; [B] is the intervening single-strand; and [A'] is the antisense strand comprising an oligonucleotide corresponding to a sequence complementary to the target sequence selected in [A].
  • composition of any one of [1] to [14], wherein the double-stranded molecule comprises RNA; [16] The composition of any one of [1] to [14], wherein the double-stranded molecule comprises DNA and RNA; [17] The composition of [16], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide; [18] The composition of [17], wherein the sense and antisense strand polynucleotides are made of DNA and RNA, respectively; [19] The composition of [18], wherein the double-stranded molecule is a chimera of DNA and RNA; [20] The composition of [19], wherein at least a region flanking to the 5'-end of one or both of the sense and antisense polynucleotides consists of RNA.
  • [21] The composition of [20], wherein the flanking region consists of 9 to 13 nucleotides; [22] The composition of any one of [1] to [21], wherein the double-stranded molecule contains one or two 3' overhang(s); [23] The composition of any one of [1] to [22], wherein the double-stranded molecule is encoded by a vector; [24] The composition of any one of [1] to [23], which further comprising a transfection-enhancing agent, cell permeable agent or pharmaceutically acceptable carrier.
  • the double-stranded molecules of the invention can be formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art.
  • Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free.
  • pharmaceutical formulations include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
  • the present pharmaceutical formulations comprise at least one of the double-stranded molecules or vectors encoding them of the present invention (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt of the molecule, mixed with a physiologically acceptable carrier medium.
  • physiologically acceptable carrier media include, for example, water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
  • the composition can contain plural kinds of the double-stranded molecules, each of the molecules can be directed to the same target sequence, or different target sequences of CDC45L or PIF1 gene.
  • the composition can contain double-stranded molecules directed to CDC45L or PIF1 gene.
  • the composition can contain double-stranded molecules directed to target sequences selected from CDC45L and PIF1 gene.
  • the present composition can contain a vector coding for one or plural double-stranded molecules.
  • the vector can encode one, two or several kinds of the present double-stranded molecules.
  • the present composition can contain plural kinds of vectors, each of the vectors coding for a different double-stranded molecule.
  • the present double-stranded molecules can be contained as liposomes in the present composition. See under the item of "(iii) Methods Of Inhibiting Or Reducing A Growth Of Cancer Cells And Treating Or Preventing Cancer Using Double-Stranded Molecules r" for details of liposomes.
  • compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives.
  • Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (for example, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
  • conventional nontoxic solid carriers can be used; for example, pharmaceutical grade of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%, for example, 25-75%, of one or more double-stranded molecule of the invention.
  • a pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, for example, 1-10% by weight, of one or more double-stranded molecule of the invention encapsulated in a liposome as described above, and propellant.
  • a carrier can also be included as desired; e.g., lecithin for intranasal delivery.
  • the present composition can contain other pharmaceutical active ingredients so long as they do not inhibit the in vivo function of the present double-stranded molecules.
  • the composition can contain chemotherapeutic agents conventionally used for treating cancers.
  • the present invention also provides the use of the double-stranded nucleic acid molecules of the present invention in manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CDC45L and/or PIF1 gene.
  • the present invention relates to the use of double-stranded nucleic acid molecule inhibiting the (over)expression of a CDC45L or PIF1 gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9, 10, 11 or 12, for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CDC45L and/or PIF1 gene.
  • the present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the CDC45L and/or PIF1 gene.
  • the present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CDC45L and/or PIF1 gene, wherein the method or process comprises step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the (over)expression of a CDC45L or PIF1 gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9, 10, 11 or 12 as active ingredients.
  • the present invention also provides a method or process for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CDC45L and/or PIF1 gene, wherein the method or process comprises step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of CDC45L or PIF1 gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9, 10, 11 or 12.
  • the present invention provides a method for diagnosing cancers mediated by CDC45L and/or PIF1 by determining the expression level of CDC45L and/or PIF1 in a subject.
  • the CDC45L and/or PIF1 -promoted cancers that can be diagnosed by the present method include lung cancers.
  • Lung cancers include non-small lung cancer, small lung cancer, a lung adenocarcinoma (ADC), a lung squamous-cell carcinoma (SCC), and a lung large-cell carcinoma (LCC).
  • ADC lung adenocarcinoma
  • SCC lung squamous-cell carcinoma
  • LCC lung large-cell carcinoma
  • Such intermediate result can be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease.
  • the present invention can be used to detect cancerous cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.
  • the present invention provides the following methods [1] to [15]: [1] A method for diagnosing cancer mediated or promoted by a CDC45L or PIF1, wherein said method comprises the steps of: (a) detecting the expression level of CDC45L gene and/or PIF1 gene in a subject-derived biological sample; and (b) relating an increase of the expression level compared to a normal control level of the gene to the disease.
  • a method of detecting or diagnosing cancer in a subject comprising determining a expression level of CDC45L and/or PIF1 in a subject-derived biological sample, wherein an increase of the level compared to a normal control level of the gene indicates that the subject suffers from or is at risk of developing cancer, or the presence of cancer in the subject, [3] The method of [1] or [2], wherein the expression level is at least 10 % greater than normal control level.
  • [4] The method of any one of [1] to [3], wherein the expression level is detected by any one of the method select from the group consisting of: (a) detecting the mRNA encoding the CDC45L or PIF1 polypeptide; (b) detecting the CDC45L or PIF1 polypeptide; and (c) detecting the biological activity of the CDC45L or PIF1 polypeptide.
  • [5] The method of any one of [1] to [3], wherein the expression level is determined by a method selected from a group consisting of: (a) detecting an mRNA of CDC45L gene and/or an mRNA of PIF1 gene, (b) detecting a protein encoded by CDC45L gene and/or a protein encoded by PIF1 gene, and (c) detecting (a) biological activity(activities) of a protein encoded by CDC45L gene and/or a protein encoded by PIF1 gene.
  • [6] The method of any one of [1] to [5], wherein the cancer results from overexpression of a CDC45L or PIF1, or is mediated or promoted by a CDC45L or PIF1.
  • [7] The method of any one of [1] to [6], wherein the cancers is lung cancer.
  • [8] The method of [7], wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.
  • the method of [4] or [5], wherein the expression level is determined by detecting a binding of an antibody against the CDC45L or PIF1 polypeptide.
  • the biological sample comprises biopsy sample, sputum or blood.
  • a subject to be diagnosed by the present method is can be a mammal.
  • exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • a biological sample is collected from a subject to be diagnosed to perform the diagnosis.
  • Any biological material can be used as the biological sample for the determination so long as it comprises the objective transcription or translation product of CDC45L and/or PIF1 gene.
  • the biological samples include, but are not limited to, bodily tissues and fluids, for example, blood, e.g. serum, sputum, urine and pleural effusion.
  • the biological sample contains a cell population comprising an epithelial cell, for example, a cancerous epithelial cell or an epithelial cell derived from tissue suspected to be cancerous. Further, if necessary, the cell can be purified from the obtained bodily tissues and fluids, and then used as the biological sample.
  • the expression level of CDC45L and/or PIF1 gene in the subject-derived biological sample is determined.
  • the expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art.
  • the mRNA of CDC45L or PIF1 gene can be quantified using probes by hybridization methods (e.g. Northern blot analysis).
  • the detection can be carried out on a chip or an array.
  • the use of an array can be for detecting the expression level of a plurality of genes (e.g., various cancer specific genes) including CDC45L and PIF1 gene.
  • Those skilled in the art can prepare such probes utilizing the sequence information of the CDC45L (SEQ ID NO: 13; GenBank Accession No.
  • the cDNA of CDC45L or PIF1 gene can be used as a probe.
  • the probe can be labeled with a suitable label, for example, dyes, fluorescent and isotopes, and the expression level of the gene can be detected as the intensity of the hybridized labels.
  • the transcription product of CDC45L or PIF1 gene can be quantified using primers by amplification-based detection methods (e.g., RT-PCR).
  • primers can also be prepared based on the available sequence information of the gene.
  • the primers (SEQ ID NO: 1 and 2 for CDC45L, SEQ ID NO: 3 and 4 for PIF1) used in the Example can be employed for the detection by RT-PCR or Northern blot, but the present invention is not restricted thereto.
  • a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of CDC45L or PIF1 gene.
  • the translation product can be detected for the diagnosis of the present invention.
  • the quantity of CDC45L or PIF1 protein can be determined.
  • a method for determining the quantity of the protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the protein.
  • the antibody can be monoclonal or polyclonal.
  • any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody can be used for the detection, so long as the fragment retains the binding ability to CDC45L or PIF1 protein.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method can be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining can be observed via immunohistochemical analysis using an antibody against CDC45L or PIF1 protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of CDC45L or PIF1 (see, Immunohistochemistry and Tissue-microarray analysis in "EXAMPLES").
  • the expression level of CDC45L and/or PIF1 can also be determined to improve the accuracy of the diagnosis.
  • the expression level of cancer marker gene including CDC45L and PIF1 in a biological sample can be considered to be increased if it increases from the control level of the corresponding cancer marker gene (e.g., in a normal or non-cancerous cell) by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
  • the control level can be determined at the same time with the test biological sample by using a sample(s) previously collected and stored from a subject/subjects whose disease state (cancerous or non-cancerous) is/are known.
  • the control level can be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of CDC45L and/or PIF1 in samples from subjects whose disease state are known.
  • the control level can be a database of expression patterns from previously tested cells.
  • the expression level of a CDC45L and/or PIF1 in a biological sample can be compared to multiple control levels, which control levels are determined from multiple reference samples.
  • a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample is used.
  • the standard value of the expression levels of CDC45L or PIF1 in a population with a known disease state is used.
  • the standard value can be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. can be used as standard value.
  • a control level determined from a biological sample that is known not to be cancerous is called "normal control level”.
  • normal tissues obtained from organ same as cancerous organs may be diagnosed.
  • normal lung tissues may be diagnosed as normal control for diagnosing lung cancer.
  • the control level is determined from a cancerous biological sample, it will be called “cancerous control level”.
  • the subject can be diagnosed to be suffering from or at a risk of developing cancer, e.g., a cancer that is mediated by or results from overexpression of CDC45L and/or PIF1.
  • a similarity in the gene expression pattern between the sample and the reference which is cancerous indicates that the subject is suffering from or at a risk of developing cancer, e.g., a cancer that is mediated by or results from overexpression of a CDC45L and/or PIF1.
  • Such cancer encompasses lung cancer.
  • control nucleic acids e.g., housekeeping genes, whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell.
  • control genes include, but are not limited to, beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
  • the present invention provides a method for detecting or identifying cancer cells in a subject-derived lung tissue sample, said method comprising the step of determining the expression level of the CDC45L and/or PIF1 gene in a subject-derived biological sample, wherein an increase in said expression level as compared to a normal control level of said gene indicates the presence or suspicion of cancer cells in the tissue.
  • the present invention may provide a doctor with useful information to diagnose a subject as afflicted with the disease.
  • clinical decisions can be reached by considering the expression level of the CDC45L and/or PIF1 gene, plus a different aspect of the disease including tissue pathology, levels of known tumor marker(s) in blood, and clinical course of the subject, etc.
  • some well-known diagnostic lung tumor markers in blood are IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1, TPA, CSLEX, SLX, STN and CYFRA.
  • the outcome of the gene expression analysis serves as an intermediate result for further diagnosis of a subject's disease state.
  • the present invention provides use of a reagent for preapring a diagnostic reagent for diagnosing cancer.
  • the reagent can be selected from the group consisting of: (a) a reagent for detecting mRNA of the CDC45L or PIF1 gene; (b) a reagent for detecting the CDC45L or PIF1 protein; and (c) a reagent for detecting the biological activity of the CDC45L or PIF1 protein.
  • such reagent is an oligonucleotide that hybridizes to the CDC45L or PIF1 polynucleotide, or an antibody that binds to the CDC45L or PIF1 polypeptide.
  • the present invention also provides a kit for use in diagnosis or detection of cancer, wherein the kit comprises a reagent which binds to a transcription or translation product of the CDC45L gene or PIF1 gene.
  • CDC45L or PIF1 is not only a useful diagnostic marker, but also suitable target for cancer therapy. Therefore, cancer treatment targeting CDC45L or PIF1 can be achieved by the present invention.
  • the cancer treatment targeting CDC45L or PIF1 refers to suppression or inhibition of CDC45L or PIF1 activity and/or expression in the cancer cells.
  • Any anti-CDC45L or anti-PIF1 agents may be used for the cancer treatment targeting CDC45L or PIF1.
  • the present agents may be used for the cancer treatment targeting CDC45L or PIF1.
  • the anti-CDC45L or anti-PIF1 agents include following substance as active ingredient: (a) a double-stranded molecule of the present invention, (b) DNA encoding thereof, or (c) a vector encoding thereof.
  • the present invention provides a method of (i) diagnosing whether a subject has the cancer to be treated, and/or (ii) selecting a subject for cancer treatment, which method includes the steps of: a) determining the expression level of CDC45L or PIF1 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of CDC45L or PIF1 with a normal control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of CDC45L or PIF1 is increased as compared to the normal control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • such a method includes the steps of: a) determining the expression level of CDC45L or PIF1 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated; b) comparing the expression level of CDC45L or PIF1 with a cancerous control level; c) diagnosing the subject as having the cancer to be treated, if the expression level of CDC45L or PIF1 is similar or equivalent to the cancerous control level; and d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
  • the present invention is based, in part, on the discovery that CDC45L (over)expression is significantly associated with poorer prognosis of patients with CDC45L-mediated cancers, e.g., lung cancers.
  • the present invention provides a method for determining or assessing the prognosis of a patient with cancer, e.g., a cancer mediated by or resulting from overexpression of a CDC45L, e.g, lung cancer, by detecting the expression level of the CDC45L gene in a biological sample of the patient; comparing the detected expression level to a control level; and determining a increased expression level to the control level as indicative of poor prognosis (poor survival).
  • cancer e.g., a cancer mediated by or resulting from overexpression of a CDC45L, e.g, lung cancer
  • prognosis refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a less favorable, negative or poor prognosis is defined by a lower post-treatment survival term or survival rate. Conversely, a positive, favorable, or good prognosis is defined by an elevated post-treatment survival term or survival rate.
  • assessing the prognosis refer to the ability of predicting, forecasting or correlating a given detection or measurement with a future outcome of cancer of the patient (e.g., malignancy, likelihood of curing cancer, estimated time of survival, and the like). For example, a determination of the expression level of CDC45L over time enables a predicting of an outcome for the patient (e.g., increase or decrease in malignancy, increase or decrease in grade of a cancer, likelihood of curing cancer, survival, and the like).
  • the phrase "assessing (or determining) the prognosis” is intended to encompass predictions and likelihood analysis of cancer, progression, particularly cancer recurrence, metastatic spread and disease relapse.
  • the present method for assessing prognosis is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria for example, disease staging, and disease monitoring and surveillance for metastasis or recurrence of neoplastic disease.
  • the present invention provides the following methods [1] to [6]:
  • [1] A method for assessing prognosis of a subject with lung cancer comprising steps of: (a) detecting an expression level of CDC45L in a subject-derived biological sample; (b) comparing the detected expression level to a control level; and (c) determining prognosis of the patient based on the comparison of (b);
  • the patient-derived biological sample used for the method can be any sample derived from the subject to be assessed so long as the CDC45L gene can be detected in the sample.
  • the biological sample comprises a lung cell (a cell obtained from lung ).
  • the biological sample includes bodily fluids for example, sputum, blood, serum, plasma, pleural effusion, and so on.
  • the sample can be cells purified or obtained from a tissue.
  • the biological samples can be obtained from a patient at various time points, including before, during, and/or after a treatment. For example, a lung cancer cell(s) obtained from a subject to be assessed is a preferable biological sample.
  • control level used for comparison can be, for example, the expression level of the CDC45L gene detected before any kind of treatment in an individual or a population of individuals who showed good or positive prognosis of cancer, after the treatment, which herein will be referred to as "good prognosis control level".
  • control level can be the expression level of the CDC45L gene detected before any kind of treatment in an individual or a population of individuals who showed poor or negative prognosis of cancer, after the treatment, which herein will be referred to as "poor prognosis control level".
  • the "control level” is a single expression pattern derived from a single reference population or from a plurality of expression patterns.
  • the control level can be determined based on the expression level of the CDC45L gene detected before any kind of treatment in a patient of cancer, or a population of the patients whose disease state (good or poor prognosis) is known.
  • the cancer is lung cancer.
  • the standard value of the expression levels of the CDC45L gene in a patient group with a known disease state is used.
  • the standard value can be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. can be used as standard value.
  • the control level can be determined at the same time with the test biological sample by using a sample(s) previously collected and stored before any kind of treatment from cancer patient(s) (control or control group) whose disease state (good prognosis or poor prognosis) are known.
  • control level can be determined by a statistical method based on the results obtained by analyzing the expression level of the CDC45L gene in samples previously collected and stored from a control group.
  • control level can be a database of expression patterns from previously tested cells or patients.
  • the expression level of the CDC45L gene in a biological sample can be compared to multiple control levels, which control levels are determined from multiple reference samples.
  • a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample is used.
  • a similarity in the expression level of the CDC45L gene to the good prognosis control level indicates a more favorable prognosis of the patient and an increase in the expression level in comparison to the good prognosis control level indicates less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome.
  • a decrease in the expression level of the CDC45L gene in comparison to the poor prognosis control level indicates a more favorable prognosis of the patient and a similarity in the expression level to the poor prognosis control level indicates less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome.
  • a lung cancer cell(s) obtained from a subject who showed good, or poor prognosis of cancer after treatment is a preferable biological sample for good, or poor prognosis control level, respectively.
  • An expression level of the CDC45L gene in a biological sample can be considered altered (i.e., increased or decreased) when the expression level differs from the control level by more than 1.0, 1.5, 2.0, 5.0, 10.0, or more fold.
  • the difference in the expression level between the test biological sample and the control level can be normalized to a control, e.g., housekeeping gene.
  • a control e.g., housekeeping gene.
  • polynucleotides whose expression levels are known not to differ between the cancerous and non-cancerous cells including those coding for beta-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1
  • CDC45L gene e.g., housekeeping gene.
  • the expression level can be determined by detecting the gene transcript in the patient-derived biological sample using techniques well known in the art.
  • the gene transcripts detected by the present method include both the transcription and translation products, for example, mRNA and protein.
  • the transcription product of the CDC45L gene can be detected by hybridization, e.g., Northern blot hybridization analyses, that use a CDC45L gene probe to the gene transcript.
  • the detection can be carried out on a chip or an array.
  • An array can be used for detecting the expression level of a plurality of genes including the CDC45L gene.
  • amplification-based detection methods for example, reverse-transcription based polymerase chain reaction (RT-PCR) which use primers specific to the CDC45L gene can be employed for the detection (see (b) Semi-quantitative RT-PCR in [EXAMPLE]).
  • the CDC45L gene-specific probe or primers can be designed and prepared using conventional techniques by referring to the whole sequence of the CDC45L (SEQ ID NO: 13).
  • the primers (SEQ ID NOs: 1 and 2) used in the Example can be employed for the detection by RT-PCR, but the present invention is not restricted thereto.
  • a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of the CDC45L gene.
  • stringent (hybridization) conditions refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH.
  • Tm thermal melting point
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30degrees Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60degrees Centigrade for longer probes or primers.
  • Stringent conditions can also be achieved with the addition of destabilizing agents, for example, formamide.
  • the translation product can be detected for the assessment of the present invention.
  • the quantity of the CDC45L protein can be determined.
  • a method for determining the quantity of the protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the CDC45L protein.
  • the antibody can be monoclonal or polyclonal.
  • any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody can be used for the detection, so long as the fragment retains the binding ability to the CDC45L protein.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method can be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the intensity of staining can be observed via immunohistochemical analysis using an antibody against CDC45L protein. Namely, the observation of strong staining indicates increased presence of the CDC45L protein and at the same time high expression level of the CDC45L gene.
  • the CDC45L protein is known to have a cell proliferating activity. Therefore, the expression level of the CDC45L gene can be determined using such cell proliferating activity as an index. For example, cells which express CDC45L are prepared and cultured in the presence of a biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability the cell proliferating activity of the biological sample can be determined.
  • the expression level of other lung cell-associated genes for example, genes known to be differentially expressed in lung cancer, can also be determined to improve the accuracy of the assessment.
  • Such other lung cancer-associated genes include those described in WO 2004/031413 and WO 2005/090603.
  • the patient to be assessed for the prognosis of cancer according to the method can be a mammal and includes human, non-human primate, mouse, rat, dog, cat, horse, and cow.
  • an intermediate result can also be provided in addition to other test results for assessing the prognosis of a subject.
  • Such intermediate result can assist a doctor, nurse, or other practitioner to assess, determine, or estimate the prognosis of a subject.
  • Additional information that can be considered, in combination with the intermediate result obtained by the present invention, to assess prognosis includes clinical symptoms and physical conditions of a subject.
  • the present invention provides use of a reagent for preapring a reagent for assessing prognosis of cancer.
  • the reagent is selected from the group consisting of: (a) a reagent for detecting mRNA of the CDC45L gene; (b) a reagent for detecting the CDC45L; and (c) a reagent for detecting the biological activity of the CDC45L protein.
  • such reagent is an oligonucleotide that hybridizes to the CDC45L polynucleotide, or an antibody that binds to the CDC45L polypeptide.
  • kits for Diagnosing Cancer or Assessing the Prognosis of Cancer provides a kit for diagnosing cancer or assessing the prognosis of cancer.
  • the present invention also provides a kit for determining a subject suffering from cancer that can be treated with the double-stranded molecule of the present invention or vector encoding thereof, which may also be useful in assessing and/or monitoring the efficacy of a cancer treatment.
  • the cancer is mediated by CDC45L and/or PIF1, or resulting from overexpression of CDC45L and/or PIF1, e.g., lung cancer.
  • the kit comprises at least one reagent for detecting the expression of the CDC45L or PIF1 gene in a patient-derived biological sample, which reagent can be selected from the group of: (a) a reagent for detecting mRNA of the CDC45L or PIF1 gene; (b) a reagent for detecting the CDC45L or PIF1 protein; and (c) a reagent for detecting the biological activity of the CDC45L or PIF1 protein.
  • Suitable reagents for detecting mRNA of the CDC45L or PIF1 gene include nucleic acids that specifically bind to or identify the CDC45L or PIF1 mRNA, for example, oligonucleotides which have a complementary sequence to a part of the CDC45L or PIF1 mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the CDC45L or PIF1 mRNA. These kinds of oligonucleotides can be prepared based on methods well known in the art. If needed, the reagent for detecting the CDC45L or PIF1 mRNA can be immobilized on a solid matrix. Moreover, more than one reagent for detecting the CDC45L or PIF1 mRNA can be included in the kit.
  • the probes or primers may be of specific sizes.
  • the sizes are selected from the group consisting of at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides and the probes and primers may range in size from 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides and 25-30 nucleotides.
  • suitable reagents for detecting the CDC45L or PIF1 protein include antibodies to the CDC45L or PIF1 protein.
  • the antibody can be monoclonal or polyclonal.
  • any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody can be used as the reagent, so long as the fragment retains the binding ability to the CDC45L or PIF1 protein.
  • Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method can be employed in the present invention to prepare such antibodies and equivalents thereof.
  • the antibody can be labeled with signal generating molecules via direct linkage or an indirect labeling technique.
  • Labels and methods for labeling antibodies and detecting the binding of antibodies to their targets are well known in the art and any labels and methods can be employed for the present invention.
  • more than one reagent for detecting the CDC45L or PIF1 protein can be included in the kit.
  • the biological activity can be determined by, for example, measuring the cell proliferating activity due to the expressed CDC45L or PIF1 protein in the biological sample.
  • the cell is cultured in the presence of a patient-derived biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability the cell proliferating activity of the biological sample can be determined.
  • the reagent for detecting the CDC45L or PIF1 mRNA can be immobilized on a solid matrix.
  • more than one reagent for detecting the biological activity of the CDC45L or PIF1 protein can be included in the kit.
  • the kit can comprise more than one of the aforementioned reagents.
  • the kit can comprise a solid matrix and reagent for binding a probe against the CDC45L or PIF1 gene or antibody against the CDC45L or PIF1 protein, a medium and container for culturing cells, positive and negative control reagents, and a secondary antibody for detecting an antibody against the CDC45L or PIF1 protein.
  • tissue samples obtained from patient with good prognosis or poor prognosis can serve as useful control reagents.
  • a kit of the present invention can further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts (e.g., written, tape, CD-ROM, etc.) with instructions for use.
  • These reagents and such can be comprised in a container with a label.
  • Suitable containers include bottles, vials, and test tubes.
  • the containers can be formed from a variety of materials, for example, glass or plastic.
  • the reagent when the reagent is a probe against the CDC45L or PIF1 mRNA, the reagent can be immobilized on a solid matrix, for example, a porous strip, to form at least one detection site.
  • the measurement or detection region of the porous strip can include a plurality of sites, each containing a nucleic acid (probe).
  • a test strip can also contain sites for negative and/or positive controls. Alternatively, control sites can be located on a strip separated from the test strip.
  • the different detection sites can contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites.
  • the number of sites displaying a detectable signal provides a quantitative indication of the amount of CDC45L or PIF1 mRNA present in the sample.
  • the detection sites can be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
  • the kit of the present invention can further comprise a positive control sample or CDC45L or PIF1 standard sample.
  • the positive control sample of the present invention can be prepared by collecting CDC45L or PIF1 positive blood samples and then those CDC45L or PIF1 level are assayed.
  • purified CDC45L or PIF1 protein or polynucleotide can be added to CDC45L or PIF1 free serum to form the positive sample or the CDC45L or PIF1 standard.
  • purified CDC45L or PIF1 can be recombinant protein.
  • the CDC45L or PIF1 level of the positive control sample is, for example more than cut off value.
  • CDC45L or PIF1 Screening Methods Using the CDC45L or PIF1 gene, polypeptide encoded by the gene or fragment thereof, or transcriptional regulatory region of the gene, it is possible to screen agents or compounds that alter the expressions of the genes or the biological activities of polypeptides encoded by the genes. Such agents or compounds may be used as pharmaceuticals for treating or preventing cancer, in particular, lung cancer.
  • the present invention provides methods of screening for candidate agents or compounds for treating or preventing cancer using the CDC45L or PIF1 gene, a polypeptide encoded by the gene or fragment thereof, or a transcriptional regulatory region of the gene.
  • Agents or compounds isolated by the screening method of the present invention is a substance that is expected to inhibit the expression of the CDC45L or PIF1 gene, or the activity of the translation product of the gene, and thus, is a candidate for treating or preventing diseases attributed to, for example, cell proliferative diseases, such as cancer (in particular, lung and esophageal cancer). Namely, the agents or compounds screened through the present methods are deemed to have a clinical benefit and can be further tested for its ability to prevent cancer cell growth in animal models or test subjects.
  • agents to be identified through the present screening methods can be any compound or composition including several compounds.
  • the test agent or compound exposed to a cell or protein according to the screening methods of the present invention can be a single compound or a combination of compounds. When a combination of compounds is used in the methods, the compounds can be contacted sequentially or simultaneously.
  • test agent or compound for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micro-molecular compounds (including nucleic acid constructs, for example, antisense DNA, siRNA, ribozymes, etc.) and natural compounds can be used in the screening methods of the present invention.
  • test agent or compound for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micro-molecular compounds (including nucleic acid constructs, for example, antisense DNA, siRNA, ribozymes, etc.) and natural compounds can be used in the screening methods of the present invention.
  • test agent or compound of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) spatially addressable parallel solid phase or solution phase libraries, (3) synthetic library methods requiring deconvolution, (4) the "one-bead one-compound” library method and (5) synthetic library methods using affinity chromatography selection.
  • the screened test agent or compound is a protein
  • for obtaining a DNA encoding the protein either the whole amino acid sequence of the protein can be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein can be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein.
  • the obtained DNA finds use in preparing the test agent or compound which is a candidate for treating or preventing cancer.
  • Test agents or compounds useful in the screening described herein can also be antibodies or non-antibody binding proteins that specifically bind to the CDC45L or PIF1 protein or partial CDC45L or PIF1 peptides that lack the activity to binding for partner.
  • Such partial protein or antibody can be prepared by the methods described herein (see (1) Cancer-related genes and cancer-related protein, and functional equivalent thereof in Definition or Antibodies) and can be tested for their ability to block binding of the protein with its binding partners.
  • test agent/compound libraries are facilitated by knowledge of the molecular structure of compounds known to have the properties sought, and/or the molecular structure of the target molecules to be inhibited.
  • One approach to preliminary screening of test agents or compounds suitable for further evaluation is computer modeling of the interaction between the test agent/compound and its target.
  • Computer modeling technology allows the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule.
  • the three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule.
  • the molecular dynamics require force field data.
  • the computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
  • CHARMm performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • test agents or compounds can be screened using the methods of the present invention to identify test agents or compounds of the library that disrupt the CDC45L or PIF1 activity for the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer.
  • Combinatorial chemical synthesis Combinatorial libraries of test agents or compounds can be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors of the CDC45L or PIF1 activity. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening.
  • simple, particularly short, polymeric molecular libraries can be constructed by simply synthesizing all permutations of the molecular family making up the library.
  • An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.
  • Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6).
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No.
  • WO 91/19735) encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers for example, hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J.
  • a second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al. (Science 1991, 251: 767-73) are examples.
  • Furka et al. 14th International Congress of Biochemistry 1988, Volume #5, Abstract FR:013; Furka, Int J Peptide Protein Res 1991, 37: 487-93
  • Houghten US Patent 4,631,211
  • Rutter et al. US Patent 5,010,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.
  • Aptamers are macromolecules composed of nucleic acid that bind tightly to a specific molecular target.
  • Tuerk and Gold discloses SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for selection of aptamers.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • a large library of nucleic acid molecules ⁇ e.g., 10 15 different molecules) can be used for screening.
  • Antagonists that binds to the CDC45Lor PIF1 polypeptide may inhibit the biological activity to mediate cell proliferation of cancer , and thus, they are candidates for treating the cancer. Therefore, the present invention provides a method for identifying potential candidates for treating or preventing cancer expressing CDC45L and/or PIF1, by identifying agents or compounds that bind to CDC45L or PIF1 polypeptide.
  • screening can be carried out as an in vitro assay system, for example, a cellular system. More specifically, first, either the CDC45L or PIF1 protein or the binding partner thereof is bound to a support, and the other protein is added together with a test compound thereto. For instance, the CDC45L or PIF1 is bound to a support, and the binding partner polypeptide is added together with a test compound thereto. Next, the mixture is incubated, washed and the other protein bound to the support is detected and/or measured.
  • inhibiting binding between two proteins refers to at least reducing binding between the proteins.
  • the percentage of binding pairs in a sample in the presence of a test agent or compound will be decreased compared to an appropriate (e.g., not treated with test compound or from a non-cancer sample, or from a cancer sample) control.
  • the reduction in the amount of proteins bound can be, e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 25%, 10%, 5%, 1% or less (e.g., 0%), than the pairs bound in a control sample.
  • supports that can be used for binding proteins include, for example, insoluble polysaccharides, for example, agarose, cellulose and dextran; and synthetic resins, for example, polyacrylamide, polystyrene and silicon; for example, commercial available beads and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials can be used.
  • beads When using beads, they can be filled into a column.
  • magnetic beads is also known in the art, and enables one to readily isolate proteins bound on the beads via magnetism.
  • binding of a protein to a support can be conducted according to routine methods, for example, chemical bonding and physical adsorption, for example.
  • a protein can be bound to a support via antibodies that specifically recognize the protein.
  • binding of a protein to a support can be also conducted by means of avidin and biotin.
  • agents or compounds that suppress the expression level of CDC45L or PIF can be also identified as candidate therapeutics for cancer.
  • the expression level of a polypeptide or functional equivalent thereof can be detected according to any method known in the art.
  • a reporter assay can be used. Suitable reporter genes and host cells are well known in the art.
  • the reporter construct required for the screening can be prepared by using the transcriptional regulatory region of CDC45L or PIF1 gene. When the transcriptional regulatory region of the gene has been known to those skilled in the art, a reporter construct can be prepared by using the previous sequence information.
  • a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library based on the nucleotide sequence information of the gene.
  • the reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of a CDC45L or PIF1 gene of interest.
  • the transcriptional regulatory region of a CDC45L or PIF1 gene is the region from a start codon to at least 500bp upstream, for example, 1000bp, for example, 5000 or 10000bp upstream.
  • a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., Chapter 17, 2001, Cold Springs Harbor Laboratory Press).
  • agents or compounds that inhibit a biological activity of CDC45L or PIF protein can be also identified as candidate therapeutics for cancer.
  • a substrate can conveniently be immobilized on a solid support. Following the reaction, the substrate converted by the polypeptide can be detected on the solid support by the methods described above. Alternatively, the contact step can be performed in solution, after which a substrate can be immobilized on a solid support, and the substrate converted by the polypeptide can be detected.
  • the solid support can be coated with streptavidin and the substrate labeled with biotin, or the solid support can be coated with antibodies against the substrate. The skilled person can determine suitable assay formats depending on the desired throughput capacity of the screen.
  • the assays of the invention are also suitable for automated procedures which facilitate high-throughput screening.
  • a number of well-known robotic systems have been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, Ltd. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.
  • a potential therapeutic effect refers to a clinical benefit with a reasonable expectation.
  • such clinical benefit includes; (a) reduction in expression of the CDC45L or PIF1 gene, (b) decrease in size, prevalence, or metastatic potential of the cancer in the subject, . (c) preventing cancers from forming, or (d) preventing or alleviating a clinical symptom of cancer.
  • the present invention provides the method of screening for an agent or compound useful in diagnosing, treating or preventing cancers using the CDC45L or PIF1 polypeptide.
  • An embodiment of this screening method comprises the steps of: (a) contacting a test agent or compound with a polypeptide selected from the group consisting of CDC45L and PIF1 protein, or fragment thereof; (b) detecting the binding level between the polypeptide or the fragment and the test agent or compound; (c) selecting the test agent or compound that binds to the polypeptide or the fragment of step (a).
  • the therapeutic effect of the test agent or compound on inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L or PIF1 associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate agent or compound that suppresses the proliferation of cancer cells, and a method for screening a candidate agent or compound for treating or preventing cancer.
  • the method includes the steps of: (a) contacting a test agent or compound with a polypeptide selected from the group consisting of CDC45L and PIF1 protein, or fragment thereof; (b) detecting the binding level between the polypeptide and said test agent or compound; (c) correlating the binding level of b) with the therapeutic effect of the test agent or compound.
  • the potential therapeutic effect of a test agent or compound on treating or preventing cancer can also be evaluated or estimated.
  • the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of CDC45L and/or PIF1, the method including steps of: (a) contacting an agent or compound with a polypeptide encoded by a polynucleotide of CDC45L or PIF1; (b) detecting the binding activity between the polypeptide and the test agent or compound; and (c) correlating the potential therapeutic effect and the test agent or compound, wherein the potential therapeutic effect is shown, when the agent or compound binds to the polypeptide.
  • the therapeutic effect may be correlated with the binding level of the CDC45L or PIF1 protein.
  • the test agent or compound may be identified or selected as the candidate agent or compound having the therapeutic effect.
  • the test agent or compound may be identified as the agent or compound having no significant therapeutic effect.
  • the CDC45L or PIF1 polypeptide to be used for screening can be a recombinant polypeptide or a protein derived from the nature or a partial peptide thereof.
  • the polypeptide to be contacted with a test compound can be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.
  • CDC45L or PIF1 polypeptide As a method of screening for proteins, for example, that bind to CDC45L or PIF1 polypeptide using CDC45L or PIF1 polypeptide, many methods well known by a person skilled in the art can be used. Such a screening can be conducted by, for example, immunoprecipitation method.
  • the gene encoding CDC45L or PIF1 polypeptide is expressed in host (e.g., animal) cells and so on by inserting the gene to an expression vector for foreign genes, for example, pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8.
  • the promoter to be used for the expression can be any promoter that can be used commonly and include, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 83-141 (1982)), the EF- alpha promoter (Kim et al., Gene 91: 217-23 (1990)), the CAG promoter (Niwa et al., Gene 108: 193 (1991)), the RSV LTR promoter (Cullen, Methods in Enzymology 152: 684-704 (1987)) the SR alpha promoter (Takebe et al., Mol Cell Biol 8: 466 (1988)), the CMV immediate early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365-9 (1987)), the SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982)), the Adenovirus late promoter (Kauf
  • the introduction of the gene into host cells to express a foreign gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 15: 1311-26 (1987)), the calcium phosphate method (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), the DEAE dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol Cell Biol 4: 1641-3 (1984)), the Lipofectin method (Derijard B., Cell 76: 1025-37 (1994); Lamb et al., Nature Genetics 5: 22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)) and so on.
  • electroporation method Chou et al., Nucleic Acids Res 15: 1311-26 (1987)
  • the calcium phosphate method Choen and Okayama, Mol Cell Biol 7
  • the polypeptide encoded by CDC45L or PIF1 gene can be expressed as a fusion protein comprising a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide.
  • a commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)).
  • Vectors which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP) and so on by the use of its multiple cloning sites are commercially available.
  • a fusion protein prepared by introducing only small epitopes consisting of several to a dozen amino acids so as not to change the property of the CDC45L or PIF1 polypeptide by the fusion is also reported.
  • Epitopes for example, polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the CDC45L or PIF1 polypeptide (Experimental Medicine 13: 85-90 (1995)).
  • an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent.
  • the immune complex consists of the CDC45L or PIF1 polypeptide, a polypeptide comprising the binding ability with the polypeptide, and an antibody. Immunoprecipitation can be also conducted using antibodies against the CDC45L or PIF1 polypeptide, besides using antibodies against the above epitopes, which antibodies can be prepared as described above.
  • An immune complex can be precipitated, for example by Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody.
  • an immune complex can be formed in the same manner as in the use of the antibody against the CDC45L or PIF1 polypeptide, using a substance specifically binding to these epitopes, for example, glutathione-Sepharose 4B.
  • Immunoprecipitation can be performed by following or according to, for example, the methods in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).
  • SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Since the protein bound to CDC45L or PIF1 polypeptide is difficult to detect by a common staining method, for example, Coomassie staining or silver staining, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35 S-methionine or 35 S-cysteine, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
  • a protein binding to the CDC45L or PIF1 polypeptide can be obtained by preparing a cDNA library from cultured cells (e.g., lung cancer cell line ) expected to express a protein binding to the CDC45L or PIF1 polypeptide using a phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing the protein expressed on a filter, reacting the purified and labeled CDC45L or PIF1 polypeptide with the above filter, and detecting the plaques expressing proteins bound to CDC45L or PIF1 polypeptide according to the label.
  • a cDNA library from cultured cells (e.g., lung cancer cell line ) expected to express a protein binding to the CDC45L or PIF1 polypeptide using a phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing the protein expressed on a filter, reacting the purified and labeled CDC45L or PIF1 polypeptide with
  • the polypeptide of the invention can be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to CDC45L or PIF1 polypeptide, or a peptide or polypeptide (for example, GST) that is fused to CDC45L OR PIF1 polypeptide. Methods using radioisotope or fluorescence and such can be also used.
  • label and “detectable label” are used herein to refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • labels include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DYNABEADS TM ), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 125I, .35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels for example colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like
  • Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,275,149; and 4,366,241.
  • Means of detecting such labels are well known to those of skill in the art.
  • radiolabels can be detected using photographic film or scintillation counters
  • fluorescent markers can be detected using a photodetector to detect emitted light.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting, the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.
  • a two-hybrid system utilizing cells can be used ("MATCHMAKER Two-Hybrid system”, “Mammalian MATCHMAKER Two-Hybrid Assay Kit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references “Dalton and Treisman, Cell 68: 597-612 (1992)", “Fields and Sternglanz, Trends Genet 10: 286-92 (1994)”).
  • the polypeptide of the invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells.
  • a cDNA library is prepared from cells expected to express a protein binding to the polypeptide of the invention, such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region.
  • the cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the polypeptide of the invention is expressed in yeast cells, the binding of the two activates a reporter gene, making positive clones detectable).
  • a protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein.
  • a reporter gene for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
  • a compound binding to the polypeptide encoded by CDC45L or PIF1 gene can also be screened using affinity chromatography.
  • the polypeptide of the invention can be immobilized on a carrier of an affinity column, and a test compound, containing a protein capable of binding to the polypeptide of the invention, is applied to the column.
  • a test compound herein can be, for example, cell extracts, cell lysates, etc. After loading the test compound, the column is washed, and compounds bound to the polypeptide of the invention can be prepared.
  • test compound When the test compound is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and cDNA libraries are screened using the oligo DNA as a probe to obtain a DNA encoding the protein.
  • a biosensor using the surface plasmon resonance phenomenon can be used as a mean for detecting or quantifying the bound compound in the present invention.
  • the interaction between the polypeptide of the invention and a test compound can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide of the invention and a test compound using a biosensor for example, BIAcore.
  • suppressing the expression level of CDC45L or PIF1 reduces cell growth.
  • candidate compounds that have the potential to treat or prevent cancers can be identified.
  • Potential of these candidate compounds to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a compound binding to CDC45L or PIF1 protein inhibits activities of cancer, it may be concluded that such compound has CDC45L or PIF1 specific therapeutic effect.
  • the present invention also provides a method for screening a candidate agents or compounds for treating or preventing cancer using a biological activity of the CDC45L or PIF1 polypeptide, or fragment thereof as an index.
  • the present invention provides the following methods of [1] to [4]: [1] A method of screening for an agent or compound useful in treating or preventing cancers expressing CDC45L and/or PIF1, said method comprising the steps of: (a) contacting a test agent or compound with a CDC45L or PIF1 polynucleotide, or functional equivalent or fragment thereof; (b) detecting a biological activity of the polypeptide of step (a); (c) comparing the level detected in the step (b) with those detected in the absence of the test agent or compound; (d) selecting the test agent or compound that reduce or inhibit the biological activity of the polypeptide.
  • the therapeutic effect of the test agent or compound on inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease e.g., lung cancer may be evaluated. Therefore, the present invention also provides a method of screening for a candidate agent or compound for inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease, e.g., lung cancer, using the CDC45L or PIF1 polypeptide or fragments thereof including the steps as follows: a) contacting a test agent or compound with the CDC45L or PIF1 polypeptide or a functional fragment thereof; and b) detecting the biological activity of the polypeptide or fragment of step (a), and c) correlating the biological activity of b) with the therapeutic effect of the test agent or compound.
  • the present invention provides a method for evaluating or estimating a therapeutic effect of a test agent or compound on treating or preventing cancer or inhibiting cancer associated with over-expression of CDC45L and/or PIF1, the method including steps of: (a) contacting a test agent or compound with a polypeptide encoded by a polynucleotide of CDC45L or PIF1 gene; (b) detecting the biological activity of the polypeptide of step (a); and (c) correlating the potential therapeutic effect and the test agent or compound, wherein the potential therapeutic effect is shown, when the test agent or compound suppresses the biological activity of the polypeptide encoded by the polynucleotide of CDC45L or PIF1 gene as compared to the biological activity of said polypeptide detected in the absence of the test agent or compound.
  • the therapeutic effect may be correlated with the biological activity of CDC45L or PIF1 polypeptide or a functional fragment thereof.
  • the test agent or compound when the test agent or compound suppresses or inhibits the biological activity of CDC45L or PIF1 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified or selected as the candidate agent or compound having the therapeutic effect.
  • the test agent or compound may identified as the agent or compound having no significant therapeutic effect.
  • any polypeptides can be used for screening so long as they comprise the biological activity of the CDC45L or PIF1 protein.
  • biological activity includes the cell proliferating activity (cell proliferation promoting activity) or the binding activity to CDC45L or PIF1 each other.
  • CDC45L protein the biological activity includes DNA replication activity.
  • PIF1 protein the biological activity includes helicase activity and telomerase inhibition activity.
  • CDC45L or PIF1 protein can be used and polypeptides functionally equivalent to these proteins can also be used.
  • Such polypeptides can be expressed endogenously or exogenously by cells.
  • the compound isolated by this screening is a candidate for antagonists of the polypeptide encoded by CDC45L or PIF1 gene.
  • antagonist refers to molecules that inhibit the function of the polypeptide by binding thereto. Said term also refers to molecules that reduce or inhibit expression of the gene encoding CDC45L or PIF1.
  • a compound isolated by this screening is a candidate for compounds which inhibit the in vivo interaction of the CDC45L or PIF1 polypeptide with molecules (including DNAs and proteins).
  • the CDC45L and PIF1 protein has the activity of promoting cell proliferation of cancer cells (Fig. 3 and Fig.5). Therefore, in the screening method of the present invention, using this biological activity, a compound which inhibits a biological activity of these protein can be screened. Such compounds would be potential candidates for treating cancer, e.g., lung cancer.
  • the biological activity to be detected in the present method is cell proliferation promoting activity
  • it can be detected, for example, by preparing cells which express the polypeptide selected from the group consisting of CDC45L and PIF1, culturing the cells in the presence of a test compound, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring the colony formation activity, e.g. MTT assay, colony formation assay or FACS shown in "EXAMPLES”.
  • suppress the biological activity refers to at least 10% suppression of the biological activity of CDC45L or PIF1 in comparison with in absence of the compound, for example, at least 25%, 50% or 75% suppression, for example, at least 90% suppression.
  • control cells which do not express CDC45L or PIF1 polypeptide are used. Accordingly, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing CDC45L or PIF1 associating disease,e.g., lung cancer, using the CDC45L or PIF1 polypeptide or fragments thereof including the steps as follows:
  • DNA replication activity or binding activity to PIF1 polypeptide may be used as an index.
  • helicase activity, telomerase inhibition activity or binding activity to CDC45L polypeptide may be used as an index.
  • suppressing the biological activity of CDC45L or PIF1 reduces cell growth.
  • candidate compounds that inhibits biological activity of CDC45L or PIF1 candidate compounds that have the potential to treat or prevent cancers can be identified. Potential of these candidate compounds to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a compound inhibits the biological activity of CDC45L or PIF1 protein inhibits activities of cancer, it may be concluded that such compound has CDC45L or PIF1 specific therapeutic effect.
  • such screening can comprise, for example, the following steps: (a) contacting a test compound with a cell expressing CDC45L and/or PIF1 gene ; (b) detecting the expression level of the CDC45L and/or PIF1 gene; and (c) selecting the test compound that reduces the expression level of CDC45L and/or PIF1 gene as compared to that detected in the absence of the test compound.
  • the therapeutic effect of the test agent or compound on inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease e.g., lung cancer may be evaluated. Therefore, the present invention also provides a method for screening a candidate agent or compound that suppresses the proliferation of cancer cells, and a method for screening a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease e.g., lung cancer,.
  • such screening may include, for example, the following steps: a) contacting a test agent or compound with a cell expressing the CDC45L and/or PIF1 gene; b) detecting the expression level of the CDC45L and/or PIF1 gene; and c) correlating the expression level of b) with the therapeutic effect of the test agent or compound.
  • the potential therapeutic effect of a test agent or compound on treating or preventing cancer can also be evaluated or estimated.
  • the present invention provides a method for evaluating or estimating a therapeutic effect of a test agent or compound on treating or preventing cancer or inhibiting cancer associated with over-expression of CDC45L and/or PIF1, the method including steps of: (a) contacting a test agent or compound with a cell expressing the CDC45L and/or PIF1 gene; (b) detecting the expression level of the CDC45L and/or PIF1 gene of step (a); and (c) correlating the potential therapeutic effect and the test agent or compound, wherein the potential therapeutic effect is shown, when the test agent or compound reduces the expression level of the CDC45L and/or PIF1 gene.
  • the therapeutic effect may be correlated with the expression level of the CDC45L or PIF1 gene.
  • the test agent or compound when the test agent or compound reduces the expression level of the CDC45L or PIF1 gene as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified or selected as the candidate agent or compound having the therapeutic effect.
  • the test agent or compound when the test agent or compound does not reduce the expression level of the CDC45L or PIF1 gene as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified as the agent or compound having no significant therapeutic effect.
  • Cells expressing the CDC45L and/or PIF1 include, for example, cell lines established from lung cancer; such cells can be used for the above screening of the present invention (e.g., A549 and SBC-3).
  • the expression level can be estimated by methods well known to one skilled in the art, for example, RT-PCR, Northern blot assay, Western blot assay, immunostaining, ELISA or flow cytometry analysis.
  • the term of "reduce the expression level” as defined herein refers to at least 10% reduction of expression level of CDC45L or PIF1 in comparison to the expression level in absence of the compound, for example, at least 25%, 50% or 75% reduced level, for example, at least 95% reduced level.
  • the compound herein includes chemical compound, double-strand nucleotide, and so on.
  • the preparation of the double-strand nucleotide is in aforementioned description.
  • a compound that reduces the expression level of CDC45L or PIF1 can be selected as candidate agents or compounds to be used for the treatment or prevention of cancers, e.g. lung cancer.
  • the screening method of the present invention can comprise the following steps: (a) contacting a candidate compound with a cell into which a vector, comprising the transcriptional regulatory region of CDC45L or PIF1 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; (b) measuring the expression level or activity of said reporter gene; and (c) selecting the candidate compound that reduces the expression level or activity of said reporter gene.
  • the therapeutic effect of the test agent or compound on inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate agent or compound that suppresses the proliferation of cancer cells, and a method for screening a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease.
  • the present invention provides a method which includes the following steps of: a) contacting a test agent or compound with a cell into which a vector, composed of the transcriptional regulatory region of the CDC45L or PIF1 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; b) detecting the expression level or activity of said reporter gene; and c) correlating the expression level of b) with the therapeutic effect of the test agent or compound.
  • the present invention also provides a method for evaluating or estimating a therapeutic effect of a test agent or compound on treating or preventing cancer or inhibiting cancer associated with over-expression of CDC45L or PIF1, the method including steps of: (a) contacting a test agent or compound with a cell into which a vector, including the transcriptional regulatory region of CDC45L or PIF1 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced; (b) measuring the expression level or activity of said reporter gene; and (c) correlating the potential therapeutic effect and the test agent or compound, wherein the potential therapeutic effect is shown, when a test agent or compound reduces the expression level or activity of said reporter gene.
  • the therapeutic effect may be correlated with the expression level or activity of said reporter gene.
  • the test agent or compound when the test agent or compound reduces the expression level or activity of said reporter gene as compared to a level detected in the absence of the test agent or compound, the test agent or compound may be identified or selected as the candidate agent or compound having the therapeutic effect.
  • the test agent or compound when the test agent or compound does not reduce the expression level or activity of said reporter gene as compared to a level detected in the absence of the test agent or compound, the test agent or compound may be identified as the agent or compound having no significant therapeutic effect.
  • reporter genes are luciferase, green florescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa and so on.
  • the reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of CX.
  • the transcriptional regulatory region of CX herein is the region from start codon to at least 500bp upstream, for example, 1000bp, for example, 5000 or 10000bp upstream, but not restricted.
  • a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).
  • the vector containing the said reporter construct is infected to host cells and the expression or activity of the reporter gene is detected by method well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on).
  • “Reduces the expression or activity” as defined herein refers to at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the compound, for example, at least 25%, 50% or 75% reduction, for example, at least 95% reduction.
  • suppressing the expression level of CDC45L or PIF1 reduces cell growth.
  • candidate compounds that inhibits expression level of CDC45L or PIF1 candidate compounds that have the potential to treat or prevent cancers can be identified. Potential of these candidate compounds to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a compound inhibits the expression level of CDC45L or PIF1 protein inhibits activities of cancer, it may be concluded that such compound has CDC45L or PIF1 specific therapeutic effect.
  • the present invention also provides a method for screening a candidate compound for inhibiting or reducing a growth of cancer cells expressing CDC45L and PIF1 gene, e.g. lung cancer cell, and a candidate compound for treating or preventing cancers, e.g. lung cancer.
  • the present invention provides the following methods of [1] to [5]: [1] A method of screening for an agent or compound that interrupts a binding between a CDC45L polypeptide and a PIF1 polypeptide, said method comprising the steps of: (a) contacting a CDC45L polypeptide or functional equivalent thereof with a PIF1 polypeptide or functional equivalent thereof in the presence of a test agent or compound; (b) detecting a binding between the polypeptides; (c) comparing the binding level detected in the step (b) with those detected in the absence of the test agent or compound; and (d) selecting the test agent or compound that reduce or inhibits the binding level.
  • a method of screening for a candidate agent or compound useful in treating or preventing cancer comprising the steps of: (a) contacting a CDC45L polypeptide or functional equivalent thereof with a PIF1 polypeptide or functional equivalent thereof in the presence of a test agent or compound; (b) detecting a binding between the polypeptides; (c) comparing the binding level detected in the step (b) with those detected in the absence of the test agent or compound; and (d) selecting the test agent or compound that reduce or inhibits the binding level.
  • [4] The method of [1] or [2], wherein the functional equivalent of PIF1 comprising the CDC45L-binding domain.
  • [5] The method of [1], wherein the cancer is lung cancer.
  • the therapeutic effect of the test agent or compound on inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L or PIF1 associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate agent or compound that suppresses the proliferation of cancer cells, and a method for screening a candidate agent or compound for treating or preventing cancer.
  • the method includes the steps of: (a) contacting a CDC45L polypeptide or functional equivalent thereof with a PIF1 polypeptide or functional equivalent thereof in the presence of a test agent or compound; (b) detecting the level of binding between the polypeptides; (c) comparing the binding level detected in the step (b) with those detected in the absence of the test agent or compound; and (d) correlating the binding level of c) with the therapeutic effect of the test agent or compound.
  • the present invention also provides a method for evaluating or estimating a therapeutic effect of a test agent or compound on treating or preventing cancer or inhibiting cancer, the method including steps of: (a) contacting a CDC45L polypeptide or functional equivalent thereof with a PIF1 polypeptide or functional equivalent thereof in the presence of a test agent or compound; (b) detecting a binding level between the polypeptides; (c) comparing the binding level detected in the step (b) with those detected in the absence of the test agent or compound; and (d) correlating the potential therapeutic effect and the test agent or compound, wherein the potential therapeutic effect is shown, when a test agent or compound reduce the binding level.
  • the therapeutic effect may be correlated with the binding level of the CDC45L and PIF1 proteins.
  • the test agent or compound when the test agent or compound reduces the binding level of CDC45L and PIF1 proteins as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified or selected as the candidate agent or compound having the therapeutic effect.
  • the test agent or compound when the test agent or compound does not reduce the binding level of CDC45L and PIF1 proteins as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified as the agent or compound having no significant therapeutic effect.
  • a functional equivalent of an CDC45L or PIF1 polypeptide is a polypeptide that has a biological activity equivalent to a CDC45L polypeptide (SEQ ID NO: 14) or PIF1 polypeptide (SEQ ID NO: 16), respectively (see, (1) Genes and Polypeptides). More specifically, the functional equivalent of PIF1 polypeptide is a fragment of polypeptide having an amino acid sequence of SEQ ID NO: 16 comprising the CDC45L-binding domain. Also, the functional equivalent of CDC45L polypeptide is a fragment of polypeptide having an amino acid sequence of SEQ ID NO: 14 comprising the PIF1-binding domain.
  • a method of screening for compounds that inhibits the binding of CDC45L to PIF1 many methods well known by one skilled in the art can be used. Such a screening can be conducted using, for example, an immunoprecipitation, West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)), a two-hybrid system utilizing cells ("MATCHMAKER Two-Hybrid system", “Mammalian MATCHMAKER Two-Hybrid Assay Kit", “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-Hybrid Vector System” (Stratagene); the references “Dalton and Treisman, Cell 68: 597-612 (1992)", “Fields and Sternglanz, Trends Genet 10: 286-92 (1994)”), affinity chromatography and A biosensor using the surface plasmon resonance phenomenon (see (i) General screening Method and (ii) Screening for Compounds
  • a polypeptide to be used for screening can be a recombinant polypeptide or a protein derived from natural sources, or a partial peptide thereof. Any test compound aforementioned can be used for screening. Any aforementioned test compound can be used (see (1) Test compounds for screening).
  • this method further comprises the step of detecting the binding of the candidate compound to CDC45L protein or PIF1 protein, or detecting the level of binding CDC45L protein to or PIF1 protein.
  • Cells expressing CDC45L protein and/or PIF1 protein include, for example, cell lines established from cancer, e.g. lung cancer, such cells can be used for the above screening of the present invention so long as the cells express these two genes.
  • cells can be transfected both or either of expression vectors of CDC45L and PIF1 protein, so as to express these two genes.
  • the binding of CDC45L protein to PIF1 protein can be detected by immunoprecipitation assay using an anti-CDC45L antibody and PIF1 antibody (Fig. 4).
  • lung adenocarcinomas NCI-H1781, NCI-H1373, LC319, A549, and PC14
  • lung squamous-cell carcinomas SCCs
  • SCCs lung squamous-cell carcinomas
  • LCC lung large-cell carcinoma
  • SCLCs small-cell lung cancers
  • Formalin-fixed primary lung tumors and adjacent normal lung tissue samples used for immunostaining on tissue microarrays had been obtained from 267 patients (159 ADCs, 89 SCCs, 16 LCCs, 3 ASCs; 90 female and 177 male patients; median age of 65.0 with a range of 26 - 84 years, 114 pT1, 125 pT2, 28 pT3 tumor size ; 208 pN0, 23 pN1, 36 pN2 node status) undergoing curative surgery at Hokkaido University and its affiliated Hospitals (Sapporo, Japan). This study and the use of all clinical materials mentioned were approved by individual institutional Ethical Committees.
  • PEF1 5'-AGGCAGTGTCCCCTTCTGTA-3'
  • SEQ ID NO: 4 5'-CCTGAAAGGAGGGATGTTCA-3'
  • ACTB 5'-GAGGTGATAGCATTGCTTTCG-3'
  • 5'-CAAGTCAGTGTACAGGTAAGC-3' SEQ ID NO: 6
  • the membrane After blocking with 5% non-fat dry milk in TBST, the membrane was incubated with primary antibodies for 1 hour at room temperature. Immunoreactive proteins were incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-sciences) for 1 hour at room temperature. After washing with TBST, the reactants were developed using the enhanced chemiluminescence kit (GE Healthcare Bio-sciences).
  • a commercially available rabbit polyclonal anti-CDC45L antibody was purchased from ATLAS Antibodies AB (Catalog No. HPA000614) and was proved to be specific to human CDC45L by western-blot analysis using lysates of lung cancer cell lines.
  • HPA000614 was added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with horseradish peroxidase-labeled anti-rabbit IgG (Histofine Simple Stain MAX PO (G), Nichirei) as the secondary antibody. Substrate-chromogen was added, and the specimens were counterstained with hematoxylin.
  • Tumor tissue microarrays were constructed with 267 formalin-fixed primary NSCLCs as described elsewhere (Chin SF, et al. Molecular Pathology 2003;56:275-9., Callagy G, et al. Diagn Mol Pathol 2003;12:27-34., Callagy G, et al. J Pathol 2005;205:388-96.).
  • the tissue area for sampling was selected based on visual alignment with the corresponding H&E-stained section on a slide.
  • Three, four, or five tissue cores (diameter, 0.6 mm; depth, 3 - 4 mm) taken from a donor tumor block were placed into a recipient paraffin block with a tissue microarrayer (Beecher Instruments).
  • each specimen was mounted with Vectashield (Vector Laboratories, Inc.) containing 4',6'-diamidine-2'-phenylindolendihydrochrolide (DAPI) and visualized with Spectral Confocal Scanning Systems (TSC SP2 AOBS: Leica Microsystems).
  • DAPI 4',6'-diamidine-2'-phenylindolendihydrochrolide
  • RNAi assay Small interfering RNA (siRNA) duplexes (100 nM) were transfected into lung cancer cell lines, A549 and SBC-3, using 24 microliter of Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. The transfected cells were cultured for 7 days, and the number of colonies was counted by Giemsa staining, and viability of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (cell counting kit-8 solution; Dojindo Laboratories), at 7 days after transfection.
  • siRNA duplexes 100 nM
  • Lipofectamine 2000 Invitrogen
  • RNAi RNAi sequences from Photinus pyralis
  • control-1 LEC: luciferase gene from Photinus pyralis
  • 5'-CGUACGCGGAAUACUUCGA-3' SEQ ID NO: 7
  • control-2 EGFP: enhanced green fluorescent protein (GFP) gene, a mutant of Aequorea victoria GFP
  • 5'-GAAGCAGCACGACUUCUUC-3' SEQ ID NO: 8
  • si-CDC45L#1, 5'-GCAAACACCUGCUCAAGUC-3' SEQ ID NO: 9
  • si-CDC45L#2, 5'-GGACGUGGAUGCUCUGUGU-3' SEQ ID NO: 10
  • si-PIF1#1, 5'-GAAAGGCCAGAGCAUCUUC-3' SEQ ID NO: 11
  • A549 cells were plated at densities of 3.5 X 10 5 cells/100 mm dish and transfected with siRNA oligonucleotide. Cells were trypsinized 48 hours after transfection, collected in PBS, and fixed in 70% cold ethanol for 30 minutes. After treatment with 100 microgram/mL RNase (Sigma-Aldrich), the cells were stained with 50 microgram/mL propidium iodide (Sigma-Aldrich) in PBS. Flow cytometry was done on a Becton Dickinson FACScan and analyzed by ModFit software (Verity Software House, Inc.). The cells selected from at least 10,000 ungated cells were analyzed for DNA content.
  • Results CDC45L expression in lung cancer and normal tissues were performed using a cDNA microarray (NPL7-12). Among 27,648 genes or ESTs screened, elevated expression (3-fold or higher) of CDC45L transcript in the great majority of the clinical lung cancer samples examined was identified. It was confirmed its overexpression by means of semi-quantitative RT-PCR experiments in 12 of 15 lung cancer tissues and in all of 15 lung-cancer cell lines (Figs. 1A and 1B).
  • CDC45L protein was confirmed in all of 6 cancer cell lines, but no band was detected in normal bronchial epithelia derived cells (SAEC) by western-blot analysis using anti-CDC45L antibody (Fig. 1C).
  • SAEC normal bronchial epithelia derived cells
  • Fig. 1C immunofluorescent analysis was performed to examine the subcellular localization of endogenous CDC45L in lung cancer cell line A549, and its strong staining in nucleus and weak staining in cytoplasm was found (Fig. 1D).
  • Northern blotting using CDC45L cDNA as a probe identified a 2.2-kb band only in testis among 16 normal tissues examined (data not shown).
  • CDC45L protein expression levels in 5 normal tissues were compared with those in lung cancers.
  • CDC45L staining with polyclonal antibody specific to CDC45L was mainly observed at nucleus and cytoplasm of tumor cells, but not detected in normal lung cells (Fig. 2B).
  • CDC45L was stained positively in 171 cases (64.0%) and negatively in 96 cases (36.0%) (details are shown in Table 1A).
  • CDC45L Effects of CDC45L on growth of cancer cells.
  • siRNA oligonucleotides specific to CDC45L sequences were constructed and transfected into lung cancer cell lines, A549 (adenocarcinoma) and SBC-3 (small cell lung cancer), that endogenously expressed CDC45L at high levels.
  • a knockdown effect was confirmed by semi-quantitative RT-PCR when si-CDC45L#1 and si-CDC45L#2 constructs were used (Fig. 3A). Subsequent MTT and colony-formation assays (Figs.
  • the present inventors subsequently re-examined primary NSCLC tissues and lung-cancer cell lines by semi-quantitative RT-PCR experiments, and found increased PIF1 expression in 9 of 15 NSCLC clinical samples as well as in all of 15 lung-cancer cell lines examined, while its expression was scarcely detected in normal lung tissues and normal bronchial epithelia derived cells, SAEC (Fig. 4A).
  • SAEC normal bronchial epithelia derived cells
  • plasmids designed to express FLAG-tagged PIF1 protein were constructed, and transfected into SBC-3 cells, and then immunoprecipitated the proteins with anti-FLAG antibody.
  • Western blot analysis of the precipitates using anti-CDC45L antibody indicated that endogenous CDC45L was co-precipitated with exogenous PIF1 (Fig. 4B).
  • Immunocytochemical analysis confirmed the co-localization of endogenous CDC45L and exogenous PIF1 in nucleus and cytoplasm (Fig. 4C).
  • CDC45L is known to be a replication-initiation protein, its involvement in clinical cancer has not been indicated. Recently advanced understanding of the regulation of replication factors has provided new sources for possible proliferation markers as well as therapeutic targets. For instance, serine-threonine kinase CDC7 is activated to phosphorylate the MCM proteins and thereby recruits additional factors to promote unwinding of the DNA as well as binding of DNA polymerase, essential for initiation of DNA replication. Treatment with potent CDC7 kinase inhibitor, PHA-767491 resulted in induction of apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models (Montagnoli A, et al. Nat Chem Biol 2008;4:357-65.).
  • MCM proteins, CDC6, and geminin (GMNN) were immunohistochemically examined as prognostic biomarkers for patients with various types of cancers including breast, prostate, lung, brain, uterus, and kidney cancers (Gonzalez MA et al., Nat Rev Cancer 2005;5:135-41.).
  • the present invention also identified the interaction between CDC45L and PIF1 proteins in lung cancers.
  • PIF1 is classified as a member of SFI 5'-to-3' DNA helicase conserved from yeast to human, which is mainly reported as a factor for DNA replication in yeast (NPL 42, 43).
  • PIF1 was also highly transactivated in the great majority of lung cancers and treatment of lung cancer cells with specific siRNA resulted in suppression of the growth activity.
  • Saccharomyces cerevisiae PIF1 helicase functions in DNA replication with the DNa2 helicase/nuclease and DNA polymerase delta (Budd ME et al., Mol Cell Biol 2006;26:2490-500.).
  • CDC45L interacts with the MCM2-7 complex, the GINS complex, and DNA polymerases delta, and plays an important role in elongation of DNA replication by bridging the processive DNA polymerases delta and epsilon with the replicative helicase in the elongating machinery (NPL39). Based on the data provided here, targeting the CDC45L-PIF1 complex as well as CDC45L expression is a new approache to suppress the cancer cell proliferation and/or survival.
  • the gene-expression analysis of cancers described herein, using the combination of laser-capture dissection and genome-wide cDNA microarray, has identified a specific gene as a target for cancer prevention and therapy. Based on the expression of this differentially expressed gene, i.e., CDC45L, the present invention provides a novel molecular diagnostic marker for identifying and detecting cancers as well as assessing the prognosis. Further, PIF1, identified as the gene that its translation product was interacted with CDC45L, was confirmed overexpression in cancers. Therefore, the present invention also provides a novel diagnostic strategy using CDC45L or PIF1.
  • CDC45L and PIF1 are involved in cancer cell survival. Therefore, the present invention also provides novel molecular targets for treating and preventing cancer. They may be useful for developing novel therapeutic drugs and preventative agents without adverse effects.
  • the methods described herein are also useful for the identification of additional molecular targets for prevention, diagnosis, and treatment of cancers.
  • the data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provides indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.

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Abstract

The present invention is based on the finding that CDC45L gene and PIF1 gene are everexpressed in cancer, and involved in cancer cell survival. The present invention features methods for diagnosing cancer or assessing/determining the prognosis of a subject with cancer, using CDC45L gene and/or PIF1 gene as diagnostic markers. The present invention also features a double-stranded molecule against CDC45L gene or PIF1 gene, a method or composition for treating and/or preventing cancer using such double-stranded molecule Also, disclosed are methods of identifying a candidate compound for treating and preventing lung cancer, using CDC45L and/or PIF1 as molecular targets.

Description

CDC45L AS TUMOR MARKER AND THERAPEUTIC TARGET FOR CANCER
PRIORITY
The present application claims the benefit of U.S. Provisional Applications No. 61/274,248, filed on August 13, 2009, the entire contents of which are incorporated by reference herein.
TECHNICAL FIELD
The present invention relates to lung cancer, more particularly the diagnosis and treatment thereof.
Primary lung cancer is the leading cause of cancer deaths worldwide (NPL 1). Many genetic alterations involved in lung carcinogenesis have been reported, but the precise molecular mechanisms still remain unclear (NPL 2). In the last few decades cytotoxic agents including paclitaxel, docetaxel, gemcitabine, and vinorelbine have emerged to offer multiple therapeutic choices for patients with advanced NSCLC, however, those regimens provide a limited survival benefit compared with cisplatin-based therapies (NPL 3). In addition to these cytotoxic drugs, several molecular-targeted agents such as monoclonal antibodies against VEGF (i.e., bevacizumab/anti-VEGF) or EGFR (i.e., cetuximab/anti-EGFR) as well as inhibitors for EGFR tyrosine kinase (i.e., gefitinib and erlotinib) were developed and are now widely used in clinical practice (NPL 4-6). Each of the new regimens can provide survival benefits to a limited proportion of the patients. Hence, new therapeutic strategies, such as the development of more effective molecular-targeted agents applicable to the great majority of patients with less toxicity, are eagerly awaited.
To isolate potential molecular targets for diagnosis, treatment, and/or prevention of lung cancer, a genome-wide analysis of gene expression profiles was performed on cancer cells purified by laser microdissection from 101 cases of lung cancer tissues using a cDNA microarray consisting of 27,648 genes or expressed sequence tags (ESTs) (NPL 7-12). To verify the biological and clinicopathological significance of the respective gene products, a screening system using a combination of the tumor-tissue microarray analysis of clinical lung cancer materials with RNA interference (RNAi) technique and cell growth/invasion assays was established (NPL 13-37).
In budding yeast (Saccharomyces cerevisiae), CDC45 cell division cycle 45-cell (CDC45) gene is an essential gene required for initiation of DNA replication, and together with MCM and CDC6 proteins it forms a complex necessary for the initiation of DNA replication in eukaryotic cells (NPL 38). A human homolog of yeast CDC45 gene termed CDC45L encodes a protein of 566 amino acids and shows 27.6% identity to yeast CDC45 (NPL 39). CDC45L interacts in HeLaS3 cells with the elongating DNA polymerases delta and -epsilon, and with Psf2, which is a component of the GINS complex as well as with MCM5 and -7, subunits of the putative replicative DNA helicase complex (NPL 40). A higher level of CDC45L protein expression in proliferating cell populations of cancer cell lines has been reported (NPL 41). However, the roles of CDC45L activation in the development and progression of cancer have not been clarified.
On the other hand, petite integration frequency 1 (PIF1) gene was first identified in Saccharomyces cerevisiae and was classified as a member of SFI 5'-to-3' DNA helicase conserved from yeast to human (NPL 42). The function of PIF1 in yeast is implicated in mtDNA repair, rDNA replication and in the regulation of telomere length (NPL 42, 43). In addition, yeast PIF1 is thought to be a region-specific DNA helicase because in vitro it requires a specific DNA structure for the efficient DNA unwinding and unwinds DNA in a 5'-to-3' direction (NPL 43). Although PIF1-like helicases exist in many organisms, most studies have been done in yeast species, and little is known about PIF1 function in mammalian cells and about its involvement in carcinogenesis.
[PTL 1] WO2007/013671
[PTL 2] 61/217,133
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In the course of screening for novel molecular targets for diagnosis, treatment and prevention of human cancers, genome-wide expression profile analyses of 267 lung cancers was performed on cDNA microarray containing 27,648 genes, coupled with laser microdissection (Kikuchi T, et al. Oncogene. 2003 Apr 10;22(14):2192-205.; Kikuchi T, et al. Int J Oncol. 2006 Apr;28(4):799-805; Kakiuchi S, et al., Mol Cancer Res. 2003 May;1(7):485-99; Kakiuchi S, et al., Hum Mol Genet. 2004 Dec 15;13(24):3029-43. Epub 2004 Oct 20; Taniwaki M, et al., Int J Oncol. 2006 Sep; 29(3):567-75.). The results demonstrate that the CDC45L gene is frequently over-expressed in the great majority of primary lung cancers. Also, PIF1 gene, identified as a gene encoding the protein that interacts with CDC45L protein, was found to be over-expressed in lung cancers. Furthermore, siRNAs against these genes suppressed cancer cell proliferation effectively. These results suggest that CDC45L gene and PIF1 gene will become good molecular targets for diagnosis or treatment of cancer.
Thus, the present invention relates to cancer-related gene CDC45L and PIF1, which are commonly up-regulated in tumors, and strategies for the development of molecular targeted drugs for cancer treatment using CDC45L and/or PIF1.
In one aspect, the present invention provides a method for diagnosing cancer, e.g., a cancer overe-expressing a CDC45L gene and/or PIF1 gene, e.g., lung cancer, using the expression level of the CDC45L and/or PIF1 gene as an index. In the methods of the present invention, the mRNA of CDC45L or PIF1 gene can be detected by appropriate primers or probes or, alternatively, the CDC45L or PIF1 protein can be detected by anti- CDC45L or PIF1 antibody in order to determine the expression level of the gene. In some embodiments, the cancer is mediated or promoted by a CDC45L and/or PIF1. In some embodiments, the cancer is lung cancer. In one embodiment, the cancer is a lung adenocarcinoma (ADC), a lung squamous-cell carcinoma (SCC), and a lung large-cell carcinoma (LCC), or a small-cell lung cancers (SCLC).
The present invention also provides a method for predicting the progress of a subject with cancer, e.g., lung cancer, using the expression level of the CDC45L as an index.
Furthermore, the present invention provides a method for predicting the prognosis of the cancer, e.g., lung cancer, patient using the expression level of the CDC45L and/or PIF1 gene or biological activity of the CDC45L and/or PIF1 protein as an index.
In another embodiment, the present invention provides a method for screening a candidate compound for treating or preventing cancer, e.g., lung cancer, using the binding to the CDC45L and/or PIF1 polypeptide, the expression level of the CDC45L and/or PIF1 gene, or biological activity of the CDC45L and/or PIF1 polypeptide as an index.
In another embodiment, the present invention provides a method for screening a candidate compound for treating or preventing cancer, e.g., lung cancer, using the interaction between CDC45L polypeptide and PIF1 polypeptide as an index.
In a further embodiment, the present invention provides double-stranded molecules, e.g., siRNA, against the CDC45L or PIF1 gene , that inhibits the expression of the gene, and vectors encoding the double stranded molecule. The double-stranded molecules of the present invention are useful for treating or preventing cancers, e.g., a cancer mediated by a CDC45L and/or PIF1 or resulting from overexpression of a CDC45L and/or PIF1, e.g., lung cancer.
In another embodiment, the present invention provides methods of treating or preventing cancer in a subject, comprising administering to said subject a pharmaceutically effective amount of a double-stranded molecule against CDC45L gene or PIF1 gene, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits cell proliferation as well as the expression of the gene.
In another embodiment, the present invention provides compositions for treating or preventing cancer, comprising a double-stranded molecule against a CDC45L or PIF1, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits cell proliferation as well as the expression of the gene, and a pharmaceutically acceptable carrier.
It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the preceding objects can be viewed in the alternative with respect to any one aspect of this invention. These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are of a preferred embodiment, and not restrictive of the invention or other alternate embodiments of the invention.
Various aspects and applications of the present invention will become apparent to the skilled artisan upon consideration of the brief description of the figures and the detailed description of the present invention and its preferred embodiments which follows:
Figure 1 depicts the expression of CDC45L in lung tumor tissues and cell lines.A, B, Expression of CDC45L in a normal lung tissue and 15 clinical lung cancer tissue samples (5 lung ADC, 5 lung SCC, and 5 SCLC) (A) and normal airway epithelia derived cells (SAEC) and 15 lung-cancer cell lines (B), detected by semi-quantitative RT-PCR analysis. C, Expression of CDC45L protein in 6 lung-cancer cell lines and SAEC cells examined by western-blot analysis. D, Subcellular localization of endogenous CDC45L protein in LC319 cells. CDC45L was stained strongly in the nucleus and weakly in the cytoplasm.
Figure 2 depicts the expression of CDC45L in normal tissues and association of CDC45L overexpression with poorer prognosis for NSCLC patients.A, Immunohistochemical analysis of CDC45L protein expression in 5 normal tissues (heart, lung, liver, kidney, and testis) and lung cancers. CDC45L expressed abundantly in testis and lung cancer cells (mainly in nucleus and/or cytoplasm), but its expression was hardly detectable in the remaining four normal tissues. B, Examples for positive and negative staining of CDC45L expression in cancer tissues (original magnification X100). C, Association of CDC45L protein expression with poor prognosis. Kaplan-Meier analysis of survival of patients with NSCLC (P = 0.0045 by the Log-rank test).
Figure 3 depicts the growth suppressive effect of siRNA against CDC45L on lung cancer cells.A, Gene knockdown effect on CDC45L expression in A549 (left) and SBC-3 (right) cells by two different siRNAs for CDC45L, si-CDC45L-#1 and si-CDC45L-#2, and two control siRNAs (si-LUC and si-EGFP), analyzed by semi-quantitative RT-PCR. B, C, Colony formation (B) and MTT (C) assays of A549 (left) and SBC-3 (right) cells transfected with si-CDC45Ls or control siRNAs. Columns, relative absorbance of triplicate assays; bars, SD. D, Flow cytometric analysis of A549 cells transfected with si-CDC45L-#2 or si-LUC (48 hours after the transfection).
Figure 4 depicts the interaction of CDC45L with PIF1.A, Expression analysis of CDC45L and PIF1 in a normal lung tissue and 15 clinical lung cancer tissue samples by semi-quantitative RT-PCR analysis. B, Interaction of endogenous CDC45L with exogenous PIF1 in lung cancer SBC-3 cells. Immunoprecipitations using cell lysates from SBC-3 cells transfected with FLAG-tagged PIF1-expression plasmids were carried out with anti-FLAG antibody. Immunoprecipitates were subjected to western-blot analysis using anti-CDC45L antibody. IB, immunoblotting; IP, immunoprecipitation. C, Immunofluorescence staining of endogenous CDC45L and exogenous PIF1 in SBC-3 cells. The CDC45L-Alexa488, PIF1-Alexa594, or cell nuclei (4'6'-diamidino-2-phenylindole dihydrochloride [DAPI]) were visualized. Co-localization of CDC45L and PIF1 in nucleus and cytoplasm was observed.
Figure 5 depicts the growth suppressive effect by siRNA for PIF1 on lung cancer cells.A, Gene knockdown effect on PIF1 expression in A549 (left) and SBC-3 (right) cells by two kinds of si-PIF1 (si-PIF1-#1 and si-PIF1-#2) and two control siRNAs (si-LUC and si-EGFP), detected by semi-quantitative RT-PCR analysis. B, C, Colony formation (B) and MTT (C) assays of A549 (left) and SBC-3 (right) cells transfected with si-PIF1s or control siRNAs. Columns, relative absorbance of triplicate assays; bars, SD.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. However, before the present materials and methods are described, it is to be understood that the present invention is not limited to the particular sizes, shapes, dimensions, materials, methodologies, protocols, etc. described herein, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
The disclosure of each publication, patent or patent application mentioned in this specification is specifically incorporated by reference herein in its entirety. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Definitions
The words "a", "an", and "the" as used herein mean "at least one" unless otherwise specifically indicated.
The terms "isolated" and "purified" used in relation with a substance (e.g., polypeptide, antibody, polynucleotide, etc.) indicates that the substance is substantially free from at least one substance that can be included in the natural source. Thus, an isolated or purified antibody refers to antibodies that are substantially free of cellular material for example, carbohydrate, lipid, or other contaminating proteins from the cell or tissue source from which the protein (antibody) is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes preparations of a polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
Thus, a polypeptide that is substantially free of cellular material includes preparations of polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the polypeptide is recombinantly produced, in some embodiments it is also substantially free of culture medium, which includes preparations of polypeptide with culture medium less than about 20%, 10%, or 5% of the volume of the protein preparation. When the polypeptide is produced by chemical synthesis, in some embodiments it is substantially free of chemical precursors or other chemicals, which includes preparations of polypeptide with chemical precursors or other chemicals involved in the synthesis of the protein less than about 30%, 20%, 10%, 5% (by dry weight) of the volume of the protein preparation. That a particular protein preparation contains an isolated or purified polypeptide can be shown, for example, by the appearance of a single band following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining or the like of the gel. In one embodiment, proteins including antibodies of the present invention are isolated or purified.
An "isolated" or "purified" nucleic acid molecule, for example, a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, nucleic acid molecules encoding proteins of the present invention are isolated or purified.
The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, for example, an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that similarly functions to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those modified after translation in cells (e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine). The phrase "amino acid analog" refers to compounds that have the same basic chemical structure (an alpha carbon bound to a hydrogen, a carboxy group, an amino group, and an R group) as a naturally occurring amino acid but have a modified R group or modified backbones (e.g., homoserine, norleucine, methionine, sulfoxide, methionine methyl sulfonium). The phrase "amino acid mimetic" refers to chemical compounds that have different structures but similar functions to general amino acids.
Amino acids can be referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
The terms "gene", "polynucleotide", "oligonucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably unless otherwise specifically indicated and are similarly to the amino acids referred to by their commonly accepted single-letter codes. Similar to the amino acids, they encompass both naturally-occurring and non-naturally occurring nucleic acid polymers. The gene, polynucleotide, oligonucleotide, nucleic acid, or nucleic acid molecule can be composed of DNA, RNA or a combination thereof.
As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "Biological sample" further refers to a homogenate, lysate, extract, cell culture or tissue culture prepared from a whole organism or a subset of its cells, tissues or component parts, or a fraction or portion thereof. Lastly, "biological sample" refers to a medium, for example, a nutrient broth or gel in which an organism has been propagated, which contains cellular components, for example, proteins or polynucleotides.
Unless otherwise defined, the terms "cancer" refers to cancers over-expressing the CDC45L and/or PIF1 gene, such as lung cancer, including adenocarcinoma (ADC), squamous-cell carcinoma (SCC), large-cell carcinoma (LCC), and small-cell lung cancer (SCLC).
(1) Genes and Polypeptides
The nucleotide sequence of human CDC45L gene is shown in SEQ ID NO: 13 and is also available as GenBank Accession No. NM_003504.3. Herein, the phrase "CDC45L gene" encompasses the human CDC45L gene as well as those of other animals including non-human primate, mouse, rat, dog, cat, horse, and cow but is not limited thereto, and includes allelic mutants and genes found in other animals as corresponding to the CDC45L gene.
The amino acid sequence encoded by the human CDC45L gene is shown as SEQ ID NO: 14 and is also available as GenBank Accession No. NM_003504.3 (NP_003495.1). In the present invention, the polypeptide encoded by the CDC45L gene is referred to as "CDC45L", and sometimes as "CDC45L polypeptide" or "CDC45L protein".
The nucleotide sequence of human PIF1 gene is shown in SEQ ID NO: 15 and is also available as GenBank Accession No. NM_025049.2. Herein, the phrase "PIF1 gene" encompasses the human PIF1 gene as well as those of other animals including non-human primate, mouse, rat, dog, cat, horse, and cow but is not limited thereto, and includes allelic mutants and genes found in other animals as corresponding to the PIF1 gene.
The amino acid sequence encoded by the human PIF1 gene is shown as SEQ ID NO: 16 and is also available as GenBank Accession No. NM_025049.2 (NP_079325.2). In the present invention, the polypeptide encoded by the PIF1 gene is referred to as "PIF1", and sometimes as "PIF1 polypeptide" or "PIF1 protein".
According to an aspect of the present invention, functional equivalents are also included in the CDC45L and PIF1. Herein, a "functional equivalent" of a protein is a polypeptide that has a biological activity equivalent to the protein. Namely, any polypeptide that retains at least one biological activity of CDC45L or PIF1 can be used as such a functional equivalent in the present invention. For example, functional equivalents of CDC45L and the functional equivalents of PIF1 retain promoting activity of cell proliferation. In addition, the biological activity of CDC45L contains binding activity to PIF1. Therefore, in a preferred embodiment, a functional equivalent of CDC45L can contain a PIF1 binding region. Also, the biological activity of PIF1 contains binding activity to CDC45L. Therefore, in a preferred embodiment, a functional equivalent of PIF1 can contain a CDC45L binding region.
Functional equivalents of CDC45L include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the CDC45L protein. Also, functional equivalents of PIF1 include those wherein one or more amino acids, e.g., 1-5 amino acids, e.g., up to 5% of amino acids, are substituted, deleted, added, or inserted to the natural occurring amino acid sequence of the PIF1 protein.
Generally, it is known that modifications of one or more amino acid in a protein do not influence the function of the protein (Mark DF, et al., Proc Natl Acad Sci U S A. 1984 Sep;81(18):5662-6; Zoller MJ & Smith M. Nucleic Acids Res. 1982 Oct 25;10(20):6487-500; Wang A, et al., Science. 1984 Jun 29;224(4656):1431-3; Dalbadie-McFarland G, et al., Proc Natl Acad Sci U S A. 1982 Nov;79(21):6409-13). One of skill in the art will recognize that individual additions, deletions, insertions, or substitutions to an amino acid sequence which alters a single amino acid or a small percentage of amino acids is a "conservative modification" wherein the alteration of a protein results in a protein with similar functions.
Examples of properties of amino acid side chains are hydrophobic amino acids (alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, valine), hydrophilic amino acids (arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, serine, threonine), and side chains having the following functional groups or characteristics in common: an aliphatic side-chain (glycine, alanine, valine, leucine, isoleucine, praline); a hydroxyl group containing side-chain (serine, threonine, tyrosine); a sulfur atom containing side-chain (C, M); a carboxylic acid and amide containing side-chain (aspartic acid, asparagine, glutamic acid, glutamine); a base containing side-chain (arginine, lysine, histidine); and an aromatic containing side-chain (histidine, phenylalanine, tyrosine, tryptophan). Furthermore, conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following eight groups each contain amino acids that are conservative substitutions for one another:
(1) Alanine (A), Glycine (G);
(2) Aspartic acid (D), Glutamic acid (E);
(3) Aspargine (N), Glutamine (Q);
(4) Arginine (R), Lysine (K);
(5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
(6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
(7) Serine (S), Threonine (T); and
(8) Cysteine (C), Methionine (M)
(see, e.g., Thomas E. Creighton, Proteins Publisher: New York: W.H. Freeman, c1984).
Such conservatively modified polypeptides are included in the CDC45L or PIF1 protein. However, the present invention is not restricted thereto and the CDC45L or PIF1 protein includes non-conservative modifications so long as they retain any one of the biological activity of the CDC45L or PIF1 protein. The number of amino acids to be mutated in such a modified protein is generally 10 amino acids of less, for example, 6 amino acids of less, for example, 3 amino acids or less.
An example of a protein modified by addition of one or more amino acids residues is a fusion protein of the CDC45L or PIF1 protein. Fusion proteins include fusions of the CDC45L or PIF1 protein and other peptides or proteins, which also can be used in the present invention. Fusion proteins can be made by techniques well known to a person skilled in the art, for example, by linking the DNA encoding the CDC45L or PIF1 gene with a DNA encoding other peptides or proteins, so that the frames match, inserting the fusion DNA into an expression vector and expressing it in a host. There is no restriction as to the peptides or proteins fused to the CDC45L or PIF1 protein so long as the resulting fusion protein retains any one of the objective biological activity of the CDC45L or PIF1 proteins.
Known peptides that can be used as peptides to be fused to the protein include, for example, FLAG (Hopp TP, et al., Biotechnology 6: 1204-10 (1988)), 6xHis containing six His (histidine) residues, 10xHis, Influenza agglutinin (HA), human c-myc fragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, alpha-tubulin fragment, B-tag, Protein C fragment, and the like. Examples of proteins that can be fused to a protein of the invention include GST (glutathione-S-transferase), Influenza agglutinin (HA), immunoglobulin constant region, beta-galactosidase, MBP (maltose-binding protein), and such.
Furthermore, the modified proteins do not exclude polymorphic variants, interspecies homologues, and those encoded by alleles of these proteins.
Methods known in the art to isolate functional equivalent proteins include, for example, hybridization techniques (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Lab. Press, 2001). One skilled in the art can readily isolate a DNA having high homology (i.e., sequence identity) with a whole or part of the human CDC45L DNA sequences (e.g., SEQ ID NO: 13) encoding the human CDC45L protein, and isolate functional equivalent proteins to the human CDC45L protein from the isolated DNA. Also, one skilled in the art can readily isolate a DNA having high homology (i.e., sequence identity) with a whole or part of the human PIF1 DNA sequences (e.g., SEQ ID NO: 15) encoding the human PIF1 protein, and isolate functional equivalent proteins to the human PIF1 protein from the isolated DNA. Thus, the proteins used for the present invention include those that are encoded by DNA that hybridize under stringent conditions with a whole or part of the DNA sequence encoding the human CDC45L protein or the human PIF1 protein and are functional equivalent to the human CDC45L protein or the human PIF1 protein. These proteins include mammal homologues corresponding to the proteins derived from human or mouse (for example, proteins encoded by monkey, rat, rabbit or bovine genes). In isolating a cDNA highly homologous to the DNA encoding the human CDC45L gene or human PIF1 gene, lung cancer tissues or cell lines, or tissues from testis can be used.
The conditions of hybridization for isolating a DNA encoding a protein functional equivalent to the human CDC45L gene or human PIF1 gene can be routinely selected by a person skilled in the art. The phrase "stringent (hybridization) conditions" refers to conditions under which a nucleic acid molecule will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but not detectably to other sequences. Stringent conditions are sequence-dependent and will differ under different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10 degrees Centigrade lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions can also be achieved with the addition of destabilizing agents for example, formamide. For selective or specific hybridization, a positive signal is at least two times of background, for example, 10 times of background hybridization.
For example, hybridization can be performed by conducting prehybridization at 68 degrees C for 30 min or longer using "Rapid-hyb buffer" (Amersham LIFE SCIENCE), adding a labeled probe, and warming at 68 degrees C for 1 hour or longer. The following washing step can be conducted, for example, in a low stringent condition. A low stringent condition is, for example, 42 degrees C, 2x SSC, 0.1% SDS, for example, 50 degrees C, 2x SSC, 0.1% SDS. In some embodiments, high stringent condition is used. A high stringent condition is, for example, washing 3 times in 2x SSC, 0.01% SDS at room temperature for 20 min, then washing 3 times in 1x SSC, 0.1% SDS at 37 degrees C for 20 min, and washing twice in 1x SSC, 0.1% SDS at 50 degrees C for 20 min. However, several factors for example, temperature and salt concentration can influence the stringency of hybridization and one skilled in the art can suitably select the factors to achieve the requisite stringency.
In place of hybridization, a gene amplification method, for example, the polymerase chain reaction (PCR) method, can be utilized to isolate a DNA encoding a protein functional equivalent to the human CDC45L or PIF1 gene, using a primer synthesized based on the sequence information of the DNA (SEQ ID NO: 13 or 15) encoding the human CDC45L or PIF1 protein (SEQ ID NO: 14 or 16), examples of primer sequences are pointed out in Semi-quantitative RT-PCR in [EXAMPLE].
Proteins that are functional equivalent to the human CDC45L or PIF1 protein encoded by the DNA isolated through the above hybridization techniques or gene amplification techniques, normally have a high homology (also referred to as sequence identity) to the amino acid sequence of the human CDC45L or PIF1 protein. "High homology" (also referred to as "high sequence identity") typically refers to the degree of identity between two optimally aligned sequences (either polypeptide or polynucleotide sequences). Typically, high homology or sequence identity refers to homology of 40% or higher, for example, 60% or higher, for example, 80% or higher, for example, 85%, 90%, 95%, 98%, 99%, or higher. The degree of homology or identity between two polypeptide or polynucleotide sequences can be determined by following the algorithm (Wilbur WJ & Lipman DJ. Proc Natl Acad Sci U S A. 1983 Feb; 80 (3):726-30).
Additional examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described (Altschul SF, et al., J Mol Biol. 1990 Oct 5; 215 (3):403-10; Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (on the worldwide web at ncbi.nlm.nih.gov/). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them.
The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff S & Henikoff JG. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10915-9).
A protein useful in the context of the present invention can have variations in amino acid sequence, molecular weight, isoelectric point, the presence or absence of sugar chains, or form, depending on the cell or host used to produce it or the purification method utilized. Nevertheless, so long as it has any one of the biological activity of the CDC45L protein (SEQ ID NO: 14) or PIF1 protein (SEQ ID NO: 16), it is useful in the present invention.
The present invention also encompasses the use of partial peptides of the CDC45L protein and the PIF1 protein. A partial peptide has an amino acid sequence specific to the CDC45L protein or the PIF1 protein and consists of less than about 400 amino acids, usually less than about 200 and often less than about 100 amino acids, and at least about 7 amino acids, for example, about 8 amino acids or more, for example, about 9 amino acids or more.
A partial CDC45L peptide used for the screenings of the present invention suitably contains at least a binding domain of CDC45L. Furthermore, a partial CDC45L peptide used for the screenings of the present invention suitably contains PIF1 binding region. Such partial peptides are also encompassed by the phrase "functional equivalent" of the CDC45L protein. Also, A partial PIF1 peptide used for the screenings of the present invention suitably contains at least a binding domain of PIF1. Furthermore, a partial PIF1 peptide used for the screenings of the present invention suitably contains CDC45L binding region. Such partial peptides are also encompassed by the phrase "functional equivalent" of the PIF1 protein.
The polypeptide or fragments used for the present method can be obtained from nature as naturally occurring proteins via conventional purification methods or through chemical synthesis based on the selected amino acid sequence. For example, conventional peptide synthesis methods that can be adopted for the synthesis include:
(1) Peptide Synthesis, Interscience, New York, 1966;
(2) The Proteins, Vol. 2, Academic Press, New York, 1976;
(3) Peptide Synthesis (in Japanese), Maruzen Co., 1975;
(4) Basics and Experiment of Peptide Synthesis (in Japanese), Maruzen Co., 1985;
(5) Development of Pharmaceuticals (second volume) (in Japanese), Vol. 14 (peptide synthesis), Hirokawa, 1991;
(6) WO99/67288; and
(7) Barany G. & Merrifield R.B., Peptides Vol. 2, "Solid Phase Peptide Synthesis", Academic Press, New York, 1980, 100-118.
Alternatively, the protein can be obtained adopting any known genetic engineering methods for producing polypeptides (e.g., Morrison DA., et al., J Bacteriol. 1977 Oct;132(1):349-51; Clark-Curtiss JE & Curtiss R 3rd. Methods Enzymol. 1983;101:347-62). For example, first, a suitable vector comprising a polynucleotide encoding the objective protein in an expressible form (e.g., downstream of a regulatory sequence comprising a promoter) is prepared, transformed into a suitable host cell, and then the host cell is cultured to produce the protein. More specifically, a gene encoding the CDC45L or PIF1 is expressed in host (e.g., animal) cells and such by inserting the gene into a vector for expressing foreign genes, for example, pSV2neo, pcDNA I, pcDNA3.1, pCAGGS, or pCD8.
A promoter can be used for the expression. Any commonly used promoters can be employed including, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic engineering, vol. 3. Academic Press, London, 1982, 83-141), the EF- alpha promoter (Kim DW, et al. Gene. 1990 Jul 16;91(2):217-23), the CAG promoter (Niwa H, et al., Gene. 1991 Dec 15;108(2):193-9), the RSV LTR promoter (Cullen BR. Methods Enzymol. 1987;152:684-704), the SR alpha promoter (Takebe Y, et al., Mol Cell Biol. 1988 Jan;8(1):466-72), the CMV immediate early promoter (Seed B & Aruffo A. Proc Natl Acad Sci U S A. 1987 May;84(10):3365-9), the SV40 late promoter (Gheysen D & Fiers W. J Mol Appl Genet. 1982;1(5):385-94), the Adenovirus late promoter (Kaufman RJ, et al., Mol Cell Biol. 1989 Mar;9(3):946-58), the HSV TK promoter, and such.
The introduction of the vector into host cells to express the CDC45L or PIF1 gene can be performed according to any methods, for example, the electroporation method (Chu G, et al., Nucleic Acids Res. 1987 Feb 11;15(3):1311-26), the calcium phosphate method (Chen C & Okayama H. Mol Cell Biol. 1987 Aug;7(8):2745-52), the DEAE dextran method (Lopata MA, et al., Nucleic Acids Res. 1984 Jul 25;12(14):5707-17; Sussman DJ & Milman G. Mol Cell Biol. 1984 Aug;4(8):1641-3), the Lipofectin method (Derijard B, et al., Cell. 1994 Mar 25;76(6):1025-37; Lamb BT, et al., Nat Genet. 1993 Sep;5(1):22-30; Rabindran SK, et al., Science. 1993 Jan 8;259(5092):230-4), and such.
The proteins can also be produced in vitro by using an in vitro translation system.
In the context of the present invention, the phrase "CDC45L gene" encompasses polynucleotides that encode the human CDC45L or any of the functional equivalents of the human CDC45L gene. Also, the phrase "PIF1 gene" encompasses polynucleotides that encode the human PIF1 or any of the functional equivalents of the human PIF1 gene.
The CDC45L gene and the PIF1 gene can be obtained from nature as naturally occurring proteins via conventional cloning methods or through chemical synthesis based on the selected nucleotide sequence. Methods for cloning genes using cDNA libraries and such are well known in the art.
(2) Antibodies
The terms "antibody" as used herein is intended to include immunoglobulins and fragments thereof which are specifically reactive to the designated protein or peptide thereof. An antibody can include human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, antibodies fused to other proteins or radiolabels, and antibody fragments. Furthermore, an antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. An "antibody" indicates all classes (e.g. IgA, IgD, IgE, IgG and IgM).
The present invention may use antibodies against CDC45L protein or PIF1 protein. These antibodies can be useful for diagnosing lung cancer. Furthermore, the present invention may use antibodies against partial peptides of CDC45L polypeptides or PIF polypeptides.
In the screening methods described bellow, antibodies against the PIF binding region of CDC45L polypeptides or the CDC45L binding region of the PIF1 polypeptides may be preferably used. These antibodies can be useful for inhibiting and/or blocking an interaction, e.g. binding, between CDC45L polypeptides and PIF1 polypeptides and can be useful for treating and/or preventing cancer (over)expressing CDC45L and/or PIF1, e.g. lung cancer. These antibodies will be provided by known methods. For techniques for the production of the antibodies used in the present invention, conventional methods can be used.
(3) Double-stranded molecules
As used herein, the term "double-stranded molecule" refers to a nucleic acid molecule that inhibits the expression of a target gene including, for example, short interfering RNA (siRNA; e.g., double-stranded ribonucleic acid (dsRNA) or small hairpin RNA (shRNA)) and short interfering DNA/RNA (siD/R-NA; e.g. double-stranded chimera of DNA and RNA (dsD/R-NA) or small hairpin chimera of DNA and RNA (shD/R-NA)).
As used herein, the term "siRNA" refers to a double-stranded RNA molecule which prevents translation of a target mRNA. Standard techniques of introducing siRNA into the cell are used, including those in which DNA is a template from which RNA is transcribed. The siRNA includes a ribonucleotide corresponding to a sense nucleic acid sequence of a target gene (also referred to as "sense strand"), a ribonucleotide corresponding to an antisense nucleic acid sequence of a target gene (also referred to as "antisense strand") or both. The siRNA can be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences of the target gene, e.g., a hairpin. The siRNA can either be a dsRNA or shRNA.
As used herein, the term "dsRNA" refers to a construct of two RNA molecules comprising complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded RNA molecule. In the present invention, the double-stranded RNA molecule may also refer to siRNA, or small interfering RNA molecule. The sequence of two strands can comprise not only the "sense" or "antisense" RNAs selected from a protein coding sequence of a target gene sequence, but also RNA molecule having a nucleotide sequence selected from non-coding region of the target gene.
As used herein the term "shRNA", as used herein, refers to an siRNA having a stem-loop structure, comprising a first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the region is sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shRNA is a single-stranded region intervening between the sense and antisense strands and can also be referred to as "intervening single-strand".
As used herein, the term "siD/R-NA" refers to a double-stranded molecule which is composed of both RNA and DNA, and includes hybrids and chimeras of RNA and DNA and prevents translation of a target mRNA. Herein, a hybrid indicates a molecule wherein an oligonucleotide composed of DNA and an oligonucleotide composed of RNA hybridize to each other to form the double-stranded molecule; whereas a chimera indicates that one or both of the strands composing the double stranded molecule can contain RNA and DNA. Standard techniques of introducing siD/R-NA into the cell are used. The siD/R-NA includes a sense nucleic acid sequence of a target gene (also referred to as "sense strand"), an antisense nucleic acid sequence of a target gene (also referred to as "antisense strand") or both. The siD/R-NA can be constructed such that a single transcript has both the sense and complementary antisense nucleic acid sequences from the target gene, e.g., a hairpin. The siD/R-NA can either be a dsD/R-NA or shD/R-NA.
As used herein, the term "dsD/R-NA" refers to a construct of two molecules comprising complementary sequences to one another and that have annealed together via the complementary sequences to form a double-stranded polynucleotide molecule. The nucleotide sequence of two strands can comprise not only the "sense" or "antisense" polynucleotides sequence selected from a protein coding sequence of target gene sequence, but also polynucleotide having a nucleotide sequence selected from non-coding region of the target gene. One or both of the two molecules constructing the dsD/R-NA are composed of both RNA and DNA (chimeric molecule), or alternatively, one of the molecules is composed of RNA and the other is composed of DNA (hybrid double-strand).
The term "shD/R-NA", as used herein, refers to an siD/R-NA having a stem-loop structure, comprising a first and second regions complementary to one another, i.e., sense and antisense strands. The degree of complementarity and orientation of the regions is sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of an shD/R-NA is a single-stranded region intervening between the sense and antisense strands and can also be referred to as "intervening single-strand".
(i) Target sequences
As used herein, the term "target sequence" is a nucleotide sequence within an mRNA or cDNA sequence of a target gene, which will result in suppress of translation of the whole mRNA of the target gene if a double-stranded molecule targeting the sequence is introduced into a cell expressing the target gene. A nucleotide sequence within an mRNA or cDNA sequence of a target gene can be determined to be a target sequence when a double-stranded molecule comprising a sequence corresponding to the target sequence inhibits expression of the target gene in a cell expressing the gene. The double stranded polynucleotide by which suppresses the gene expression may consist of the target sequence and 3' overhang (e.g., uu).
When a target sequence is shown by cDNA sequence, the sense strand of the double-stranded cDNA, i.e., a sequence that mRNA sequence is converted into DNA sequence, is used for defining a target sequence. A double-stranded molecule is composed of a sense strand that has a sequence corresponding to a target sequence and an antisense strand that has a complementary sequence to the target sequence, and the antisense strand hybridizes with the sense strand at the complementary sequence to form a double-stranded molecule.
Herein, the phrase "corresponding to" means converting a target sequence according to the kind of nucleic acid that constitutes a sense strand of a double-stranded molecule. For example, when a target sequence is shown in DNA sequence and a sense strand of a double-stranded molecule has an RNA region, base "t"s within the RNA region is replaced with base "u"s. On the other hand, when a target sequence is shown in RNA sequence and a sense strand of a double-stranded molecule has a DNA region, base "u"s within the DNA region is replaced with "t"s. For example, when a target sequence is shown in the RNA sequence of SEQ ID NO: 9 and the sense strand of the double-stranded molecule has the 3' side half region composed of DNA, "a sequence corresponding to a target sequence" is "5'- GCAAACACCUGCTCAAGTC-3'".
Also, a complementary sequence to a target sequence for an antisense strand of a double-stranded molecule can be defined according to the kind of nucleic acid that constitutes the antisense strand. For example, when a target sequence is shown in the RNA sequence of SEQ ID NO: 9 and the antisense strand of the double-stranded molecule has the 5' side half region composed of DNA, "a complementary sequence to a target sequence" is 3'- CGUUUGUGGACGAGTTCAG -5'".
On the other hand, when a double-stranded molecule is composed of RNA, the sequence corresponding to a target sequence of SEQ ID NO: 9 is the RNA sequence of SEQ ID NO: 9, and the complementary sequence corresponding to a target sequence of SEQ ID NO: 9 is the RNA sequence of "3'- CGUUUGUGGACGAGUUCAG -5'".
A double-stranded molecule may has one or two 3'overhangs having 2 to 5 nucleotides in length (e.g., uu) and/or a loop sequence that links a sense strand and an antisense strand to form hairpin structure, in addition to a sequence corresponding to a target sequence and complementary sequence thereto.
A double-stranded molecule against CDC45L gene, which molecule hybridizes to a CDC45L mRNA, inhibits or reduces production of CDC45L protein encoded by the gene by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and thus, inhibiting expression of the CDC45L protein. Also, A double-stranded molecule against PIF1 gene, which molecule hybridizes to a PIF1 mRNA, inhibits or reduces production of PIF1 protein encoded by the genes by associating with the normally single-stranded mRNA transcript of the gene, thereby interfering with translation and thus, inhibiting expression of the PIF1 protein.
The expression of CDC45L gene and PIF1 gene in cancer cell lines, was inhibited by each two double-stranded molecules (Fig. 3 and Fig.5).
Therefore, the present invention provides isolated double-stranded molecules having the property to inhibit or reduce the expression of CDC45L gene or PIF1 gene in cancer cells when introduced into a cell. The target sequences of double-stranded molecules may be designed by siRNA design algorithm mentioned below.
In preferred embodiments, target sequences for CDC45L include, for example,
5'- GCAAACACCUGCUCAAGUC -3' (SEQ ID NO: 9) or
5'- GGACGUGGAUGCUCUGUGU -3' (SEQ ID NO: 10).
Also, in preferred embodiments, target sequences for PIF1 include, for example,
5'- GAAAGGCCAGAGCAUCUUC-3' (SEQ ID NO: 11) or
5'- GGCAUGACCCUGGAUUGUG-3' (SEQ ID NO: 12).
In other words, the present invention also provides a double-stranded molecule whose target sequence comprises or consisting of SEQ ID NO: 9, 10, 11 or 12.
Specifically, the present invention provides the following double-stranded molecules [1] to [18]:
[1] An isolated double-stranded molecule, which, when introduced into a cell, inhibits in vivo expression of a CDC45L gene or PIF1 gene and cell proliferation, wherein the double-stranded molecule acts at mRNA which matches a target sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:12;
[2] An isolated double-stranded molecule, which, when introduced into a cell, inhibits in vivo expression of a CDC45L gene or PIF1 gene and cell proliferation, wherein the double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form a double strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO:12;.
[3] The double-stranded molecule of [1] ot [2], wherein the target sequence comprises from about 19 to about 25 contiguous nucleotide from the nucleotide sequence selected from SEQ ID NO: 13 or 15.
[4] The double-stranded molecule of any one of [1] to [2], which has a length of less than about 100 nucleotides.
[5] The double-stranded molecule of [4], which has a length of less than about 75 nucleotides.
[6] The double-stranded molecule of [5], which has a length of less than about 50 nucleotides.
[7] The double-stranded molecule of [6] which has a length of less than about 25 nucleotides.
[8] The double-stranded molecule of [7], which has a length of between about 19 and about 25 nucleotides.
[9] The double-stranded molecule of any one of [1] to [8], which consists of a single oligonucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
[10] The double-stranded molecule of [9], which has a general formula 5'-[A]-[B]-[A']-3', wherein
[A] is a sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12
[B] is an intervening single-strand; and
[A'] is an antisense strand comprising a nucleotide sequence corresponding to a sequence complementary to the target sequence selected in [A].
[11] The double-stranded molecule of any one of [1] to [10], which comprises RNA.
[12] The double-stranded molecule of any one of [1] to [11], which comprises both DNA and RNA.
[13] The double-stranded molecule of [12], which is a hybrid of a DNA polynucleotide and an RNA polynucleotide.
[14] The double-stranded molecule of [13] wherein the sense and the antisense strands are made of DNA and RNA, respectively.
[15] The double-stranded molecule of [12], which is a chimera of DNA and RNA.
[16] The double-stranded molecule of [15], wherein a 5'-end region of the target sequence in the sense strand, and/or a 3'-end region of the complementary sequence of the target sequence in the antisense strand consists of RNA.
[17] The double-stranded molecule of [16], wherein the RNA region consists of 9 to 13 nucleotides; and
[18] The double-stranded molecule of any one of [1] to [2], which contains one or two 3' overhang(s).
The double-stranded molecule of the present invention will be described in more detail below.
Methods for designing double-stranded molecules having the ability to inhibit target gene expression in cells are known. (See, for example, US Pat No. 6,506,559, herein incorporated by reference in its entirety). For example, a computer program for designing siRNAs is available from the Ambion website (on the worldwide web at ambion.com/techlib/misc/siRNA_finder.html).
The computer program selects target nucleotide sequences for double-stranded molecules based on the following protocol.
Design of Target Sites
1. Beginning with the AUG start codon of the transcript, scan downstream for AA di-nucleotide sequences. Record the occurrence of each AA and the 3' adjacent 19 nucleotides as potential siRNA target sites. Tuschl et al. recommend to avoid designing siRNA to the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases) as these can be richer in regulatory protein binding sites, and UTR-binding proteins and/or translation initiation complexes can interfere with binding of the siRNA endonuclease complex.
2. Compare the potential target sites to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. Basically, BLAST, which can be found on the NCBI server at: on the worldwide web at ncbi.nlm.nih.gov/BLAST/, is used (Altschul SF, et al., Nucleic Acids Res. 1997 Sep 1;25(17):3389-402).
3. Select qualifying target sequences for synthesis. Selecting several target sequences along the length of the gene to evaluate is typical.
Preferred target sequences for CDC45L gene designed in "Examples" include
5'- GCAAACACCUGCUCAAGUC -3' (SEQ ID NO: 9) or
5'- GGACGUGGAUGCUCUGUGU -3' (SEQ ID NO: 10).
Also, preferred target sequences for PIF1 gene designed in "Examples" include,
5'- GAAAGGCCAGAGCAUCUUC-3' (SEQ ID NO: 11) or
5'- GGCAUGACCCUGGAUUGUG-3' (SEQ ID NO: 12).
Specifically, the present invention provides the double-stranded molecules targeting any one of above-mentioned target sequences that were respectively examined for their ability to inhibit and reduce the growth of cancer cells expressing the target genes. The growth of cancer cells expressing CDC45L gene or PIF1 gene, were inhibited and reduced by double-stranded molecules of the present invention (Fig. 3 and Fig.5).
Therefore, the present invention provides double-stranded molecules targeting a target sequence for CDC45L gene selected from the group consisting of
5'- GCAAACACCUGCUCAAGUC -3' (SEQ ID NO: 9) and
5'- GGACGUGGAUGCUCUGUGU -3' (SEQ ID NO: 10).
Also, the present invention provides double-stranded molecules targeting a target sequence for PIF1 gene selected from the group consisting of
5'- GAAAGGCCAGAGCAUCUUC-3' (SEQ ID NO: 11) and
5'- GGCAUGACCCUGGAUUGUG-3' (SEQ ID NO: 12).
The double-stranded molecules of the present invention targeting the above-mentioned target sequence of CDC45L or PIF1 gene include isolated polynucleotide(s) that comprises any one of the nucleic acid sequences of target sequences and/or complementary sequences to the target sequences. Examples of a double-stranded molecule targeting CDC45L or PIF1 gene include an oligonucleotide comprising the sequence corresponding to SEQ ID NO: 9, 10, 11 or 12, and complementary sequences thereto. However, the present invention is not limited to these examples, and minor modifications in the aforementioned nucleic acid sequences are acceptable so long as the modified molecule retains the ability to suppress the expression of CDC45L or PIF1 gene. Herein, "minor modification" in a nucleic acid sequence indicates one, two or several substitution, deletion, addition or insertion of nucleic acids to the sequence.
In an embodiment, a double-stranded molecule is composed of two polynucleotides, one polynucleotide has a sequence corresponding to a target sequence, i.e., sense strand, and another polypeptide has a complementary sequence to the target sequence, i.e., antisense strand. The sense strand polynucleotide and the antisense strand polynucleotide hybridize to each other to form double-stranded molecule. Examples of such double-stranded molecules include dsRNA and dsD/R-NA.
In an another embodiment, a double-stranded molecule is composed of a polynucleotide that has both a sequence corresponding to a target sequence, i.e., sense strand, and a complementary sequence to the target sequence, i.e., antisense strand. Generally, the sense strand and the antisense strand are linked by an intervening strand, and hybridize to each other to form a hairpin loop structure. Examples of such double-stranded molecule include shRNA and shD/R-NA.
In other words, a double-stranded molecule of the present invention comprises a sense strand polynucleotide having a nucleotide sequence of the target sequence and anti-sense strand polynucleotide having a nucleotide sequence complementary to the target sequence, and both of polynucleotides hybridize to each other to form the double-stranded molecule. In the double-stranded molecule comprising the polynucleotides, a part of the polynucleotide of either or both of the strands may be RNA, and when the target sequence is defined with a DNA sequence, the nucleotide "t" within the target sequence and complementary sequence thereto is replaced with "u".
In one embodiment of the present invention, such a double-stranded molecule of the present invention comprises a stem-loop structure, composed of the sense and antisense strands. The sense and antisense strands may be joined by a loop. Accordingly, the present invention also provides the double-stranded molecule comprising a single polynucleotide containing both the sense strand and the antisense strand linked or flanked by an intervening single-strand.
In the present invention, double-stranded molecules targeting the CDC45L gene or PIF1 gene may have a sequence selected from among SEQ ID NOs: 9, 10, 11 and 12 as a target sequence. Accordingly, preferable examples of the double-stranded molecule of the present invention include polynucleotides that hybridize to each other at a sequence corresponding to SEQ ID NO: 9, 10, 11 or 12 and a complementary sequence thereto, and a polynucleotide that has a sequence corresponding to SEQ ID NO: 9, 10, 11 or 12 and a complementary sequence thereto.
According to the present invention, a double-stranded molecule of the present invention can be tested for its ability using the methods utilized in the Examples (see, RNA interference assay in "EXAMPLES"). In the Examples, the double-stranded molecules comprising sense strands and antisense strands complementary thereto of various portions of mRNA of CDC45L or PIF1 genes were tested in vitro for their ability to decrease production of CDC45L or PIF1 gene product in cancers cell lines (e.g., using SBC-3 and A549) according to standard methods. Furthermore, for example, reduction in CDC45L or PIF1 gene product in cells contacted with the candidate double-stranded molecule compared to cells cultured in the absence of the candidate molecule can be detected by, e.g. RT-PCR using primers for CDC45L or PIF1 gene mRNA mentioned (see,(b)) Semi-quantitative RT-PCR in "EXAMPLES"). Sequences which decrease the production of CDC45L or PIF1 gene product in vitro cell-based assays can then be tested for there inhibitory effects on cell growth. Sequences which inhibit cell growth in vitro cell-based assay can then be tested for their in vivo ability using animals with cancer, e.g. nude mouse xenograft models, to confirm decreased production of CDC45L or PIF1 gene product and decreased cancer cell growth.
When the isolated polynucleotide is RNA or derivatives thereof, base "t" should be replaced with "u" in the nucleotide sequences. As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a polynucleotide, and the term "binding" means the physical or chemical interaction between two polynucleotides. When the polynucleotide comprises modified nucleotides and/or non-phosphodiester linkages, these polynucleotides can also bind each other as same manner. Generally, complementary polynucleotide sequences hybridize under appropriate conditions to form stable duplexes containing few or no mismatches. Furthermore, the sense strand and antisense strand of the isolated polynucleotide of the present invention can form double-stranded molecule or hairpin loop structure by the hybridization. In one embodiment, such duplexes contain no more than 1 mismatch for every 10 matches. In some embodiments, where the strands of the duplex are fully complementary, such duplexes contain no mismatches.
The polynucleotide of the invention are typically less than 500, 200, 100, 75, 50, or 25 nucleotides in length. The isolated polynucleotides of the present invention are useful for forming double-stranded molecules against CDC45L or PIF1 gene or preparing template DNAs encoding the double-stranded molecules. When the polynucleotides are used for forming double-stranded molecules, the sense strand of the polynucleotide can be longer than 19 nucleotides, for example, longer than 21 nucleotides, for example, between about 19 and 25 nucleotides.
Accordingly, the present invention provides the double-stranded molecules comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence. In preferable embodiments, the sense strand hybridizes with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
The double-stranded molecule serves as a guide for identifying homologous sequences in mRNA for the RISC complex, when the double-stranded molecule is introduced into cells. The identified target RNA is cleaved and degraded by the nuclease activity of Dicer, through which the double-stranded molecule eventually decreases or inhibits production (expression) of the polypeptide encoded by the RNA. Thus, a double-stranded molecule of the invention can be defined by its ability to generate a single-strand that specifically hybridizes to the mRNA of the CDC45L or PIF1 gene under stringent conditions. Herein, the portion of the mRNA that hybridizes with the single-strand generated from the double-stranded molecule is referred to as "target sequence" or "target nucleic acid" or "target nucleotide". In the present invention, nucleotide sequence of the "target sequence" can be shown using not only the RNA sequence of the mRNA, but also the DNA sequence of cDNA synthesized from the mRNA.
The double-stranded molecules of the invention can contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the double-stranded molecule. The skilled person will be aware of other types of chemical modification which can be incorporated into the present molecules (WO03/070744; WO2005/045037). In one embodiment, modifications can be used to provide improved resistance to degradation or improved uptake. Examples of such modifications include phosphorothioate linkages, 2'-O-methyl ribonucleotides (especially on the sense strand of a double-stranded molecule), 2'-deoxy-fluoro ribonucleotides, 2'-deoxy ribonucleotides, "universal base" nucleotides, 5'-C- methyl nucleotides, and inverted deoxyabasic residue incorporation (US Pat Appl. No. 20060122137).
In another embodiment, modifications can be used to enhance the stability or to increase targeting efficiency of the double-stranded molecule. Modifications include chemical cross linking between the two complementary strands of a double-stranded molecule, chemical modification of a 3' or 5' terminus of a strand of a double-stranded molecule, sugar modifications, nucleobase modifications and/or backbone modifications, 2 -fluoro modified ribonucleotides and 2'-deoxy ribonucleotides (WO2004/029212).
In another embodiment, modifications can be used to increased or decreased affinity for the complementary nucleotides in the target mRNA and/or in the complementary double-stranded molecule strand (WO2005/044976). For example, an unmodified pyrimidine nucleotide can be substituted for a 2-thio, 5-alkynyl, 5-methyl, or 5-propynyl pyrimidine. Additionally, an unmodified purine can be substituted with a 7-deaza, 7-alkyl, or 7-alkenyl purine. In another embodiment, when the double-stranded molecule is a double-stranded molecule with a 3' overhang, the 3'- terminal nucleotide overhanging nucleotides can be replaced by deoxyribonucleotides (Elbashir SM et al., Genes Dev 2001 Jan 15, 15(2): 188-200). For further details, published documents for example, US Pat Appl. No.20060234970 are available. The present invention is not limited to these examples and any known chemical modifications can be employed for the double-stranded molecules of the present invention so long as the resulting molecule retains the ability to inhibit the expression of the target gene.
Furthermore, the double-stranded molecules of the invention can comprise both DNA and RNA, e.g., dsD/R-NA or shD/R-NA. Specifically, a hybrid polynucleotide of a DNA strand and an RNA strand or a DNA-RNA chimera polynucleotide shows increased stability. Mixing of DNA and RNA, i.e., a hybrid type double-stranded molecule made of a DNA strand (polynucleotide) and an RNA strand (polynucleotide), a chimera type double-stranded molecule comprising both DNA and RNA on any or both of the single strands (polynucleotides), or the like can be formed for enhancing stability of the double-stranded molecule. The hybrid of a DNA strand and an RNA strand can be either where the sense strand is DNA and the antisense strand is RNA, or the opposite so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene.
In some embodiments, the sense strand polynucleotide is DNA and the antisense strand polynucleotide is RNA. Also, the chimera type double-stranded molecule may have the structure either where both of the sense and antisense strands are composed of DNA and RNA, or where any one of the sense and antisense strands is composed of DNA and RNA so long as it has an activity to inhibit expression of the target gene when introduced into a cell expressing the gene. In order to enhance stability of the double-stranded molecule, in some embodiments, the molecule contains as much DNA as possible, whereas to induce inhibition of the target gene expression, the molecule is required to be RNA within a range to induce sufficient inhibition of the expression. In one example of the chimera type double-stranded molecule, an upstream partial region (i.e., a region flanking to the target sequence or complementary sequence thereof within the sense or antisense strands) of the double-stranded molecule is RNA.
The upstream partial region means the 5' side (5'-end) of the sense strand and the 3' side (3'-end) of the antisense strand. That is, in some embodiments, a region flanking to the 3'-end of the antisense strand, or both of a region flanking to the 5'-end of sense strand and a region flanking to the 3'-end of antisense strand consists of RNA. For instance, the chimera or hybrid type double-stranded molecule of the present invention comprise following combinations.
sense strand: 5'-[DNA]-3'
3'-(RNA)-[DNA]-5': antisense strand,
sense strand: 5'-(RNA)-[DNA]-3'
3'-(RNA)-[DNA]-5': antisense strand, and
sense strand: 5'-(RNA)-[DNA]-3'
3'-(RNA)-5': antisense strand.
The upstream partial region can be a domain of about 9 to 13 nucleotides counted from the terminus of the target sequence or complementary sequence thereto within the sense or antisense strands of the double-stranded molecules. Moreover, examples of such chimera type double-stranded molecules include those having a strand length of 19 to 21 nucleotides in which at least the upstream half region (5' side region for the sense strand and 3' side region for the antisense strand) of the polynucleotide is RNA and the other half is DNA. In such a chimera type double-stranded molecule, the effect to inhibit expression of the target gene is much higher when the entire antisense strand is RNA (US Pat Appl. No. 20050004064).
In the present invention, the double-stranded molecule can form a hairpin, for example, a short hairpin RNA (shRNA) and short hairpin made of DNA and RNA (shD/R-NA). The shRNA or shD/R-NA is a sequence of RNA or mixture of RNA and DNA making a tight hairpin turn that can be used to silence gene expression via RNA interference. The shRNA or shD/R-NA comprises the sense target sequence and the antisense target sequence on a single strand wherein the sequences are separated by a loop sequence. Generally, the hairpin structure is cleaved by the cellular machinery into dsRNA or dsD/R-NA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the target sequence of the dsRNA or dsD/R-NA.
A loop sequence composed of an arbitrary nucleotide sequence can be located between the sense and antisense sequence in order to form the hairpin loop structure. Thus, the present invention also provides a double-stranded molecule having the general formula 5'-[A]-[B]-[A']-3', wherein [A] is the sense strand containing a sequence corresponding to a target sequence, [B] is an intervening single-strand and [A'] is the antisense strand containing a complementary sequence to [A]. The target sequence can be selected from the group consisting of, for example, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.
The present invention is not limited to these examples, and the target sequence in [A] can be modified sequences from these examples so long as the double-stranded molecule retains the ability to suppress the expression of the targeted CDC45L or PIF1 gene and result in inhibits or reduces the cell expressing these genes. The region [A] hybridizes to [A'] to form a loop comprising the region [B]. The intervening single-stranded portion [B], i.e., the loop sequence can be 3 to 23 nucleotides in length. The loop sequence, for example, can be selected from group consisting of following sequences (on the worldwide web at ambion.com/techlib/tb/tb_506.html). Furthermore, loop sequence consisting of 23 nucleotides also provides active siRNA (Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26):
CCC, CCACC, or CCACACC: Jacque JM et al., Nature 2002 Jul 25, 418(6896): 435-8, Epub 2002 Jun 26;
UUCG: Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5; Fruscoloni P et al., Proc Natl Acad Sci USA 2003 Feb 18, 100(4): 1639-44, Epub 2003 Feb 10; and
UUCAAGAGA: Dykxhoorn DM et al., Nat Rev Mol Cell Biol 2003 Jun, 4(6): 457-67.
Exemplary double-stranded molecules having hairpin loop structure of the present invention are shown below. In the following structure, the loop sequence can be selected from group consisting of AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA; however, the present invention is not limited thereto:
GCAAACACCUGCUCAAGUC-[B]- GACUUGAGCAGGUGUUUGC (for target sequence of SEQ ID NO: 9);
GGACGUGGAUGCUCUGUGU-[B]-ACACAGAGCAUCCACGUCC (for target sequence of SEQ ID NO: 10);
GAAAGGCCAGAGCAUCUUC-[B]-GAAGAUGCUCUGGCCUUUC (for target sequence of SEQ ID NO: 11);and
GGCAUGACCCUGGAUUGUG-[B]- CACAAUCCAGGGUCAUGCC (for target sequence of SEQ ID NO: 12).
Furthermore, in order to enhance the inhibition activity of the double-stranded molecules, several nucleotides can be added to 3'end of the sense strand and/or the antisense strand, as 3' overhangs. The number of nycleotides to be added is at least 2, generally 2 to 10, for example, 2 to 5. The added nucleotides form single strand at the 3'end of sense strand and/or the antisense strand of the double-stranded molecule. The nucleotides for 3' overhang are preferably "u" or "t", but are not limited to. When the double-stranded molecule has a harpin loop structure, a 3' overhang is added to the 3' end of the antisense strand.
The method of preparing the double-stranded molecule can use any chemical synthetic method known in the art. According to the chemical synthesis method, sense and antisense single-stranded polynucleotides are separately synthesized and then annealed together via an appropriate method to obtain a double-stranded molecule. Alternatively, a double stranded molecule or siRNA molecule of the present invention may also be synthesized with in vitro translation. In this embodiment, DNA encoding a nucleotide sequence that comprises the target sequence and antisense thereof is transcribed into the double stranded molecule in vitro. In one embodiment for the annealing, the synthesized single-stranded polynucleotides are mixed in a molar ratio of at least about 3:7, for example, about 4:6, for example, substantially equimolar amount (i.e., a molar ratio of about 5:5). Next, the mixture is heated to a temperature at which double-stranded molecules dissociate and then is gradually cooled down. The annealed double-stranded polynucleotide can be purified by usually employed methods known in the art. Example of purification methods include methods utilizing agarose gel electrophoresis or wherein remaining single-stranded polynucleotides are optionally removed by, e.g., degradation with appropriate enzyme.
The regulatory sequences flanking target sequences can be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. The double-stranded molecules can be transcribed intracellularly by cloning CDC45L or PIF1 gene templates into a vector containing, e.g., a RNA pol III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.
Alternatively, the double-stranded molecules may be transcribed intracellularly by cloning its coding sequence into a vector containing a regulatory sequence that directs the expression of the double-stranded molecule in an adequate cell (e.g., a RNA poly III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter) adjacent to the coding sequence. The regulatory sequences flanking the coding sequences of double-stranded molecule may be identical or different, such that their expression can be modulated independently, or in a temporal or spatial manner. Details of vectors which are capable of producing the double-stranded molecules will be described below.
(ii) Vector
Also included in the invention is a vector containing one or more of the double-stranded molecules described herein, and a cell containing the vector. A vector of the present invention encodes a double-stranded molecule of the present invention in an expressible form. Herein, the phrase "in an expressible form" indicates that the vector, when introduced into a cell, will express the molecule. In one embodiment, the vector includes regulatory elements necessary for expression of the double-stranded molecule. Accordingly, in one embodiment, the expression vector encodes the double-stranded molecule of the present invention and is adapted for expression of the double-stranded molecule. Such vectors of the present invention can be used for producing the present double-stranded molecules, or directly as an active ingredient for treating cancer.
Alternatively, the present invention provides vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to SEQ ID NOs: 9, 10, 11 or 12, and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits expression of said gene. Preferably, the polynucleotide is an oligonucleotide of between about 19 and 25 nucleotides in length (e.g., contiguous nucleotides from the nucleotide sequence of SEQ ID NO: 13, or 15). More preferably, the combination of polynucleotide comprises a single nucleotide transcript comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence. More preferably, the combination of polynucleotide has the general formula 5'-[A]-[B]-[A']-3', wherein [A] is a nucleotide sequence comprising SEQ ID NO: 9, 10, 11 or 12; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotide; and [A'] is a nucleotide sequence complementary to [A].
Vectors of the present invention can be produced, for example, by cloning a sequence comprising target sequence into an expression vector so that regulatory sequences are operatively-linked to the sequence in a manner to allow expression (by transcription of the DNA molecule) of both strands (Lee NS et al., Nat Biotechnol 2002 May, 20(5): 500-5). For example, RNA molecule that is the antisense to mRNA is transcribed by a first promoter (e.g., a promoter sequence flanking to the 3' end of the cloned DNA) and RNA molecule that is the sense strand to the mRNA is transcribed by a second promoter (e.g., a promoter sequence flanking to the 5' end of the cloned DNA). The sense and antisense strands hybridize in vivo to generate a double-stranded molecule constructs for silencing of the gene. Alternatively, two vectors constructs respectively encoding the sense and antisense strands of the double-stranded molecule are utilized to respectively express the sense and antisense strands and then forming a double-stranded molecule construct. Furthermore, the cloned sequence can encode a construct having a secondary structure (e.g., hairpin); namely, a single transcript of a vector contains both the sense and complementary antisense sequences of the target gene.
The vectors of the present invention can also be equipped so to achieve stable insertion into the genome of the target cell (see, e.g., Thomas KR & Capecchi MR, Cell 1987, 51: 503-12 for a description of homologous recombination cassette vectors). See, e.g., Wolff et al., Science 1990, 247: 1465-8; US Pat Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., US Pat No. 5,922,687).
The vectors of the present invention can be, for example, viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts, for example, vaccinia or fowlpox (see, e.g., US Pat No. 4,722,848). This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode the double-stranded molecule. Upon introduction into a cell expressing the target gene, the recombinant vaccinia virus expresses the molecule and thereby suppresses the proliferation of the cell. Another example of useable vector includes Bacille Calmette Guerin (BCG). BCG vectors are described in Stover et al., Nature 1991, 351: 456-60. A wide variety of other vectors are useful for therapeutic administration and production of the double-stranded molecules; examples include adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata et al., Mol Med Today 2000, 6: 66-71; Shedlock et al., J Leukoc Biol 2000, 68: 793-806; and Hipp et al., In Vivo 2000, 14: 571-85.
(iii) Methods Of Inhibiting Or Reducing A Growth Of Cancer Cells And Treating Or Preventing Cancer Using Double-Stranded Molecules
In the present invention, double-stranded molecules targeting the above-mentioned target sequences were respectively examined for their ability to inhibit or reduce the growth of cells (over)expressing the target genes. The growth of cancer cells (over)expressing CDC45L and/or PIF1 gene, was inhibited or reduced by double-stranded molecules of the present invention (Fig. 3 and Fig.5).
Therefore, the present invention provides methods for inhibiting cell growth, i.e., cancerous cell growth of a cell from a cancer resulting from overexpression of a CDC45L and/or PIF1 gene, or that is mediated by a CDC45L and/or PIF1 gene, by inhibiting the expression of the CDC45L and/or PIF1 gene. CDC45L or PIF1 gene expression can be inhibited by any of the aforementioned double-stranded molecules of the present invention which specifically target the expression of CDC45L or PIF1 gene or the vectors of the present invention that can express any of the double-stranded molecules of the present invention.
Such ability of the present double-stranded molecules and vectors to inhibit cell growth of cancerous cells indicates that they can be used for methods for treating cancer, a cancer resulting from overexpression of a CDC45L and/or PIF1 gene, or that is mediated by a CDC45L and/or PIF1 gene. Thus, the present invention provides methods to treat patients with a cancer resulting from overexpression of a CDC45L and/or PIF1 gene, or that is mediated by a CDC45L and/or PIF1 gene by administering a double-stranded molecule, i.e., an inhibitory nucleic acid, against a CDC45L or PIF1 gene or a vector expressing the molecule without adverse effect because those genes were hardly detected in normal organs.
Specifically, the present invention provides the following methods [1] to [23]:
[1] A method for inhibiting or reducing a growth of a cell (over)expressing a CDC45L and/or PIF1 gene or a method for treating or preventing cancer (over)expressing CDC45L and/or PIF1 gene, wherein said method comprising the step of administering to a subject at least one double-stranded molecule or vector encoding the double-stranded molecule, wherein said double-stranded molecule, when introduced into a cell, inhibits or reduces in vivo expression of said CDC45L or PIF1 gene.
[2] The method of [1], wherein said double-stranded molecule acts at mRNA which shares sequence identity with or is complementary to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12.
[3] The method of [1], wherein said double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form a double strand, wherein said sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12.
[4] The method of any one of [1] to [3], wherein a plurality of double-stranded molecules are administered;
[5] The method of [4], wherein the plurality of double-stranded molecules target the same gene;
[6] The method of any one of [1] to [5], wherein the double-stranded molecule has a length of less than about 100 nucleotides;
[7] The method of [6], wherein the double-stranded molecule has a length of less than about 75 nucleotides;
[8] The method of [7], wherein the double-stranded molecule has a length of less than about 50 nucleotides;
[9] The method of [8], wherein the double-stranded molecule has a length of less than about 25 nucleotides;
[10] The method of any one of [1] to [9], wherein the sense strand hybridize with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length;
[11] The method of any one of [1] to [10], wherein said double-stranded molecule consists of a single oligonucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
[12] The method of [11], wherein said double-stranded molecule has a general formula 5'-[A]-[B]-[A']-3', wherein
[A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12;
[B] is the intervening single-strand; and
[A'] is the antisense strand comprising an oligonucleotide corresponding to a sequence complementary to the target sequence selected in [A].
[13] The method of any one of [1] to [12], wherein the double-stranded molecule comprises RNA.
[14] The method of any one of [1] to [12], wherein the double-stranded molecule comprises both DNA and RNA.
[15] The method of [14], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide.
[16] The method of [15] wherein the sense and antisense strand polynucleotides a made of DNA and RNA, respectively.
[17] The method of [14], wherein the double-stranded molecule is a chimera of DNA and RNA.
[18] The method of [17], wherein a region flanking to the 5'-end of one or both of the sense and antisense polynucleotides a made of RNA.
[19] The method of [18], wherein the flanking region consists of 9 to 13 nucleotides.
[20] The method of any one of [1] to [19], wherein the double-stranded molecule contains one or two 3' overhang(s).
[21] The method of any one of [1] to [20], wherein the double-stranded molecule is encoded by a vector.
[22] The method of [21], wherein said double-stranded molecule has a general formula 5'-[A]-[B]-[A']-3', wherein
[A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12;
[B] is the intervening single-strand; and
[A'] is the antisense strand comprising an oligonucleotide corresponding to a sequence complementary to the target sequence selected in [A].
[23] The method of any one of [1] to [22], wherein the double-stranded molecule is contained in a composition which comprises in addition to the molecule a transfection-enhancing agent and/or cell permeable agent.
The method of the present invention will be described in more detail below.
The growth of cells (over)expressing a CDC45L and/or PIF1 gene is inhibited by contacting the cells with a double-stranded molecule against CDC45L or PIF1 gene, a vector expressing the molecule or a composition comprising the same. The cell may be further contacted with a transfection agent. Suitable transfection agents are known in the art. The phrase "inhibition of cell growth" indicates that the cell proliferates at a lower rate or has decreased viability compared to a cell not exposed to the molecule. Cell growth can be measured by methods known in the art, e.g., using the MTT cell proliferation assay.
The growth of any kind of cell can be suppressed according to the present method so long as the cell expresses or over-expresses the target gene of the double-stranded molecule of the present invention. Exemplary cells include cancers cells.
Thus, patients suffering from or at risk of developing disease related to CDC45L and/or PIF1 gene can be treated by administering at least one of the present double-stranded molecules, at least one vector expressing at least one of the molecules or at least one composition comprising at least one of the molecules. For example, patients of cancers can be treated according to the present methods. The type of cancer can be identified by standard methods according to the particular type of tumor to be diagnosed. In some embodiments, patients treated by the methods of the present invention are selected by detecting the (over)expression of a CDC45L and/or PIF1 gene in a biopsy from the patient by RT-PCR, hybridization or immunoassay. In some embodiments, before the treatment of the present invention, the biopsy specimen from the subject is confirmed for CDC45L and/or PIF1 gene over-expression by methods known in the art, for example, immunohistochemical analysis, hybridization or RT-PCR (see, Semi-quantitative RT-PCR, Western-blotting or Immunohistochemistry in "EXAMPLES").
According to the present method to inhibit or reduce cell growth and thereby treating cancer, when administering plural kinds of the double-stranded molecules (or vectors expressing or compositions containing the same), each of the molecules can direct to the different target sequence of same gene, or different target sequences of different gene. For example, the method can utilize different double-stranded molecules directing to CDC45L or PIF1 gene transcript. Alternatively, for example, the method can utilize double-stranded molecules directed to one, two or more target sequences selected from same gene.
For inhibiting cell growth, a double-stranded molecule of present invention can be directly introduced into the cells in a form to achieve binding of the molecule with corresponding mRNA transcripts. Alternatively, as described above, a DNA encoding the double-stranded molecule can be introduced into cells as a vector. For introducing the double-stranded molecules and vectors into the cells, transfection-enhancing agent, for example, FuGENE (Roche diagnostics), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical), can be employed.
A treatment is determined efficacious if it leads to clinical benefit for example, reduction in expression of a CDC45L or PIF1 gene, or a decrease in size, prevalence, or metastatic potential of the cancer in the subject. When the treatment is applied prophylactically, "efficacious" means that it retards or prevents cancers from forming or prevents or alleviates a clinical symptom of cancer. Efficaciousness is determined in association with any known method for diagnosing or treating the particular tumor type.
To the extent that the methods and compositions of the present invention find utility in the context of "prevention" and "prophylaxis", such terms are interchangeably used herein to refer to any activity that reduces the burden of mortality or morbidity from disease. Prevention and prophylaxis can occur "at primary, secondary and tertiary prevention levels." While primary prevention and prophylaxis avoid the development of a disease, secondary and tertiary levels of prevention and prophylaxis encompass activities aimed at the prevention and prophylaxis of the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications. Alternatively, prevention and prophylaxis can include a wide range of prophylactic therapies aimed at alleviating the severity of the particular disorder, e.g. reducing the proliferation and metastasis of tumors.
The treatment and/or prophylaxis of cancer and/or the prevention of postoperative recurrence thereof include any of the following steps, such as the surgical removal of cancer cells, the inhibition of the growth of cancerous cells, the involution or regression of a tumor, the induction of remission and suppression of occurrence of cancer, the tumor regression, and the reduction or inhibition of metastasis. Effectively treating and/or the prophylaxis of cancer decreases mortality and improves the prognosis of individuals having cancer, decreases the levels of tumor markers in the blood, and alleviates detectable symptoms accompanying cancer. For example, reduction or improvement of symptoms constitutes effectively treating and/or the prophylaxis include 10%, 20%, 30% or more reduction, or stable disease.
It is understood that the double-stranded molecule of the invention degrades the target mRNA (gene transcripts) in substoichiometric amounts. Without wishing to be bound by any theory, it is believed that the double-stranded molecule of the invention causes degradation of the target mRNA in a catalytic manner. Thus, compared to standard cancer therapies, significantly less a double-stranded molecule needs to be delivered at or near the site of cancer to exert therapeutic effect.
One skilled in the art can readily determine an effective amount of the double-stranded molecule of the invention to be administered to a given subject, by taking into account factors for example, body weight, age, sex, type of disease, symptoms and other conditions of the subject; the route of administration; and whether the administration is regional or systemic. Generally, an effective amount of the double-stranded molecule of the invention comprises an intercellular concentration at or near the cancer site of from about 1 nanomolar (nM) to about 100 nM, for example, from about 2 nM to about 50 nM, for example, from about 2.5 nM to about 10 nM. It is contemplated that greater or smaller amounts of the double-stranded molecule can be administered.
The present methods can be used to inhibit the growth or metastasis of cancer; for example, a cancer resulting from overexpression of a CDC45L and/or PIF1 gene or that is mediated by a CDC45L and/or PIF1 gene, e.g., lung cancer. In particular, a double-stranded molecule directed to a target sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10 for CDC45L, and SEQ ID NO: 11 and SEQ ID NO: 12 for PIF1 find use for the treatment of cancers.
For treating cancer, e.g., a cancer promoted by a CDC45L and/or PIF1 gene, the double-stranded molecule of the invention can also be administered to a subject in combination with a pharmaceutical agent different from the double-stranded molecule. Alternatively, the double-stranded molecule of the invention can be administered to a subject in combination with another therapeutic method designed to treat cancer. For example, the double-stranded molecule of the invention can be administered in combination with therapeutic methods currently employed for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery and treatment using chemotherapeutic agents, for example, cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen).
In the present methods, the double-stranded molecule can be administered to the subject either as a naked double-stranded molecule, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the double-stranded molecule.
Suitable delivery reagents for administration in conjunction with the present a double-stranded molecule include the Mirus Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes. In one embodiment, the delivery reagent is a liposome.
Liposomes can aid in the delivery of the double-stranded molecule to a particular tissue, for example, retinal or tumor tissue, and can also increase the blood half-life of the double-stranded molecule. Liposomes suitable for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol, for example, cholesterol. The selection of lipids is generally guided by consideration of factors for example, the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes, for example as described in Szoka et al., Ann Rev Biophys Bioeng 1980, 9: 467; and US Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 5,019,369, the entire disclosures of which are herein incorporated by reference.
In some embodiments, the liposomes encapsulating the present double-stranded molecule comprises a ligand molecule that can deliver the liposome to the cancer site. Ligands which bind to receptors prevalent in tumor or vascular endothelial cells, for example, monoclonal antibodies that bind to tumor antigens or endothelial cell surface antigens, find use.
In some embodiments, the liposomes encapsulating the present double-stranded molecule are modified so as to avoid clearance by the mononuclear macrophage and reticuloendothelial systems, for example, by having opsonization-inhibition moieties bound to the surface of the structure. In one embodiment, a liposome of the invention can comprise both opsonization-inhibition moieties and a ligand.
Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the macrophage-monocyte system ("MMS") and reticuloendothelial system ("RES"); e.g., as described in US Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference. Liposomes modified with opsonization-inhibition moieties thus remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes.
Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" microvasculature. Thus, target tissue characterized by such microvasculature defects, for example, solid tumors, will efficiently accumulate these liposomes; see Gabizon et al., Proc Natl Acad Sci USA 1988, 18: 6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation in liver and spleen. Thus, liposomes of the invention that are modified with opsonization-inhibition moieties can deliver the present double-stranded molecule to tumor cells.
Opsonization inhibiting moieties suitable for modifying liposomes can be water-soluble polymers with a molecular weight from about 500 to about 40,000 daltons, for example, from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers for example, polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, for example, ganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
In some embodiments, the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes".
The opsonization inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl-ethanolamine lipid-soluble anchor, and then bound to a membrane. Similarly, a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive amination using Na(CN)BH3 and a solvent mixture for example, tetrahydrofuran and water in a 30:12 ratio at 60 degrees C.
Vectors expressing a double-stranded molecule of the invention are discussed above. Such vectors expressing at least one double-stranded molecule of the invention can also be administered directly or in conjunction with a suitable delivery reagent, including the Mirus Transit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine) or liposomes. Methods for delivering recombinant viral vectors, which express a double-stranded molecule of the invention, to an area of cancer in a patient are within the skill of the art.
The double-stranded molecule of the invention can be administered to the subject by any means suitable for delivering the double-stranded molecule into cancer sites. For example, the double-stranded molecule can be administered by gene gun, electroporation, or by other suitable parenteral or enteral administration routes.
Suitable enteral administration routes include oral, rectal, or intranasal delivery.
Suitable parenteral administration routes include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection); subcutaneous injection or deposition including subcutaneous infusion (for example, by osmotic pumps); direct application to the area at or near the site of cancer, for example by a catheter or other placement device (e.g., a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation. In some embodiments, injections or infusions of the double-stranded molecule or vector can be given at or near the site of cancer.
The double-stranded molecule of the invention can be administered in a single dose or in multiple doses. Where the administration of the double-stranded molecule of the invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Injection of the agent can be directly into the tissue or near the site of cancer. Multiple injections of the agent into the tissue at or near the site of cancer can be administered.
One skilled in the art can also readily determine an appropriate dosage regimen for administering the double-stranded molecule of the invention to a given subject. For example, the double-stranded molecule can be administered to the subject once, for example, as a single injection or deposition at or near the cancer site. Alternatively, the double-stranded molecule can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, for example, from about seven to about ten days. In one exemplary dosage regimen, the double-stranded molecule is injected at or near the site of cancer once a day for seven days. When a dosage regimen comprises multiple administrations, it is understood that the effective amount of a double-stranded molecule administered to the subject can comprise the total amount of a double-stranded molecule administered over the entire dosage regimen.
In the present invention, a cancer overexpressing CDC45L and/or PIF1 can be treated with at least one active ingredient selected from the group consisting of:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof.
The cancer includes, but is not limited to, lung cancer. Accordingly, prior to the administration of the double-stranded molecule of the present invention as active ingredient, it is preferable to confirm whether the expression level of CDC45L and/or PIF1 in the cancer cells or tissues to be treated is enhanced as compared with normal cells of the same organ. Thus, in one embodiment, the present invention provides a method for treating a cancer (over)expressing CDC45L and/or PIF1, which method may include the steps of:
i) determining the expression level of CDC45L and/or PIF1 in cancer cells or tissue(s) obtained from a subject with the cancer to be treated;
ii) comparing the expression level of CDC45L and/or PIF1 with normal control; and
iii) administrating at least one component selected from the group consisting of
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof,
to a subject with a cancer overexpressing CDC45L and/or PIF1 as compared with normal control.
Alternatively, the present invention also provides a pharmaceutical composition comprising at least one component selected from the group consisting of:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, and
(c) a vector encoding thereof,
for use in administrating to a subject having a cancer overexpressing CDC45L and/or PIF1. In other words, the present invention further provides a method for identifying a subject to be treated with:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, or
(c) a vector encoding thereof,
which method may include the step of determining an expression level of CDC45L and/or PIF1 in subject-derived cancer cells or tissue(s), wherein an increase of the level compared to a normal control level of the gene indicates that the subject has cancer which may be treated with:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, or
(c) a vector encoding thereof.
The method of treating a cancer of the present invention will be described in more detail below.
A subject to be treated by the present method is preferably a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
According to the present invention, the expression level of CDC45L and/or PIF1 in cancer cell in cancer cells or tissues obtained from a subject is determined. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, the mRNA of CDC45L or PIF1 may be quantified using probes by hybridization methods (e.g., Northern hybridization). The detection may be carried out on a chip or an array. The use of an array is preferable for detecting the expression level of CDC45L and/or PIF1. Those skilled in the art can prepare such probes utilizing the sequence information of CDC45L or PIF1. For example, the cDNA of CDC45L or PIF1 may be used as the probes. If necessary, the probes may be labeled with a suitable label, such as dyes, fluorescent substances and isotopes, and the expression level of the gene may be detected as the intensity of the hybridized labels.
Furthermore, the transcription product of CDC45L or PIF1 (e.g., SEQ ID NO: 13, or 15) may be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers may be prepared based on the available sequence information of the gene.
Specifically, a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of CDC45L or PIF1. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but not to other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degree Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under a defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to their target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 degree Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60 degree Centigrade for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Alternatively, the translation product may be detected for the diagnosis of the present invention. For example, the quantity of CDC45L or PIF1 protein (SEQ ID NO: 14, or 16) may be determined. Methods for determining the quantity of the protein as the translation product include immunoassay methods that use an antibody specifically recognizing the protein. The antibody may be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody may be used for the detection, so long as the fragment or modified antibody retains the binding ability to the CDC45L or PIF1 protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method may be employed in the present invention to prepare such antibodies and equivalents thereof.
As another method to detect the expression level of CDC45L and/or PIF1 gene based on its translation product, the intensity of staining may be measured via immunohistochemical analysis using an antibody against the CDC45L or PIF1 protein. Namely, in this measurement, strong staining indicates increased presence/level of the protein and, at the same time, high expression level of CDC45L or PIF1 gene.
The expression level of a target gene, i.e., the CDC45L or PIF1 gene, in cancer cells can be determined to be increased if the level increases from the control level (e.g., the level in normal cells) of the target gene by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
The control level may be determined at the same time with the cancer cells by using a sample(s) previously collected and stored from a subject/subjects whose disease state(s) (cancerous or non-cancerous) is/are known. In addition, normal cells obtained from non-cancerous regions of an organ that has the cancer to be treated may be used as normal control. Alternatively, the control level may be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of CDC45L and/or PIF1 gene in samples from subjects whose disease states are known. Furthermore, the control level can be derived from a database of expression patterns from previously tested cells. Moreover, according to an aspect of the present invention, the expression level of CDC45L and/or PIF1 gene in a biological sample may be compared to multiple control levels, which are determined from multiple reference samples. It is preferred to use a control level determined from a reference sample derived from a tissue type similar to that of the subject-derived biological sample. Moreover, it is preferred to use the standard value of the expression levels of CDC45L and/or PIF1 gene in a population with a known disease state. The standard value may be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. may be used as the standard value.
In the context of the present invention, a control level determined from a biological sample that is known to be non-cancerous is referred to as a "normal control level". On the other hand, if the control level is determined from a cancerous biological sample, it is referred to as a "cancerous control level".
When the expression level of CDC45L and/or PIF1 gene is increased as compared to the normal control level, or is similar/equivalent to the cancerous control level, the subject may be diagnosed with cancer to be treated.
(iv) Compositions
Furthermore, the present invention provides pharmaceutical compositions comprising at least one of the present double-stranded molecules or the vectors coding for the molecules. Specifically, the present invention provides the following compositions [1] to [24]:
[1] A composition for inhibiting or reducing a growth of cell expressing CDC45L and/or PIF1 gene, or for treating or preventing a cancer expressing a CDC45L and/or PIF1 gene, which comprises at least one double-stranded molecule or vector encoding the double-stranded molecule, wherein said double-stranded molecule, when introduced into a cell, inhibits or reduces in vivo expression of said gene.
[2] The composition of [1], wherein said double-stranded molecule acts at mRNA which matched a target sequence selected from the group SEQ ID NO: 9 and 10 for CDC45L and SEQ ID NO: 11 and 12 for PIF1.
[3] The composition of [1], wherein said double-stranded molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form a double strand, wherein said sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12.
[4] The composition of any one of [1] to [3], wherein the cancer to be treated is lung cancer;
[5] The composition of [4], wherein the lung cancer is small cell lung cancer or non-small cell lung cancer;
[6] The composition of any one of [1] to [5], wherein the composition contains plural kinds of the double-stranded molecules;
[7] The composition of [6], wherein the plural kinds of the double-stranded molecules target the same gene;
[8] The composition of any one of [1] to [7], wherein the double-stranded molecule has a length of less than about 100 nucleotides;
[9] The composition of [8], wherein the double-stranded molecule has a length of less than about 75 nucleotides;
[10] The composition of [9], wherein the double-stranded molecule has a length of less than about 50 nucleotides;
[11] The composition of [10], wherein the double-stranded molecule has a length of less than about 25 nucleotides;
[12] The composition of any one of [1] to [11], wherein the sense strand hybridize with antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length;
[13] The composition of any one of [1] to [12], wherein said double-stranded molecule consists of a single oligonucleotide comprising both the sense and antisense strands linked by an intervening single-strand.
[14] The composition of [13], wherein said double-stranded molecule has a general formula 5'-[A]-[B]-[A']-3', wherein
[A] is the sense strand comprising a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12;
[B] is the intervening single-strand; and
[A'] is the antisense strand comprising an oligonucleotide corresponding to a sequence complementary to the target sequence selected in [A].
[15] The composition of any one of [1] to [14], wherein the double-stranded molecule comprises RNA;
[16] The composition of any one of [1] to [14], wherein the double-stranded molecule comprises DNA and RNA;
[17] The composition of [16], wherein the double-stranded molecule is a hybrid of a DNA polynucleotide and an RNA polynucleotide;
[18] The composition of [17], wherein the sense and antisense strand polynucleotides are made of DNA and RNA, respectively;
[19] The composition of [18], wherein the double-stranded molecule is a chimera of DNA and RNA;
[20] The composition of [19], wherein at least a region flanking to the 5'-end of one or both of the sense and antisense polynucleotides consists of RNA.
[21] The composition of [20], wherein the flanking region consists of 9 to 13 nucleotides;
[22] The composition of any one of [1] to [21], wherein the double-stranded molecule contains one or two 3' overhang(s);
[23] The composition of any one of [1] to [22], wherein the double-stranded molecule is encoded by a vector;
[24] The composition of any one of [1] to [23], which further comprising a transfection-enhancing agent, cell permeable agent or pharmaceutically acceptable carrier.
The method of the present invention will be described in more detail below.
The double-stranded molecules of the invention can be formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference.
The present pharmaceutical formulations comprise at least one of the double-stranded molecules or vectors encoding them of the present invention (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt of the molecule, mixed with a physiologically acceptable carrier medium. Exemplary physiologically acceptable carrier media include, for example, water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
According to the present invention, the composition can contain plural kinds of the double-stranded molecules, each of the molecules can be directed to the same target sequence, or different target sequences of CDC45L or PIF1 gene. For example, the composition can contain double-stranded molecules directed to CDC45L or PIF1 gene. Alternatively, for example, the composition can contain double-stranded molecules directed to target sequences selected from CDC45L and PIF1 gene.
Furthermore, the present composition can contain a vector coding for one or plural double-stranded molecules. For example, the vector can encode one, two or several kinds of the present double-stranded molecules. Alternatively, the present composition can contain plural kinds of vectors, each of the vectors coding for a different double-stranded molecule.
Moreover, the present double-stranded molecules can be contained as liposomes in the present composition. See under the item of "(iii) Methods Of Inhibiting Or Reducing A Growth Of Cancer Cells And Treating Or Preventing Cancer Using Double-Stranded Molecules r" for details of liposomes.
Pharmaceutical compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (for example, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
For solid compositions, conventional nontoxic solid carriers can be used; for example, pharmaceutical grade of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
For example, a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%, for example, 25-75%, of one or more double-stranded molecule of the invention. A pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, for example, 1-10% by weight, of one or more double-stranded molecule of the invention encapsulated in a liposome as described above, and propellant. A carrier can also be included as desired; e.g., lecithin for intranasal delivery.
In addition to the above, the present composition can contain other pharmaceutical active ingredients so long as they do not inhibit the in vivo function of the present double-stranded molecules. For example, the composition can contain chemotherapeutic agents conventionally used for treating cancers.
The present invention also provides the use of the double-stranded nucleic acid molecules of the present invention in manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CDC45L and/or PIF1 gene. For example, the present invention relates to the use of double-stranded nucleic acid molecule inhibiting the (over)expression of a CDC45L or PIF1 gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9, 10, 11 or 12, for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CDC45L and/or PIF1 gene.
Alternatively, the present invention further provides the double-stranded nucleic acid molecules of the present invention for use in treating a cancer expressing the CDC45L and/or PIF1 gene.
The present invention further provides a method or process for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CDC45L and/or PIF1 gene, wherein the method or process comprises step for formulating a pharmaceutically or physiologically acceptable carrier with a double-stranded nucleic acid molecule inhibiting the (over)expression of a CDC45L or PIF1 gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9, 10, 11 or 12 as active ingredients.
The present invention also provides a method or process for manufacturing a pharmaceutical composition for treating a cancer (over)expressing the CDC45L and/or PIF1 gene, wherein the method or process comprises step for admixing an active ingredient with a pharmaceutically or physiologically acceptable carrier, wherein the active ingredient is a double-stranded nucleic acid molecule inhibiting the expression of CDC45L or PIF1 gene in a cell, which over-expresses the gene, which molecule comprises a sense strand and an antisense strand complementary thereto, hybridized to each other to form the double-stranded nucleic acid molecule and targets a sequence of SEQ ID NOs: 9, 10, 11 or 12.
(4) Method for Diagnosing CDC45L -Mediated Cancers
The expression of CDC45L gene were found to be specifically elevated in lung cancer tissues compared with corresponding normal tissues (Fig.1, Fig. 2). Furthermore, the expression of PIF1 gene were also found to be specifically elevated in lung cancer tissues compared with corresponding normal lung tissues (Fig. 4). Therefore, the genes identified herein as well as its transcription and translation products have diagnostic utility as markers for cancers mediated by CDC45L and/or PIF1 gene and by measuring the expression of the CDC45L and/or PIF1 gene in a sample derived from a patient suspected to be suffering from cancers, these cancers can be diagnosed. Specifically, the present invention provides a method for diagnosing cancers mediated by CDC45L and/or PIF1 by determining the expression level of CDC45L and/or PIF1 in a subject. The CDC45L and/or PIF1 -promoted cancers that can be diagnosed by the present method include lung cancers. Lung cancers include non-small lung cancer, small lung cancer, a lung adenocarcinoma (ADC), a lung squamous-cell carcinoma (SCC), and a lung large-cell carcinoma (LCC). According to the present invention, an intermediate result for examining the condition of a subject can be provided. Such intermediate result can be combined with additional information to assist a doctor, nurse, or other practitioner to diagnose that a subject suffers from the disease. Alternatively, the present invention can be used to detect cancerous cells in a subject-derived tissue, and provide a doctor with useful information to diagnose that the subject suffers from the disease.
Specifically, the present invention provides the following methods [1] to [15]:
[1] A method for diagnosing cancer mediated or promoted by a CDC45L or PIF1, wherein said method comprises the steps of:
(a) detecting the expression level of CDC45L gene and/or PIF1 gene in a subject-derived biological sample; and
(b) relating an increase of the expression level compared to a normal control level of the gene to the disease.
[2] A method of detecting or diagnosing cancer in a subject, comprising determining a expression level of CDC45L and/or PIF1 in a subject-derived biological sample, wherein an increase of the level compared to a normal control level of the gene indicates that the subject suffers from or is at risk of developing cancer, or the presence of cancer in the subject,
[3] The method of [1] or [2], wherein the expression level is at least 10 % greater than normal control level.
[4] The method of any one of [1] to [3], wherein the expression level is detected by any one of the method select from the group consisting of:
(a) detecting the mRNA encoding the CDC45L or PIF1 polypeptide;
(b) detecting the CDC45L or PIF1 polypeptide; and
(c) detecting the biological activity of the CDC45L or PIF1 polypeptide.
[5] The method of any one of [1] to [3], wherein the expression level is determined by a method selected from a group consisting of:
(a) detecting an mRNA of CDC45L gene and/or an mRNA of PIF1 gene,
(b) detecting a protein encoded by CDC45L gene and/or a protein encoded by PIF1 gene, and
(c) detecting (a) biological activity(activities) of a protein encoded by CDC45L gene and/or a protein encoded by PIF1 gene.
[6] The method of any one of [1] to [5], wherein the cancer results from overexpression of a CDC45L or PIF1, or is mediated or promoted by a CDC45L or PIF1.
[7] The method of any one of [1] to [6], wherein the cancers is lung cancer.
[8] The method of [7], wherein the lung cancer is non-small cell lung cancer or small cell lung cancer.
[9] The method of [4] ot [5], wherein the expression level is determined by detecting a hybridization of probe to the gene transcript encoding the CDC45L or PIF1 polypeptide.
[10] The method of [4] or [5], wherein the expression level is determined by detecting a binding of an antibody against the CDC45L or PIF1 polypeptide.
[11] The method of any one of [1] to [10], wherein the biological sample comprises biopsy sample, sputum or blood.
[12] The method of any one of [1] to [10], wherein the subject-derived biological sample comprises an epithelial cell, serum or pleural effusion.
[13] The method of [1] to [12], wherein the subject-derived biological sample comprises a cancer cell.
[14] The method of [1] to [13], wherein the subject-derived biological sample comprises a cancerous epithelial cell.
[15] The method of [1] to [10], wherein the subject-derived biological sample comprises a lung tissue.
The method of diagnosing cancers will be described in more detail below.
A subject to be diagnosed by the present method is can be a mammal. Exemplary mammals include, but are not limited to, e.g., human, non-human primate, mouse, rat, dog, cat, horse, and cow.
In performing the present methods, a biological sample is collected from a subject to be diagnosed to perform the diagnosis. Any biological material can be used as the biological sample for the determination so long as it comprises the objective transcription or translation product of CDC45L and/or PIF1 gene. The biological samples include, but are not limited to, bodily tissues and fluids, for example, blood, e.g. serum, sputum, urine and pleural effusion. In some embodiments, the biological sample contains a cell population comprising an epithelial cell, for example, a cancerous epithelial cell or an epithelial cell derived from tissue suspected to be cancerous. Further, if necessary, the cell can be purified from the obtained bodily tissues and fluids, and then used as the biological sample.
According to the present invention, the expression level of CDC45L and/or PIF1 gene in the subject-derived biological sample is determined. The expression level can be determined at the transcription (nucleic acid) product level, using methods known in the art. For example, the mRNA of CDC45L or PIF1 gene can be quantified using probes by hybridization methods (e.g. Northern blot analysis). The detection can be carried out on a chip or an array. The use of an array can be for detecting the expression level of a plurality of genes (e.g., various cancer specific genes) including CDC45L and PIF1 gene. Those skilled in the art can prepare such probes utilizing the sequence information of the CDC45L (SEQ ID NO: 13; GenBank Accession No. NM_003504.3) or PIF1 (SEQ ID NO: 15; GenBank Accession No. NM_025049.2). For example, the cDNA of CDC45L or PIF1 gene can be used as a probe. If necessary, the probe can be labeled with a suitable label, for example, dyes, fluorescent and isotopes, and the expression level of the gene can be detected as the intensity of the hybridized labels.
Furthermore, the transcription product of CDC45L or PIF1 gene can be quantified using primers by amplification-based detection methods (e.g., RT-PCR). Such primers can also be prepared based on the available sequence information of the gene. For example, the primers (SEQ ID NO: 1 and 2 for CDC45L, SEQ ID NO: 3 and 4 for PIF1) used in the Example can be employed for the detection by RT-PCR or Northern blot, but the present invention is not restricted thereto.
Specifically, a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of CDC45L or PIF1 gene.
Alternatively, the translation product can be detected for the diagnosis of the present invention. For example, the quantity of CDC45L or PIF1 protein can be determined. A method for determining the quantity of the protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the protein. The antibody can be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody can be used for the detection, so long as the fragment retains the binding ability to CDC45L or PIF1 protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method can be employed in the present invention to prepare such antibodies and equivalents thereof.
As another method to detect the expression level of CDC45L and/or PIF1 based on its translation product, the intensity of staining can be observed via immunohistochemical analysis using an antibody against CDC45L or PIF1 protein. Namely, the observation of strong staining indicates increased presence of the protein and at the same time high expression level of CDC45L or PIF1 (see, Immunohistochemistry and Tissue-microarray analysis in "EXAMPLES").
Moreover, in addition to the expression level of CDC45L and/or PIF1, the expression level of other cancer-associated genes, for example, genes known to be differentially expressed in cancers can also be determined to improve the accuracy of the diagnosis.
The expression level of cancer marker gene including CDC45L and PIF1 in a biological sample can be considered to be increased if it increases from the control level of the corresponding cancer marker gene (e.g., in a normal or non-cancerous cell) by, for example, 10%, 25%, or 50%; or increases to more than 1.1 fold, more than 1.5 fold, more than 2.0 fold, more than 5.0 fold, more than 10.0 fold, or more.
The control level can be determined at the same time with the test biological sample by using a sample(s) previously collected and stored from a subject/subjects whose disease state (cancerous or non-cancerous) is/are known. Alternatively, the control level can be determined by a statistical method based on the results obtained by analyzing previously determined expression level(s) of CDC45L and/or PIF1 in samples from subjects whose disease state are known. Furthermore, the control level can be a database of expression patterns from previously tested cells. Moreover, according to an aspect of the present invention, the expression level of a CDC45L and/or PIF1 in a biological sample can be compared to multiple control levels, which control levels are determined from multiple reference samples. In some embodiments, a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample is used. In some embodiments, the standard value of the expression levels of CDC45L or PIF1 in a population with a known disease state is used. The standard value can be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. can be used as standard value.
In the context of the present invention, a control level determined from a biological sample that is known not to be cancerous is called "normal control level". For example, normal tissues obtained from organ same as cancerous organs may be diagnosed. Accordingly, normal lung tissues may be diagnosed as normal control for diagnosing lung cancer. On the other hand, if the control level is determined from a cancerous biological sample, it will be called "cancerous control level".
When the expression level of CDC45L and/or PIF1 is increased compared to the normal control level or is similar to the cancerous control level, the subject can be diagnosed to be suffering from or at a risk of developing cancer, e.g., a cancer that is mediated by or results from overexpression of CDC45L and/or PIF1. Furthermore, in case where the expression levels of CDC45L and/or PIF1 gene are compared, a similarity in the gene expression pattern between the sample and the reference which is cancerous indicates that the subject is suffering from or at a risk of developing cancer, e.g., a cancer that is mediated by or results from overexpression of a CDC45L and/or PIF1. Such cancer encompasses lung cancer.
Difference between the expression levels of a test biological sample and the control level can be normalized to the expression level of control nucleic acids, e.g., housekeeping genes, whose expression levels are known not to differ depending on the cancerous or non-cancerous state of the cell. Exemplary control genes include, but are not limited to, beta-actin, glyceraldehyde 3 phosphate dehydrogenase, and ribosomal protein P1.
Alternatively, the present invention provides a method for detecting or identifying cancer cells in a subject-derived lung tissue sample, said method comprising the step of determining the expression level of the CDC45L and/or PIF1 gene in a subject-derived biological sample, wherein an increase in said expression level as compared to a normal control level of said gene indicates the presence or suspicion of cancer cells in the tissue.
Such result may be combined with additional information to assist a doctor, nurse, or other healthcare practitioner in diagnosing a subject as afflicted with the disease. In other words, the present invention may provide a doctor with useful information to diagnose a subject as afflicted with the disease. For example, according to the present invention, when there is doubt regarding the presence of cancer cells in the tissue obtained from a subject, clinical decisions can be reached by considering the expression level of the CDC45L and/or PIF1 gene, plus a different aspect of the disease including tissue pathology, levels of known tumor marker(s) in blood, and clinical course of the subject, etc. For example, some well-known diagnostic lung tumor markers in blood are IAP, ACT, BFP, CA19-9, CA50, CA72-4, CA130, CEA, KMO-1, NSE, SCC, SP1, Span-1, TPA, CSLEX, SLX, STN and CYFRA. Namely, in this particular embodiment of the present invention, the outcome of the gene expression analysis serves as an intermediate result for further diagnosis of a subject's disease state.
Alternatively, the present invention provides use of a reagent for preapring a diagnostic reagent for diagnosing cancer. In some embodiments, the reagent can be selected from the group consisting of:
(a) a reagent for detecting mRNA of the CDC45L or PIF1 gene;
(b) a reagent for detecting the CDC45L or PIF1 protein; and
(c) a reagent for detecting the biological activity of the CDC45L or PIF1 protein.
Specifically, such reagent is an oligonucleotide that hybridizes to the CDC45L or PIF1 polynucleotide, or an antibody that binds to the CDC45L or PIF1 polypeptide.
In other words, the present invention also provides a kit for use in diagnosis or detection of cancer, wherein the kit comprises a reagent which binds to a transcription or translation product of the CDC45L gene or PIF1 gene.
In the present invention, it is revealed that CDC45L or PIF1 is not only a useful diagnostic marker, but also suitable target for cancer therapy. Therefore, cancer treatment targeting CDC45L or PIF1 can be achieved by the present invention. In the present invention, the cancer treatment targeting CDC45L or PIF1 refers to suppression or inhibition of CDC45L or PIF1 activity and/or expression in the cancer cells. Any anti-CDC45L or anti-PIF1 agents may be used for the cancer treatment targeting CDC45L or PIF1. In the present agents may be used for the cancer treatment targeting CDC45L or PIF1. In the present invention, the anti-CDC45L or anti-PIF1 agents include following substance as active ingredient:
(a) a double-stranded molecule of the present invention,
(b) DNA encoding thereof, or
(c) a vector encoding thereof.
Accordingly, in a preferred embodiment, the present invention provides a method of (i) diagnosing whether a subject has the cancer to be treated, and/or (ii) selecting a subject for cancer treatment, which method includes the steps of:
a) determining the expression level of CDC45L or PIF1 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of CDC45L or PIF1 with a normal control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of CDC45L or PIF1 is increased as compared to the normal control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
Alternatively, such a method includes the steps of:
a) determining the expression level of CDC45L or PIF1 in cancer cells or tissue(s) obtained from a subject who is suspected to have the cancer to be treated;
b) comparing the expression level of CDC45L or PIF1 with a cancerous control level;
c) diagnosing the subject as having the cancer to be treated, if the expression level of CDC45L or PIF1 is similar or equivalent to the cancerous control level; and
d) selecting the subject for cancer treatment, if the subject is diagnosed as having the cancer to be treated, in step c).
(5) Method for Assessing the Prognosis of CDC45L Mediated Cancer
The present invention is based, in part, on the discovery that CDC45L (over)expression is significantly associated with poorer prognosis of patients with CDC45L-mediated cancers, e.g., lung cancers. Thus, the present invention provides a method for determining or assessing the prognosis of a patient with cancer, e.g., a cancer mediated by or resulting from overexpression of a CDC45L, e.g, lung cancer, by detecting the expression level of the CDC45L gene in a biological sample of the patient; comparing the detected expression level to a control level; and determining a increased expression level to the control level as indicative of poor prognosis (poor survival).
Herein, the term "prognosis" refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a less favorable, negative or poor prognosis is defined by a lower post-treatment survival term or survival rate. Conversely, a positive, favorable, or good prognosis is defined by an elevated post-treatment survival term or survival rate.
The terms "assessing the prognosis" refer to the ability of predicting, forecasting or correlating a given detection or measurement with a future outcome of cancer of the patient (e.g., malignancy, likelihood of curing cancer, estimated time of survival, and the like). For example, a determination of the expression level of CDC45L over time enables a predicting of an outcome for the patient (e.g., increase or decrease in malignancy, increase or decrease in grade of a cancer, likelihood of curing cancer, survival, and the like).
In the context of the present invention, the phrase "assessing (or determining) the prognosis" is intended to encompass predictions and likelihood analysis of cancer, progression, particularly cancer recurrence, metastatic spread and disease relapse. The present method for assessing prognosis is intended to be used clinically in making decisions concerning treatment modalities, including therapeutic intervention, diagnostic criteria for example, disease staging, and disease monitoring and surveillance for metastasis or recurrence of neoplastic disease.
Specifically, the present invention provides the following methods [1] to [6]:
[1] A method for assessing prognosis of a subject with lung cancer, wherein the method comprises steps of:
(a) detecting an expression level of CDC45L in a subject-derived biological sample;
(b) comparing the detected expression level to a control level; and
(c) determining prognosis of the patient based on the comparison of (b);
[2] The method of [1], wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level indicates poor prognosis;
[3] The method of [1], wherein the control level is a poor prognosis control level and a similar expression level to the control level indicates poor prognosis;
[4] The method of [2], wherein the increase is at least 10% greater than said control level; and
[5] The method of any one of [1] to [4], wherein the expression level is determined by a method selected from a group consisting of:
(a) detecting an mRNA of CDC45L gene;
(b) detecting a protein encoded by CDC45L gene; and
(c) detecting a biological activity of the protein encoded by CDC45L gene;
[6] The method of any one of [1] to [5], wherein the subject-derived biological sample comprises a lung cancer tissue or lung cancer cells.
The patient-derived biological sample used for the method can be any sample derived from the subject to be assessed so long as the CDC45L gene can be detected in the sample. In some embodiments, the biological sample comprises a lung cell (a cell obtained from lung ). Furthermore, the biological sample includes bodily fluids for example, sputum, blood, serum, plasma, pleural effusion, and so on. Moreover, the sample can be cells purified or obtained from a tissue. The biological samples can be obtained from a patient at various time points, including before, during, and/or after a treatment. For example, a lung cancer cell(s) obtained from a subject to be assessed is a preferable biological sample.
According to the present invention, it was shown that the higher the expression level of the CDC45L gene measured in the patient-derived biological sample, the poorer the prognosis for post-treatment remission, recovery, and/or survival and the higher the likelihood of poor clinical outcome. Thus, according to the present method, the "control level" used for comparison can be, for example, the expression level of the CDC45L gene detected before any kind of treatment in an individual or a population of individuals who showed good or positive prognosis of cancer, after the treatment, which herein will be referred to as "good prognosis control level". Alternatively, the "control level" can be the expression level of the CDC45L gene detected before any kind of treatment in an individual or a population of individuals who showed poor or negative prognosis of cancer, after the treatment, which herein will be referred to as "poor prognosis control level". The "control level" is a single expression pattern derived from a single reference population or from a plurality of expression patterns. Thus, the control level can be determined based on the expression level of the CDC45L gene detected before any kind of treatment in a patient of cancer, or a population of the patients whose disease state (good or poor prognosis) is known. In some embodiments, the cancer is lung cancer. In some embodiments, the standard value of the expression levels of the CDC45L gene in a patient group with a known disease state is used. The standard value can be obtained by any method known in the art. For example, a range of mean +/- 2 S.D. or mean +/- 3 S.D. can be used as standard value.
The control level can be determined at the same time with the test biological sample by using a sample(s) previously collected and stored before any kind of treatment from cancer patient(s) (control or control group) whose disease state (good prognosis or poor prognosis) are known.
Alternatively, the control level can be determined by a statistical method based on the results obtained by analyzing the expression level of the CDC45L gene in samples previously collected and stored from a control group. Furthermore, the control level can be a database of expression patterns from previously tested cells or patients. Moreover, according to an aspect of the present invention, the expression level of the CDC45L gene in a biological sample can be compared to multiple control levels, which control levels are determined from multiple reference samples. In some embodiments, a control level determined from a reference sample derived from a tissue type similar to that of the patient-derived biological sample is used.
According to the present invention, a similarity in the expression level of the CDC45L gene to the good prognosis control level indicates a more favorable prognosis of the patient and an increase in the expression level in comparison to the good prognosis control level indicates less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome. On the other hand, a decrease in the expression level of the CDC45L gene in comparison to the poor prognosis control level indicates a more favorable prognosis of the patient and a similarity in the expression level to the poor prognosis control level indicates less favorable, poorer prognosis for post-treatment remission, recovery, survival, and/or clinical outcome. For example, a lung cancer cell(s) obtained from a subject who showed good, or poor prognosis of cancer after treatment is a preferable biological sample for good, or poor prognosis control level, respectively.
An expression level of the CDC45L gene in a biological sample can be considered altered (i.e., increased or decreased) when the expression level differs from the control level by more than 1.0, 1.5, 2.0, 5.0, 10.0, or more fold.
The difference in the expression level between the test biological sample and the control level can be normalized to a control, e.g., housekeeping gene. For example, polynucleotides whose expression levels are known not to differ between the cancerous and non-cancerous cells, including those coding for beta-actin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein P1, can be used to normalize the expression levels of the CDC45L gene.
The expression level can be determined by detecting the gene transcript in the patient-derived biological sample using techniques well known in the art. The gene transcripts detected by the present method include both the transcription and translation products, for example, mRNA and protein.
For instance, the transcription product of the CDC45L gene can be detected by hybridization, e.g., Northern blot hybridization analyses, that use a CDC45L gene probe to the gene transcript. The detection can be carried out on a chip or an array. An array can be used for detecting the expression level of a plurality of genes including the CDC45L gene. As another example, amplification-based detection methods, for example, reverse-transcription based polymerase chain reaction (RT-PCR) which use primers specific to the CDC45L gene can be employed for the detection (see (b) Semi-quantitative RT-PCR in [EXAMPLE]). The CDC45L gene-specific probe or primers can be designed and prepared using conventional techniques by referring to the whole sequence of the CDC45L (SEQ ID NO: 13). For example, the primers (SEQ ID NOs: 1 and 2) used in the Example can be employed for the detection by RT-PCR, but the present invention is not restricted thereto.
Specifically, a probe or primer used for the present method hybridizes under stringent, moderately stringent, or low stringent conditions to the mRNA of the CDC45L gene. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different under different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of a stringent condition is selected to be about 5 degrees Centigrade lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30degrees Centigrade for short probes or primers (e.g., 10 to 50 nucleotides) and at least about 60degrees Centigrade for longer probes or primers. Stringent conditions can also be achieved with the addition of destabilizing agents, for example, formamide.
Alternatively, the translation product can be detected for the assessment of the present invention. For example, the quantity of the CDC45L protein can be determined. A method for determining the quantity of the protein as the translation product includes immunoassay methods that use an antibody specifically recognizing the CDC45L protein. The antibody can be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody can be used for the detection, so long as the fragment retains the binding ability to the CDC45L protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method can be employed in the present invention to prepare such antibodies and equivalents thereof.
As another method to detect the expression level of the CDC45L gene based on its translation product, the intensity of staining can be observed via immunohistochemical analysis using an antibody against CDC45L protein. Namely, the observation of strong staining indicates increased presence of the CDC45L protein and at the same time high expression level of the CDC45L gene.
Furthermore, the CDC45L protein is known to have a cell proliferating activity. Therefore, the expression level of the CDC45L gene can be determined using such cell proliferating activity as an index. For example, cells which express CDC45L are prepared and cultured in the presence of a biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability the cell proliferating activity of the biological sample can be determined.
Moreover, in addition to the expression level of the CDC45L gene, the expression level of other lung cell-associated genes, for example, genes known to be differentially expressed in lung cancer, can also be determined to improve the accuracy of the assessment. Such other lung cancer-associated genes include those described in WO 2004/031413 and WO 2005/090603.
The patient to be assessed for the prognosis of cancer according to the method can be a mammal and includes human, non-human primate, mouse, rat, dog, cat, horse, and cow.
Alternatively, according to the present invention, an intermediate result can also be provided in addition to other test results for assessing the prognosis of a subject. Such intermediate result can assist a doctor, nurse, or other practitioner to assess, determine, or estimate the prognosis of a subject. Additional information that can be considered, in combination with the intermediate result obtained by the present invention, to assess prognosis includes clinical symptoms and physical conditions of a subject.
Alternatively, the present invention provides use of a reagent for preapring a reagent for assessing prognosis of cancer. In some embodiments, the reagent is selected from the group consisting of:
(a) a reagent for detecting mRNA of the CDC45L gene;
(b) a reagent for detecting the CDC45L; and
(c) a reagent for detecting the biological activity of the CDC45L protein.
Specifically, such reagent is an oligonucleotide that hybridizes to the CDC45L polynucleotide, or an antibody that binds to the CDC45L polypeptide.
(6) Kits for Diagnosing Cancer or Assessing the Prognosis of Cancer
The present invention provides a kit for diagnosing cancer or assessing the prognosis of cancer. The present invention also provides a kit for determining a subject suffering from cancer that can be treated with the double-stranded molecule of the present invention or vector encoding thereof, which may also be useful in assessing and/or monitoring the efficacy of a cancer treatment. In some embodiments, the cancer is mediated by CDC45L and/or PIF1, or resulting from overexpression of CDC45L and/or PIF1, e.g., lung cancer. Specifically, the kit comprises at least one reagent for detecting the expression of the CDC45L or PIF1 gene in a patient-derived biological sample, which reagent can be selected from the group of:
(a) a reagent for detecting mRNA of the CDC45L or PIF1 gene;
(b) a reagent for detecting the CDC45L or PIF1 protein; and
(c) a reagent for detecting the biological activity of the CDC45L or PIF1 protein.
Suitable reagents for detecting mRNA of the CDC45L or PIF1 gene include nucleic acids that specifically bind to or identify the CDC45L or PIF1 mRNA, for example, oligonucleotides which have a complementary sequence to a part of the CDC45L or PIF1 mRNA. These kinds of oligonucleotides are exemplified by primers and probes that are specific to the CDC45L or PIF1 mRNA. These kinds of oligonucleotides can be prepared based on methods well known in the art. If needed, the reagent for detecting the CDC45L or PIF1 mRNA can be immobilized on a solid matrix. Moreover, more than one reagent for detecting the CDC45L or PIF1 mRNA can be included in the kit.
The probes or primers may be of specific sizes. The sizes are selected from the group consisting of at least 10 nucleotides, at least 12 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides and the probes and primers may range in size from 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides and 25-30 nucleotides.
On the other hand, suitable reagents for detecting the CDC45L or PIF1 protein include antibodies to the CDC45L or PIF1 protein. The antibody can be monoclonal or polyclonal. Furthermore, any fragment or modification (e.g., chimeric antibody, scFv, Fab, F(ab')2, Fv, etc.) of the antibody can be used as the reagent, so long as the fragment retains the binding ability to the CDC45L or PIF1 protein. Methods to prepare these kinds of antibodies for the detection of proteins are well known in the art, and any method can be employed in the present invention to prepare such antibodies and equivalents thereof. Furthermore, the antibody can be labeled with signal generating molecules via direct linkage or an indirect labeling technique. Labels and methods for labeling antibodies and detecting the binding of antibodies to their targets are well known in the art and any labels and methods can be employed for the present invention. Moreover, more than one reagent for detecting the CDC45L or PIF1 protein can be included in the kit.
Furthermore, the biological activity can be determined by, for example, measuring the cell proliferating activity due to the expressed CDC45L or PIF1 protein in the biological sample. For example, the cell is cultured in the presence of a patient-derived biological sample, and then by detecting the speed of proliferation, or by measuring the cell cycle or the colony forming ability the cell proliferating activity of the biological sample can be determined. If needed, the reagent for detecting the CDC45L or PIF1 mRNA can be immobilized on a solid matrix. Moreover, more than one reagent for detecting the biological activity of the CDC45L or PIF1 protein can be included in the kit.
The kit can comprise more than one of the aforementioned reagents. Furthermore, the kit can comprise a solid matrix and reagent for binding a probe against the CDC45L or PIF1 gene or antibody against the CDC45L or PIF1 protein, a medium and container for culturing cells, positive and negative control reagents, and a secondary antibody for detecting an antibody against the CDC45L or PIF1 protein. For example, tissue samples obtained from patient with good prognosis or poor prognosis can serve as useful control reagents. A kit of the present invention can further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts (e.g., written, tape, CD-ROM, etc.) with instructions for use. These reagents and such can be comprised in a container with a label. Suitable containers include bottles, vials, and test tubes. The containers can be formed from a variety of materials, for example, glass or plastic.
As an embodiment of the present invention, when the reagent is a probe against the CDC45L or PIF1 mRNA, the reagent can be immobilized on a solid matrix, for example, a porous strip, to form at least one detection site. The measurement or detection region of the porous strip can include a plurality of sites, each containing a nucleic acid (probe). A test strip can also contain sites for negative and/or positive controls. Alternatively, control sites can be located on a strip separated from the test strip. Optionally, the different detection sites can contain different amounts of immobilized nucleic acids, i.e., a higher amount in the first detection site and lesser amounts in subsequent sites. Upon the addition of test sample, the number of sites displaying a detectable signal provides a quantitative indication of the amount of CDC45L or PIF1 mRNA present in the sample. The detection sites can be configured in any suitably detectable shape and are typically in the shape of a bar or dot spanning the width of a test strip.
The kit of the present invention can further comprise a positive control sample or CDC45L or PIF1 standard sample. The positive control sample of the present invention can be prepared by collecting CDC45L or PIF1 positive blood samples and then those CDC45L or PIF1 level are assayed. Alternatively, purified CDC45L or PIF1 protein or polynucleotide can be added to CDC45L or PIF1 free serum to form the positive sample or the CDC45L or PIF1 standard. In the present invention, purified CDC45L or PIF1 can be recombinant protein. The CDC45L or PIF1 level of the positive control sample is, for example more than cut off value.
(7) Screening Methods
Using the CDC45L or PIF1 gene, polypeptide encoded by the gene or fragment thereof, or transcriptional regulatory region of the gene, it is possible to screen agents or compounds that alter the expressions of the genes or the biological activities of polypeptides encoded by the genes. Such agents or compounds may be used as pharmaceuticals for treating or preventing cancer, in particular, lung cancer. Thus, the present invention provides methods of screening for candidate agents or compounds for treating or preventing cancer using the CDC45L or PIF1 gene, a polypeptide encoded by the gene or fragment thereof, or a transcriptional regulatory region of the gene.
Agents or compounds isolated by the screening method of the present invention is a substance that is expected to inhibit the expression of the CDC45L or PIF1 gene, or the activity of the translation product of the gene, and thus, is a candidate for treating or preventing diseases attributed to, for example, cell proliferative diseases, such as cancer (in particular, lung and esophageal cancer). Namely, the agents or compounds screened through the present methods are deemed to have a clinical benefit and can be further tested for its ability to prevent cancer cell growth in animal models or test subjects.
(I) Test compounds for screening
In the context of the present invention, agents to be identified through the present screening methods can be any compound or composition including several compounds. Furthermore, the test agent or compound exposed to a cell or protein according to the screening methods of the present invention can be a single compound or a combination of compounds. When a combination of compounds is used in the methods, the compounds can be contacted sequentially or simultaneously.
Any test agent or compound, for example, cell extracts, cell culture supernatant, products of fermenting microorganism, extracts from marine organism, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic micro-molecular compounds (including nucleic acid constructs, for example, antisense DNA, siRNA, ribozymes, etc.) and natural compounds can be used in the screening methods of the present invention. The test agent or compound of the present invention can be also obtained using any of the numerous approaches in combinatorial library methods known in the art, including
(1) biological libraries,
(2) spatially addressable parallel solid phase or solution phase libraries,
(3) synthetic library methods requiring deconvolution,
(4) the "one-bead one-compound" library method and
(5) synthetic library methods using affinity chromatography selection.
The biological library methods using affinity chromatography selection is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des 1997, 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al., Proc Natl Acad Sci USA 1993, 90: 6909-13; Erb et al., Proc Natl Acad Sci USA 1994, 91: 11422-6; Zuckermann et al., J Med Chem 37: 2678-85, 1994; Cho et al., Science 1993, 261: 1303-5; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2059; Carell et al., Angew Chem Int Ed Engl 1994, 33: 2061; Gallop et al., J Med Chem 1994, 37: 1233-51). Libraries of compounds can be presented in solution (see Houghten, Bio/Techniques 1992, 13: 412-21) or on beads (Lam, Nature 1991, 354: 82-4), chips (Fodor, Nature 1993, 364: 555-6), bacteria (US Pat. No. 5,223,409), spores (US Pat. No. 5,571,698; 5,403,484 and 5,223,409), plasmids (Cull et al., Proc Natl Acad Sci USA 1992, 89: 1865-9) or phage (Scott and Smith, Science 1990, 249: 386-90; Devlin, Science 1990, 249: 404-6; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Felici, J Mol Biol 1991, 222: 301-10; US Pat. Application 2002-103360).
A compound in which a part of the structure of the compound screened by any of the present screening methods is converted by addition, deletion and/or replacement, is included in the compounds obtained by the screening methods of the present invention.
Furthermore, when the screened test agent or compound is a protein, for obtaining a DNA encoding the protein, either the whole amino acid sequence of the protein can be determined to deduce the nucleic acid sequence coding for the protein, or partial amino acid sequence of the obtained protein can be analyzed to prepare an oligo DNA as a probe based on the sequence, and screen cDNA libraries with the probe to obtain a DNA encoding the protein. The obtained DNA finds use in preparing the test agent or compound which is a candidate for treating or preventing cancer.
Test agents or compounds useful in the screening described herein can also be antibodies or non-antibody binding proteins that specifically bind to the CDC45L or PIF1 protein or partial CDC45L or PIF1 peptides that lack the activity to binding for partner. Such partial protein or antibody can be prepared by the methods described herein (see (1) Cancer-related genes and cancer-related protein, and functional equivalent thereof in Definition or Antibodies) and can be tested for their ability to block binding of the protein with its binding partners.
(i) Molecular modeling
Construction of test agent/compound libraries is facilitated by knowledge of the molecular structure of compounds known to have the properties sought, and/or the molecular structure of the target molecules to be inhibited. One approach to preliminary screening of test agents or compounds suitable for further evaluation is computer modeling of the interaction between the test agent/compound and its target.
Computer modeling technology allows the visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule. The three-dimensional construct typically depends on data from x-ray crystallographic analysis or NMR imaging of the selected molecule. The molecular dynamics require force field data. The computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
An example of the molecular modeling system described generally above includes the CHARMm and QUANTA programs, Polygen Corporation, Waltham, Mass. CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
A number of articles review computer modeling of drugs interactive with specific proteins, for example, Rotivinen et al. Acta Pharmaceutica Fennica 1988, 97: 159-66; Ripka, New Scientist 1988, 54-8; McKinlay & Rossmann, Annu Rev Pharmacol Toxiciol 1989, 29: 111-22; Perry & Davies, Prog Clin Biol Res 1989, 291: 189-93; Lewis & Dean, Proc R Soc Lond 1989, 236: 125-40, 141-62; and, with respect to a model receptor for nucleic acid components, Askew et al., J Am Chem Soc 1989, 111: 1082-90.
Other computer programs that screen and graphically depict chemicals are available from companies for example, BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario. See, e.g., DesJarlais et al., J Med Chem 1988, 31: 722-9; Meng et al., J Computer Chem 1992, 13: 505-24; Meng et al., Proteins 1993, 17: 266-78; Shoichet et al., Science 1993, 259: 1445-50.
Once an inhibitor of the CDC45L or PIF1 activity has been identified, combinatorial chemistry techniques can be employed to construct any number of variants based on the chemical structure of the identified inhibitor, as detailed below. The resulting library of candidate inhibitors, or "test agents or compounds" can be screened using the methods of the present invention to identify test agents or compounds of the library that disrupt the CDC45L or PIF1 activity for the treatment and/or prophylaxis of cancer and/or the prevention of post-operative recurrence of cancer.
(ii) Combinatorial chemical synthesis
Combinatorial libraries of test agents or compounds can be produced as part of a rational drug design program involving knowledge of core structures existing in known inhibitors of the CDC45L or PIF1 activity. This approach allows the library to be maintained at a reasonable size, facilitating high throughput screening. Alternatively, simple, particularly short, polymeric molecular libraries can be constructed by simply synthesizing all permutations of the molecular family making up the library. An example of this latter approach would be a library of all peptides six amino acids in length. Such a peptide library could include every 6 amino acid sequence permutation. This type of library is termed a linear combinatorial chemical library.
Preparation of combinatorial chemical libraries is well known to those of skill in the art, and can be generated by either chemical or biological synthesis. Combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., US Patent 5,010,175; Furka, Int J Pept Prot Res 1991, 37: 487-93; Houghten et al., Nature 1991, 354: 84-6). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptides (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., WO 93/20242), random bio-oligomers (e.g., WO 92/00091), benzodiazepines (e.g., US Patent 5,288,514), diversomers for example, hydantoins, benzodiazepines and dipeptides (DeWitt et al., Proc Natl Acad Sci USA 1993, 90:6909-13), vinylogous polypeptides (Hagihara et al., J Amer Chem Soc 1992, 114: 6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J Amer Chem Soc 1992, 114: 9217-8), analogous organic syntheses of small compound libraries (Chen et al., J. Amer Chem Soc 1994, 116: 2661), oligocarbamates (Cho et al., Science 1993, 261: 1303), and/or peptidylphosphonates (Campbell et al., J Org Chem 1994, 59: 658), nucleic acid libraries (see Ausubel, Current Protocols in Molecular Biology, 1990-2008, John Wiley Interscience; Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., 2001, Cold Spring Harbor Laboratory, New York, USA), peptide nucleic acid libraries (see, e.g., US Patent 5,539,083), antibody libraries (see, e.g., Vaughan et al., Nature Biotechnology 1996, 14(3):309-14 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 1996, 274: 1520-22; US Patent 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Gordon EM. Curr Opin Biotechnol. 1995 Dec 1;6(6):624-31.; isoprenoids, US Patent 5,569,588; thiazolidinones and metathiazanones, US Patent 5,549,974; pyrrolidines, US Patents 5,525,735 and 5,519,134; morpholino compounds, US Patent 5,506,337; benzodiazepines, 5,288,514, and the like).
(iii) Other candidates
Another approach uses recombinant bacteriophage to produce libraries. Using the "phage method" (Scott & Smith, Science 1990, 249: 386-90; Cwirla et al., Proc Natl Acad Sci USA 1990, 87: 6378-82; Devlin et al., Science 1990, 249: 404-6), very large libraries can be constructed (e.g., 106 -108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23: 709-15; Geysen et al., J Immunologic Method 1987, 102: 259-74); and the method of Fodor et al. (Science 1991, 251: 767-73) are examples. Furka et al. (14th International Congress of Biochemistry 1988, Volume #5, Abstract FR:013; Furka, Int J Peptide Protein Res 1991, 37: 487-93), Houghten (US Patent 4,631,211) and Rutter et al. (US Patent 5,010,175) describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.
Aptamers are macromolecules composed of nucleic acid that bind tightly to a specific molecular target. Tuerk and Gold (Science. 249:505-510 (1990)) discloses SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method for selection of aptamers. In the SELEX method, a large library of nucleic acid molecules {e.g., 1015 different molecules) can be used for screening.
(II) Screening methods
(i) General screening Method
Using the CDC45L or PIF1 gene, proteins encoded by the gene or transcriptional regulatory region of the gene, compounds that alter the expression of the gene or the biological activity of a polypeptide encoded by the gene can be identified. In the present invention, CDC45L and PIF1gene were found to be over-expressed in cancer, and demonstrated to be involved in cancer cell growth and/or survival. Therefore, compounds that alter the expressions of these genes or the biological activities of polypeptides by those genes can be candidate therapeutics for cancer.
Antagonists that binds to the CDC45Lor PIF1 polypeptide may inhibit the biological activity to mediate cell proliferation of cancer , and thus, they are candidates for treating the cancer. Therefore, the present invention provides a method for identifying potential candidates for treating or preventing cancer expressing CDC45L and/or PIF1, by identifying agents or compounds that bind to CDC45L or PIF1 polypeptide.
As a method of screening for compounds that inhibit the binding between a CDC45L or PIF1 protein and a binding partner thereof (e.g., CDC45L and PIF1 each other), many methods well known by one skilled in the art can be used. For example, screening can be carried out as an in vitro assay system, for example, a cellular system. More specifically, first, either the CDC45L or PIF1 protein or the binding partner thereof is bound to a support, and the other protein is added together with a test compound thereto. For instance, the CDC45L or PIF1 is bound to a support, and the binding partner polypeptide is added together with a test compound thereto. Next, the mixture is incubated, washed and the other protein bound to the support is detected and/or measured.
In the context of the present invention, "inhibition of binding" between two proteins refers to at least reducing binding between the proteins. Thus, in some cases, the percentage of binding pairs in a sample in the presence of a test agent or compound will be decreased compared to an appropriate (e.g., not treated with test compound or from a non-cancer sample, or from a cancer sample) control. The reduction in the amount of proteins bound can be, e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 25%, 10%, 5%, 1% or less (e.g., 0%), than the pairs bound in a control sample.
Examples of supports that can be used for binding proteins include, for example, insoluble polysaccharides, for example, agarose, cellulose and dextran; and synthetic resins, for example, polyacrylamide, polystyrene and silicon; for example, commercial available beads and plates (e.g., multi-well plates, biosensor chip, etc.) prepared from the above materials can be used. When using beads, they can be filled into a column. Alternatively, the use of magnetic beads is also known in the art, and enables one to readily isolate proteins bound on the beads via magnetism.
The binding of a protein to a support can be conducted according to routine methods, for example, chemical bonding and physical adsorption, for example. Alternatively, a protein can be bound to a support via antibodies that specifically recognize the protein. Moreover, binding of a protein to a support can be also conducted by means of avidin and biotin.
The methods of screening for molecules that bind when the immobilized polypeptide is exposed to synthetic chemical compounds, or natural substance banks, or a random phage peptide display library, and the methods of screening using high-throughput based on combinatorial chemistry techniques (Wrighton et al., Science 273: 458-63 (1996); Verdine, Nature 384: 11-3 (1996)) to isolate not only proteins but chemical compounds that bind to the protein (including agonist and antagonist) are well known to one skilled in the art.
Furthermore, in the screening method of the present invention, agents or compounds that suppress the expression level of CDC45L or PIF can be also identified as candidate therapeutics for cancer. The expression level of a polypeptide or functional equivalent thereof can be detected according to any method known in the art. For example, a reporter assay can be used. Suitable reporter genes and host cells are well known in the art. The reporter construct required for the screening can be prepared by using the transcriptional regulatory region of CDC45L or PIF1 gene. When the transcriptional regulatory region of the gene has been known to those skilled in the art, a reporter construct can be prepared by using the previous sequence information. When the transcriptional regulatory region remains unidentified, a nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library based on the nucleotide sequence information of the gene. Specifically, the reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of a CDC45L or PIF1 gene of interest. The transcriptional regulatory region of a CDC45L or PIF1 gene is the region from a start codon to at least 500bp upstream, for example, 1000bp, for example, 5000 or 10000bp upstream. A nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., Chapter 17, 2001, Cold Springs Harbor Laboratory Press).
Furthermore, in the screening method of the present invention, agents or compounds that inhibit a biological activity of CDC45L or PIF protein can be also identified as candidate therapeutics for cancer.
Various low-throughput and high-throughput enzyme assay formats are known in the art and can be readily adapted for detection or measuring of a biological activity of the CDC45L or PIF1 polypeptide. For high-throughput assays, a substrate can conveniently be immobilized on a solid support. Following the reaction, the substrate converted by the polypeptide can be detected on the solid support by the methods described above. Alternatively, the contact step can be performed in solution, after which a substrate can be immobilized on a solid support, and the substrate converted by the polypeptide can be detected. To facilitate such assays, the solid support can be coated with streptavidin and the substrate labeled with biotin, or the solid support can be coated with antibodies against the substrate. The skilled person can determine suitable assay formats depending on the desired throughput capacity of the screen.
The assays of the invention are also suitable for automated procedures which facilitate high-throughput screening. A number of well-known robotic systems have been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, Ltd. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).
In the present invention, it is revealed that suppression of the expression level and/or biological activity of CDC45L or PIF1 lead to suppression of the growth of cancer cells. Therefore, when a compound or agent suppresses the expression and/or activity of CDC45L or PIF1, the suppression is indicative of a potential therapeutic effect in a subject. In the present invention, a potential therapeutic effect refers to a clinical benefit with a reasonable expectation. In the present invention, such clinical benefit includes;
(a) reduction in expression of the CDC45L or PIF1 gene,
(b) decrease in size, prevalence, or metastatic potential of the cancer in the subject, .
(c) preventing cancers from forming, or
(d) preventing or alleviating a clinical symptom of cancer.
(ii) Screening for Compounds that Bind to CDC45L or PIF1 protein(s)
In present invention, over-expression of CDC45L or PIF1 was detected in cancer, but not in normal tissues. Further, polypeptides encoded by these genes were demonstrated to be involved in cancer cell growth. Therefore, those genes can be good molecular targets for cancer therapy and diagnosis. Agents or compounds that bind to CDC45L or PIF1 polypeptide may inhibit biological activities of these polypeptides. Such compounds are used as pharmaceuticals for treating or preventing lung cancer or detecting agents for diagnosing lung cancer and assessing a prognosis of lung cancer patient.
Specifically, the present invention provides the method of screening for an agent or compound useful in diagnosing, treating or preventing cancers using the CDC45L or PIF1 polypeptide. An embodiment of this screening method comprises the steps of:
(a) contacting a test agent or compound with a polypeptide selected from the group consisting of CDC45L and PIF1 protein, or fragment thereof;
(b) detecting the binding level between the polypeptide or the fragment and the test agent or compound;
(c) selecting the test agent or compound that binds to the polypeptide or the fragment of step (a).
According to the present invention, the therapeutic effect of the test agent or compound on inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L or PIF1 associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate agent or compound that suppresses the proliferation of cancer cells, and a method for screening a candidate agent or compound for treating or preventing cancer.
More specifically, the method includes the steps of:
(a) contacting a test agent or compound with a polypeptide selected from the group consisting of CDC45L and PIF1 protein, or fragment thereof;
(b) detecting the binding level between the polypeptide and said test agent or compound;
(c) correlating the binding level of b) with the therapeutic effect of the test agent or compound.
Alternatively, according to the present invention, the potential therapeutic effect of a test agent or compound on treating or preventing cancer can also be evaluated or estimated. In some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test substance on treating or preventing cancer or inhibiting cancer associated with over-expression of CDC45L and/or PIF1, the method including steps of:
(a) contacting an agent or compound with a polypeptide encoded by a polynucleotide of CDC45L or PIF1;
(b) detecting the binding activity between the polypeptide and the test agent or compound; and
(c) correlating the potential therapeutic effect and the test agent or compound, wherein the potential therapeutic effect is shown, when the agent or compound binds to the polypeptide.
In the present invention, the therapeutic effect may be correlated with the binding level of the CDC45L or PIF1 protein. For example, when the test agent or compound binds to CDC45L or PIF1 protein, the test agent or compound may be identified or selected as the candidate agent or compound having the therapeutic effect. Alternatively, when the test agent or compound does not bind to CDC45L or PIF1 protein, the test agent or compound may be identified as the agent or compound having no significant therapeutic effect.
The method of the present invention will be described in more detail below.
The CDC45L or PIF1 polypeptide to be used for screening can be a recombinant polypeptide or a protein derived from the nature or a partial peptide thereof. The polypeptide to be contacted with a test compound can be, for example, a purified polypeptide, a soluble protein, a form bound to a carrier or a fusion protein fused with other polypeptides.
As a method of screening for proteins, for example, that bind to CDC45L or PIF1 polypeptide using CDC45L or PIF1 polypeptide, many methods well known by a person skilled in the art can be used. Such a screening can be conducted by, for example, immunoprecipitation method. The gene encoding CDC45L or PIF1 polypeptide is expressed in host (e.g., animal) cells and so on by inserting the gene to an expression vector for foreign genes, for example, pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8.
The promoter to be used for the expression can be any promoter that can be used commonly and include, for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic Engineering, vol. 3. Academic Press, London, 83-141 (1982)), the EF- alpha promoter (Kim et al., Gene 91: 217-23 (1990)), the CAG promoter (Niwa et al., Gene 108: 193 (1991)), the RSV LTR promoter (Cullen, Methods in Enzymology 152: 684-704 (1987)) the SR alpha promoter (Takebe et al., Mol Cell Biol 8: 466 (1988)), the CMV immediate early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365-9 (1987)), the SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982)), the Adenovirus late promoter (Kaufman et al., Mol Cell Biol 9: 946 (1989)), the HSV TK promoter and so on.
The introduction of the gene into host cells to express a foreign gene can be performed according to any methods, for example, the electroporation method (Chu et al., Nucleic Acids Res 15: 1311-26 (1987)), the calcium phosphate method (Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), the DEAE dextran method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and Milman, Mol Cell Biol 4: 1641-3 (1984)), the Lipofectin method (Derijard B., Cell 76: 1025-37 (1994); Lamb et al., Nature Genetics 5: 22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)) and so on.
The polypeptide encoded by CDC45L or PIF1 gene can be expressed as a fusion protein comprising a recognition site (epitope) of a monoclonal antibody by introducing the epitope of the monoclonal antibody, whose specificity has been revealed, to the N- or C- terminus of the polypeptide. A commercially available epitope-antibody system can be used (Experimental Medicine 13: 85-90 (1995)). Vectors which can express a fusion protein with, for example, beta-galactosidase, maltose binding protein, glutathione S-transferase, green florescence protein (GFP) and so on by the use of its multiple cloning sites are commercially available. Also, a fusion protein prepared by introducing only small epitopes consisting of several to a dozen amino acids so as not to change the property of the CDC45L or PIF1 polypeptide by the fusion is also reported. Epitopes, for example, polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (an epitope on monoclonal phage) and such, and monoclonal antibodies recognizing them can be used as the epitope-antibody system for screening proteins binding to the CDC45L or PIF1 polypeptide (Experimental Medicine 13: 85-90 (1995)).
In immunoprecipitation, an immune complex is formed by adding these antibodies to cell lysate prepared using an appropriate detergent. The immune complex consists of the CDC45L or PIF1 polypeptide, a polypeptide comprising the binding ability with the polypeptide, and an antibody. Immunoprecipitation can be also conducted using antibodies against the CDC45L or PIF1 polypeptide, besides using antibodies against the above epitopes, which antibodies can be prepared as described above. An immune complex can be precipitated, for example by Protein A sepharose or Protein G sepharose when the antibody is a mouse IgG antibody. If the polypeptide encoded by CDC45L or PIF1 gene is prepared as a fusion protein with an epitope, for example, GST, an immune complex can be formed in the same manner as in the use of the antibody against the CDC45L or PIF1 polypeptide, using a substance specifically binding to these epitopes, for example, glutathione-Sepharose 4B.
Immunoprecipitation can be performed by following or according to, for example, the methods in the literature (Harlow and Lane, Antibodies, 511-52, Cold Spring Harbor Laboratory publications, New York (1988)).
SDS-PAGE is commonly used for analysis of immunoprecipitated proteins and the bound protein can be analyzed by the molecular weight of the protein using gels with an appropriate concentration. Since the protein bound to CDC45L or PIF1 polypeptide is difficult to detect by a common staining method, for example, Coomassie staining or silver staining, the detection sensitivity for the protein can be improved by culturing cells in culture medium containing radioactive isotope, 35S-methionine or 35S-cysteine, labeling proteins in the cells, and detecting the proteins. The target protein can be purified directly from the SDS-polyacrylamide gel and its sequence can be determined, when the molecular weight of a protein has been revealed.
As a method of screening for proteins binding to CDC45L or PIF1 polypeptide using the polypeptide, for example, West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)) can be used. Specifically, a protein binding to the CDC45L or PIF1 polypeptide can be obtained by preparing a cDNA library from cultured cells (e.g., lung cancer cell line ) expected to express a protein binding to the CDC45L or PIF1 polypeptide using a phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing the protein expressed on a filter, reacting the purified and labeled CDC45L or PIF1 polypeptide with the above filter, and detecting the plaques expressing proteins bound to CDC45L or PIF1 polypeptide according to the label. The polypeptide of the invention can be labeled by utilizing the binding between biotin and avidin, or by utilizing an antibody that specifically binds to CDC45L or PIF1 polypeptide, or a peptide or polypeptide (for example, GST) that is fused to CDC45L OR PIF1 polypeptide. Methods using radioisotope or fluorescence and such can be also used.
The terms "label" and "detectable label" are used herein to refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 125I, .35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels for example colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,275,149; and 4,366,241. Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting, the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.
Alternatively, in another embodiment of the screening method of the present invention, a two-hybrid system utilizing cells can be used ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); the references "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)").
In the two-hybrid system, the polypeptide of the invention is fused to the SRF-binding region or GAL4-binding region and expressed in yeast cells. A cDNA library is prepared from cells expected to express a protein binding to the polypeptide of the invention, such that the library, when expressed, is fused to the VP16 or GAL4 transcriptional activation region. The cDNA library is then introduced into the above yeast cells and the cDNA derived from the library is isolated from the positive clones detected (when a protein binding to the polypeptide of the invention is expressed in yeast cells, the binding of the two activates a reporter gene, making positive clones detectable). A protein encoded by the cDNA can be prepared by introducing the cDNA isolated above to E. coli and expressing the protein. As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene, luciferase gene and such can be used in addition to the HIS3 gene.
A compound binding to the polypeptide encoded by CDC45L or PIF1 gene can also be screened using affinity chromatography. For example, the polypeptide of the invention can be immobilized on a carrier of an affinity column, and a test compound, containing a protein capable of binding to the polypeptide of the invention, is applied to the column. A test compound herein can be, for example, cell extracts, cell lysates, etc. After loading the test compound, the column is washed, and compounds bound to the polypeptide of the invention can be prepared. When the test compound is a protein, the amino acid sequence of the obtained protein is analyzed, an oligo DNA is synthesized based on the sequence, and cDNA libraries are screened using the oligo DNA as a probe to obtain a DNA encoding the protein.
A biosensor using the surface plasmon resonance phenomenon can be used as a mean for detecting or quantifying the bound compound in the present invention. When such a biosensor is used, the interaction between the polypeptide of the invention and a test compound can be observed real-time as a surface plasmon resonance signal, using only a minute amount of polypeptide and without labeling (for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate the binding between the polypeptide of the invention and a test compound using a biosensor for example, BIAcore.
The methods of screening for molecules that bind when the immobilized CDC45L or PIF1 polypeptide is exposed to synthetic chemical compounds, or natural substance banks or a random phage peptide display library, and the methods of screening using high-throughput based on combinatorial chemistry techniques (Wrighton et al., Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature 384: 17-9 (1996)) to isolate not only proteins but chemical compounds that bind to the CDC45L or PIF1 protein (including agonist and antagonist) are well known to one skilled in the art.
In the present invention, it is revealed that suppressing the expression level of CDC45L or PIF1, reduces cell growth. Thus, by screening for candidate compounds that bind to CDC45L or PIF1, candidate compounds that have the potential to treat or prevent cancers can be identified. Potential of these candidate compounds to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a compound binding to CDC45L or PIF1 protein inhibits activities of cancer, it may be concluded that such compound has CDC45L or PIF1 specific therapeutic effect.
(iii) Screening for Compound that Suppress the Biological Activity of CDC45L or PIF1 Protein
The present invention also provides a method for screening a candidate agents or compounds for treating or preventing cancer using a biological activity of the CDC45L or PIF1 polypeptide, or fragment thereof as an index.
Specifically, the present invention provides the following methods of [1] to [4]:
[1] A method of screening for an agent or compound useful in treating or preventing cancers expressing CDC45L and/or PIF1, said method comprising the steps of:
(a) contacting a test agent or compound with a CDC45L or PIF1 polynucleotide, or functional equivalent or fragment thereof;
(b) detecting a biological activity of the polypeptide of step (a);
(c) comparing the level detected in the step (b) with those detected in the absence of the test agent or compound;
(d) selecting the test agent or compound that reduce or inhibit the biological activity of the polypeptide.
[2] The method of [1], wherein the biological activity is a cell proliferation promoting activity;
[3] The method of [1], wherein the biological activity of CDC45L polypeptide is DNA replication activity or binding activity to PIF1 polypeptide; and
[4] The method of [1], wherein the biological activity of PIF1 polypeptide is helicase activity, telomerase inhibition activity or binding activity to CDC45L polypeptide.
According to the present invention, the therapeutic effect of the test agent or compound on inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease e.g., lung cancer, may be evaluated. Therefore, the present invention also provides a method of screening for a candidate agent or compound for inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease, e.g., lung cancer, using the CDC45L or PIF1 polypeptide or fragments thereof including the steps as follows:
a) contacting a test agent or compound with the CDC45L or PIF1 polypeptide or a functional fragment thereof; and
b) detecting the biological activity of the polypeptide or fragment of step (a), and
c) correlating the biological activity of b) with the therapeutic effect of the test agent or compound.
Alternatively, in some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test agent or compound on treating or preventing cancer or inhibiting cancer associated with over-expression of CDC45L and/or PIF1, the method including steps of:
(a) contacting a test agent or compound with a polypeptide encoded by a polynucleotide of CDC45L or PIF1 gene;
(b) detecting the biological activity of the polypeptide of step (a); and
(c) correlating the potential therapeutic effect and the test agent or compound, wherein the potential therapeutic effect is shown, when the test agent or compound suppresses the biological activity of the polypeptide encoded by the polynucleotide of CDC45L or PIF1 gene as compared to the biological activity of said polypeptide detected in the absence of the test agent or compound.
In the present invention, the therapeutic effect may be correlated with the biological activity of CDC45L or PIF1 polypeptide or a functional fragment thereof. For example, when the test agent or compound suppresses or inhibits the biological activity of CDC45L or PIF1 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified or selected as the candidate agent or compound having the therapeutic effect. Alternatively, when the test agent or compound does not suppress or inhibit the biological activity of CDC45L or PIF1 polypeptide or a functional fragment thereof as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified as the agent or compound having no significant therapeutic effect.
The method of the present invention will be described in more detail below.
Any polypeptides can be used for screening so long as they comprise the biological activity of the CDC45L or PIF1 protein. Such biological activity includes the cell proliferating activity (cell proliferation promoting activity) or the binding activity to CDC45L or PIF1 each other. For CDC45L protein, the biological activity includes DNA replication activity. For PIF1 protein, the biological activity includes helicase activity and telomerase inhibition activity. For example, CDC45L or PIF1 protein can be used and polypeptides functionally equivalent to these proteins can also be used. Such polypeptides can be expressed endogenously or exogenously by cells.
The compound isolated by this screening is a candidate for antagonists of the polypeptide encoded by CDC45L or PIF1 gene. The term "antagonist" refers to molecules that inhibit the function of the polypeptide by binding thereto. Said term also refers to molecules that reduce or inhibit expression of the gene encoding CDC45L or PIF1. Moreover, a compound isolated by this screening is a candidate for compounds which inhibit the in vivo interaction of the CDC45L or PIF1 polypeptide with molecules (including DNAs and proteins).
In the present invention, the CDC45L and PIF1 protein has the activity of promoting cell proliferation of cancer cells (Fig. 3 and Fig.5). Therefore, in the screening method of the present invention, using this biological activity, a compound which inhibits a biological activity of these protein can be screened. Such compounds would be potential candidates for treating cancer, e.g., lung cancer.
When the biological activity to be detected in the present method is cell proliferation promoting activity, it can be detected, for example, by preparing cells which express the polypeptide selected from the group consisting of CDC45L and PIF1, culturing the cells in the presence of a test compound, and determining the speed of cell proliferation, measuring the cell cycle and such, as well as by measuring the colony formation activity, e.g. MTT assay, colony formation assay or FACS shown in "EXAMPLES".
The term of "suppress the biological activity" as defined herein refers to at least 10% suppression of the biological activity of CDC45L or PIF1 in comparison with in absence of the compound, for example, at least 25%, 50% or 75% suppression, for example, at least 90% suppression.
In the preferred embodiments, control cells which do not express CDC45L or PIF1 polypeptide are used. Accordingly, the present invention also provides a method of screening for a candidate substance for inhibiting the cell growth or a candidate substance for treating or preventing CDC45L or PIF1 associating disease,e.g., lung cancer, using the CDC45L or PIF1 polypeptide or fragments thereof including the steps as follows:
a) culturing cells which express a CDC45L or PIF1 polypeptide or a functional fragment thereof, and control cells that do not express a CDC45L or PIF1 polypeptide or a functional fragment thereof in the presence of a test agent or compound;
b) detecting the biological activity of the cells which express the protein and control cells; and
c) selecting the test compound that inhibits the biological activity in the cells which express the protein as compared to the proliferation detected in the control cells and in the absence of the test agent or compound.
In addition to the cell proliferation promoting activity, when CDC45L polypeptide or fragment thereof is used in the screening, DNA replication activity or binding activity to PIF1 polypeptide may be used as an index. Also, when PIF1 polypeptide or fragment thereof is used in the screening, helicase activity, telomerase inhibition activity or binding activity to CDC45L polypeptide may be used as an index. These biological activities can be detected by methods well-known in the art.
In the present invention, it is revealed that suppressing the biological activity of CDC45L or PIF1, reduces cell growth. Thus, by screening for candidate compounds that inhibits biological activity of CDC45L or PIF1, candidate compounds that have the potential to treat or prevent cancers can be identified. Potential of these candidate compounds to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a compound inhibits the biological activity of CDC45L or PIF1 protein inhibits activities of cancer, it may be concluded that such compound has CDC45L or PIF1 specific therapeutic effect.
(iv) Screening for Compounds that Alter the Expression of CDC45L or PIF1
In the present invention, the decrease of the expression of CDC45L or PIF1 by a double-stranded molecule specific for CDC45L or PIF1 caused inhibition of cancer cell proliferation (Fig. 3 and Fig.5). Therefore, compounds that can be used in the treatment or prevention of cancer can be identified through screenings that use the expression levels of CDC45L or PIF1 as indices. In the context of the present invention, such screening can comprise, for example, the following steps:
(a) contacting a test compound with a cell expressing CDC45L and/or PIF1 gene ;
(b) detecting the expression level of the CDC45L and/or PIF1 gene; and
(c) selecting the test compound that reduces the expression level of CDC45L and/or PIF1 gene as compared to that detected in the absence of the test compound.
According to the present invention, the therapeutic effect of the test agent or compound on inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease e.g., lung cancer, may be evaluated. Therefore, the present invention also provides a method for screening a candidate agent or compound that suppresses the proliferation of cancer cells, and a method for screening a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease e.g., lung cancer,.
In the context of the present invention, such screening may include, for example, the following steps:
a) contacting a test agent or compound with a cell expressing the CDC45L and/or PIF1 gene;
b) detecting the expression level of the CDC45L and/or PIF1 gene; and
c) correlating the expression level of b) with the therapeutic effect of the test agent or compound.
Alternatively, according to the present invention, the potential therapeutic effect of a test agent or compound on treating or preventing cancer can also be evaluated or estimated. In some embodiments, the present invention provides a method for evaluating or estimating a therapeutic effect of a test agent or compound on treating or preventing cancer or inhibiting cancer associated with over-expression of CDC45L and/or PIF1, the method including steps of:
(a) contacting a test agent or compound with a cell expressing the CDC45L and/or PIF1 gene;
(b) detecting the expression level of the CDC45L and/or PIF1 gene of step (a); and
(c) correlating the potential therapeutic effect and the test agent or compound, wherein the potential therapeutic effect is shown, when the test agent or compound reduces the expression level of the CDC45L and/or PIF1 gene.
In the present invention, the therapeutic effect may be correlated with the expression level of the CDC45L or PIF1 gene. For example, when the test agent or compound reduces the expression level of the CDC45L or PIF1 gene as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified or selected as the candidate agent or compound having the therapeutic effect. Alternatively, when the test agent or compound does not reduce the expression level of the CDC45L or PIF1 gene as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified as the agent or compound having no significant therapeutic effect.
The method of the present invention will be described in more detail below.
Cells expressing the CDC45L and/or PIF1 include, for example, cell lines established from lung cancer; such cells can be used for the above screening of the present invention (e.g., A549 and SBC-3). The expression level can be estimated by methods well known to one skilled in the art, for example, RT-PCR, Northern blot assay, Western blot assay, immunostaining, ELISA or flow cytometry analysis. The term of "reduce the expression level" as defined herein refers to at least 10% reduction of expression level of CDC45L or PIF1 in comparison to the expression level in absence of the compound, for example, at least 25%, 50% or 75% reduced level, for example, at least 95% reduced level. The compound herein includes chemical compound, double-strand nucleotide, and so on. The preparation of the double-strand nucleotide is in aforementioned description. In the method of screening, a compound that reduces the expression level of CDC45L or PIF1 can be selected as candidate agents or compounds to be used for the treatment or prevention of cancers, e.g. lung cancer.
Alternatively, the screening method of the present invention can comprise the following steps:
(a) contacting a candidate compound with a cell into which a vector, comprising the transcriptional regulatory region of CDC45L or PIF1 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression level or activity of said reporter gene; and
(c) selecting the candidate compound that reduces the expression level or activity of said reporter gene.
According to the present invention, the therapeutic effect of the test agent or compound on inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate agent or compound that suppresses the proliferation of cancer cells, and a method for screening a candidate agent or compound for treating or preventing CDC45L and/or PIF1 associating disease.
According to another aspect, the present invention provides a method which includes the following steps of:
a) contacting a test agent or compound with a cell into which a vector, composed of the transcriptional regulatory region of the CDC45L or PIF1 gene and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
b) detecting the expression level or activity of said reporter gene; and
c) correlating the expression level of b) with the therapeutic effect of the test agent or compound.
Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test agent or compound on treating or preventing cancer or inhibiting cancer associated with over-expression of CDC45L or PIF1, the method including steps of:
(a) contacting a test agent or compound with a cell into which a vector, including the transcriptional regulatory region of CDC45L or PIF1 and a reporter gene that is expressed under the control of the transcriptional regulatory region, has been introduced;
(b) measuring the expression level or activity of said reporter gene; and
(c) correlating the potential therapeutic effect and the test agent or compound, wherein the potential therapeutic effect is shown, when a test agent or compound reduces the expression level or activity of said reporter gene.
In the present invention, the therapeutic effect may be correlated with the expression level or activity of said reporter gene. For example, when the test agent or compound reduces the expression level or activity of said reporter gene as compared to a level detected in the absence of the test agent or compound, the test agent or compound may be identified or selected as the candidate agent or compound having the therapeutic effect. Alternatively, when the test agent or compound does not reduce the expression level or activity of said reporter gene as compared to a level detected in the absence of the test agent or compound, the test agent or compound may be identified as the agent or compound having no significant therapeutic effect.
Suitable reporter genes and host cells are well known in the art. For example, reporter genes are luciferase, green florescence protein (GFP), Discosoma sp. Red Fluorescent Protein (DsRed), Chrolamphenicol Acetyltransferase (CAT), lacZ and beta-glucuronidase (GUS), and host cell is COS7, HEK293, HeLa and so on. The reporter construct required for the screening can be prepared by connecting reporter gene sequence to the transcriptional regulatory region of CX. The transcriptional regulatory region of CX herein is the region from start codon to at least 500bp upstream, for example, 1000bp, for example, 5000 or 10000bp upstream, but not restricted. A nucleotide segment containing the transcriptional regulatory region can be isolated from a genome library or can be propagated by PCR. Methods for identifying a transcriptional regulatory region, and also assay protocol are well known (Molecular Cloning third edition chapter 17, 2001, Cold Springs Harbor Laboratory Press).
The vector containing the said reporter construct is infected to host cells and the expression or activity of the reporter gene is detected by method well known in the art (e.g., using luminometer, absorption spectrometer, flow cytometer and so on). "Reduces the expression or activity" as defined herein refers to at least 10% reduction of the expression or activity of the reporter gene in comparison with in absence of the compound, for example, at least 25%, 50% or 75% reduction, for example, at least 95% reduction.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
In the present invention, it is revealed that suppressing the expression level of CDC45L or PIF1, reduces cell growth. Thus, by screening for candidate compounds that inhibits expression level of CDC45L or PIF1, candidate compounds that have the potential to treat or prevent cancers can be identified. Potential of these candidate compounds to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a compound inhibits the expression level of CDC45L or PIF1 protein inhibits activities of cancer, it may be concluded that such compound has CDC45L or PIF1 specific therapeutic effect.
(v) Screening using the binding of CDC45L and PIF1 as an index
In the present invention, it was confirmed that the CDC45L protein interacts with PIF11 protein (Fig. 4B). Thus, a compound that inhibits the binding between CDC45L protein and PIF1 protein can be screened using such a binding of CDC45L protein and PIF1 protein as an index. Such compounds may have potential therapeutic effect on cancer treatment as those protein are involved in cancer cell growth. Therefore, the present invention provides a method for screening a compound for inhibiting the binding between CDC45L protein and PIF1 protein using such a binding of CDC45L protein and PIF1 protein as an index. Furthermore, the present invention also provides a method for screening a candidate compound for inhibiting or reducing a growth of cancer cells expressing CDC45L and PIF1 gene, e.g. lung cancer cell, and a candidate compound for treating or preventing cancers, e.g. lung cancer.
Specifically, the present invention provides the following methods of [1] to [5]:
[1] A method of screening for an agent or compound that interrupts a binding between a CDC45L polypeptide and a PIF1 polypeptide, said method comprising the steps of:
(a) contacting a CDC45L polypeptide or functional equivalent thereof with a PIF1 polypeptide or functional equivalent thereof in the presence of a test agent or compound;
(b) detecting a binding between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test agent or compound; and
(d) selecting the test agent or compound that reduce or inhibits the binding level.
[2] A method of screening for a candidate agent or compound useful in treating or preventing cancer, said method comprising the steps of:
(a) contacting a CDC45L polypeptide or functional equivalent thereof with a PIF1 polypeptide or functional equivalent thereof in the presence of a test agent or compound;
(b) detecting a binding between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test agent or compound; and
(d) selecting the test agent or compound that reduce or inhibits the binding level.
[3] The method of [1] or [2], wherein the functional equivalent of CDC45L comprising the PIF1-binding domain.
[4] The method of [1] or [2], wherein the functional equivalent of PIF1 comprising the CDC45L-binding domain.
[5] The method of [1], wherein the cancer is lung cancer.
According to the present invention, the therapeutic effect of the test agent or compound on inhibiting the cell growth or a candidate agent or compound for treating or preventing CDC45L or PIF1 associating disease may be evaluated. Therefore, the present invention also provides a method for screening a candidate agent or compound that suppresses the proliferation of cancer cells, and a method for screening a candidate agent or compound for treating or preventing cancer.
More specifically, the method includes the steps of:
(a) contacting a CDC45L polypeptide or functional equivalent thereof with a PIF1 polypeptide or functional equivalent thereof in the presence of a test agent or compound;
(b) detecting the level of binding between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test agent or compound; and
(d) correlating the binding level of c) with the therapeutic effect of the test agent or compound.
Alternatively, in some embodiments, the present invention also provides a method for evaluating or estimating a therapeutic effect of a test agent or compound on treating or preventing cancer or inhibiting cancer, the method including steps of:
(a) contacting a CDC45L polypeptide or functional equivalent thereof with a PIF1 polypeptide or functional equivalent thereof in the presence of a test agent or compound;
(b) detecting a binding level between the polypeptides;
(c) comparing the binding level detected in the step (b) with those detected in the absence of the test agent or compound; and
(d) correlating the potential therapeutic effect and the test agent or compound, wherein the potential therapeutic effect is shown, when a test agent or compound reduce the binding level.
In the present invention, the therapeutic effect may be correlated with the binding level of the CDC45L and PIF1 proteins. For example, when the test agent or compound reduces the binding level of CDC45L and PIF1 proteins as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified or selected as the candidate agent or compound having the therapeutic effect. Alternatively, when the test agent or compound does not reduce the binding level of CDC45L and PIF1 proteins as compared to a level detected in the absence of the test agent or compound, the test agent or compound may identified as the agent or compound having no significant therapeutic effect.
In the context of the present invention, a functional equivalent of an CDC45L or PIF1 polypeptide is a polypeptide that has a biological activity equivalent to a CDC45L polypeptide (SEQ ID NO: 14) or PIF1 polypeptide (SEQ ID NO: 16), respectively (see, (1) Genes and Polypeptides). More specifically, the functional equivalent of PIF1 polypeptide is a fragment of polypeptide having an amino acid sequence of SEQ ID NO: 16 comprising the CDC45L-binding domain. Also, the functional equivalent of CDC45L polypeptide is a fragment of polypeptide having an amino acid sequence of SEQ ID NO: 14 comprising the PIF1-binding domain.
As a method of screening for compounds that inhibits the binding of CDC45L to PIF1, many methods well known by one skilled in the art can be used. Such a screening can be conducted using, for example, an immunoprecipitation, West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991)), a two-hybrid system utilizing cells ("MATCHMAKER Two-Hybrid system", "Mammalian MATCHMAKER Two-Hybrid Assay Kit", "MATCHMAKER one-Hybrid system" (Clontech); "HybriZAP Two-Hybrid Vector System" (Stratagene); the references "Dalton and Treisman, Cell 68: 597-612 (1992)", "Fields and Sternglanz, Trends Genet 10: 286-92 (1994)"), affinity chromatography and A biosensor using the surface plasmon resonance phenomenon (see (i) General screening Method and (ii) Screening for Compounds that Bind to CDC45L or PIF1 protein(s)).
A polypeptide to be used for screening can be a recombinant polypeptide or a protein derived from natural sources, or a partial peptide thereof. Any test compound aforementioned can be used for screening.
Any aforementioned test compound can be used (see (1) Test compounds for screening).
In some embodiments, this method further comprises the step of detecting the binding of the candidate compound to CDC45L protein or PIF1 protein, or detecting the level of binding CDC45L protein to or PIF1 protein. Cells expressing CDC45L protein and/or PIF1 protein include, for example, cell lines established from cancer, e.g. lung cancer, such cells can be used for the above screening of the present invention so long as the cells express these two genes. Alternatively cells can be transfected both or either of expression vectors of CDC45L and PIF1 protein, so as to express these two genes. The binding of CDC45L protein to PIF1 protein can be detected by immunoprecipitation assay using an anti-CDC45L antibody and PIF1 antibody (Fig. 4).
In the present invention, it is revealed that suppressing the binding between CDC45L protein and PIF1 protein, reduces cell growth. Thus, by screening for candidate compounds that inhibits the binding between CDC45L protein and PIF1 protein, candidate compounds that have the potential to treat or prevent cancers can be identified. Potential of these candidate compounds to treat or prevent cancers may be evaluated by second and/or further screening to identify therapeutic agent for cancers. For example, when a compound inhibits the binding between CDC45L protein and PIF1 protein inhibits activities of cancer, it may be concluded that such compound has CDC45L or PIF1 specific therapeutic effect.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.
Materials and Methods
Cancer cell lines and tissue samples. The human lung-cancer cell lines used in this study were as follows: lung adenocarcinomas (ADCs) NCI-H1781, NCI-H1373, LC319, A549, and PC14; lung squamous-cell carcinomas (SCCs) SK-MES-1, NCI-H2170, NCI-H520, NCI-H1703, and LU61; a lung large-cell carcinoma (LCC) LX1; and small-cell lung cancers (SCLCs) SBC-3, SBC-5, DMS273, and DMS114. All cells were grown in monolayer in appropriate media supplemented with 10% fetal calf serum (FCS) and were maintained at 37 degrees C in humidified air with 5% CO2. Human small airway epithelial cells (SAEC) used as normal control were grown in optimized medium (SAGM) from Cambrex Bio Science Inc. Primary lung cancer samples had been obtained earlier with informed consent as described elsewhere (NPL 8, 12). Clinical stage was judged according to the UICC TNM classification (Sobin L WC. TNM Classification of Malignant Tumours, 6th edition. Anonymous. New York: Wiley-Liss. 2002). Formalin-fixed primary lung tumors and adjacent normal lung tissue samples used for immunostaining on tissue microarrays had been obtained from 267 patients (159 ADCs, 89 SCCs, 16 LCCs, 3 ASCs; 90 female and 177 male patients; median age of 65.0 with a range of 26 - 84 years, 114 pT1, 125 pT2, 28 pT3 tumor size ; 208 pN0, 23 pN1, 36 pN2 node status) undergoing curative surgery at Hokkaido University and its affiliated Hospitals (Sapporo, Japan). This study and the use of all clinical materials mentioned were approved by individual institutional Ethical Committees.
Semi-quantitative RT-PCR. A total of 3 microgram aliquot of mRNA from each sample was reversely transcribed to single-stranded cDNAs using random primer (Roche Diagnostics) and Superscript II (Invitrogen). Semi-quantitative RT-PCR experiments were carried out with the following sets of synthesized primers specific to CDC45L, PIF1 or with beta-actin (ACTB)-specific primers as an internal control: CDC45 cell division cycle 45-like (CDC45L), 5'-CACAACCATTTTGACCTCTCAGT-3' (SEQ ID NO: 1) and 5'-GCTTCTACATCTCAAATCATGTCC-3' (SEQ ID NO: 2), PIF1 5'-to-3' DNA helicase homolog (S. cerevisiae) (PIF1), 5'-AGGCAGTGTCCCCTTCTGTA-3' (SEQ ID NO: 3) and 5'-CCTGAAAGGAGGGATGTTCA-3' (SEQ ID NO: 4), ACTB, 5'-GAGGTGATAGCATTGCTTTCG-3' (SEQ ID NO: 5) and 5'-CAAGTCAGTGTACAGGTAAGC-3' (SEQ ID NO: 6). PCR reactions were optimized for the number of cycles to ensure product intensity to be within the linear phase of amplification.
Western-blotting. Cells were lysed in lysis buffer; 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.5% NP40, 0.5% sodium deoxycholate, and Protease Inhibitor Cocktail Set III (Calbiochem). The protein content of each lysate was determined by a Bio-Rad protein assay (Bio-Rad) with bovine serum albumin (BSA) as a standard. Ten micrograms of each lysate were resolved on 10 - 12% denaturing polyacrylamide gels (with 3% polyacrylamide stacking gel) and transferred electrophoretically to a nitrocellulose membrane (GE Healthcare Bio-sciences). After blocking with 5% non-fat dry milk in TBST, the membrane was incubated with primary antibodies for 1 hour at room temperature. Immunoreactive proteins were incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-sciences) for 1 hour at room temperature. After washing with TBST, the reactants were developed using the enhanced chemiluminescence kit (GE Healthcare Bio-sciences). A commercially available rabbit polyclonal anti-CDC45L antibody was purchased from ATLAS Antibodies AB (Catalog No. HPA000614) and was proved to be specific to human CDC45L by western-blot analysis using lysates of lung cancer cell lines.
Immunohistochemistry and Tissue microarray. To investigate the CDC45L protein in clinical samples that had been embedded in paraffin blocks, the sections using ENVISION+ Kit/HRP (pH9) (DakoCytomation) were stained in the following manner. Briefly, slides were immersed in Target Retrieval Solution and boiled at 108 degrees C for 15 minutes in an autoclave for antigen retrieval. 3 microgram/ml of a rabbit polyclonal anti-human CDC45L antibody (ATLAS Antibodies AB; Catalog No. HPA000614) was added to each slide after blocking of endogenous peroxidase and proteins, and the sections were incubated with horseradish peroxidase-labeled anti-rabbit IgG (Histofine Simple Stain MAX PO (G), Nichirei) as the secondary antibody. Substrate-chromogen was added, and the specimens were counterstained with hematoxylin.
Tumor tissue microarrays were constructed with 267 formalin-fixed primary NSCLCs as described elsewhere (Chin SF, et al. Molecular Pathology 2003;56:275-9., Callagy G, et al. Diagn Mol Pathol 2003;12:27-34., Callagy G, et al. J Pathol 2005;205:388-96.). The tissue area for sampling was selected based on visual alignment with the corresponding H&E-stained section on a slide. Three, four, or five tissue cores (diameter, 0.6 mm; depth, 3 - 4 mm) taken from a donor tumor block were placed into a recipient paraffin block with a tissue microarrayer (Beecher Instruments). A core of normal tissue was punched from each case, and 5-micrometer sections of the resulting microarray block were used for immunohistochemical analysis. Three independent investigators semi-quantitatively assessed CDC45L positivity without prior knowledge of clinicopathological data. Since the intensity of staining within each tumor tissue core was mostly homogenous, the intensity of CDC45L staining in the nucleus and cytoplasm was evaluated by recording as negative (no appreciable staining in tumor cells) or positive (brown staining appreciable in the nucleus and cytoplasm of tumor cells). Cases were accepted as positive only if all three reviewers independently defined them as such.
Statistical analysis. Statistical analyses were performed using the StatView statistical program (SaS). Tumor-specific survival curves were calculated from the date of surgery to the time of death related to NSCLC, or to the last follow-up observation. Kaplan-Meier curves were calculated for each relevant variable and for CDC45L expression; differences in survival times among patient subgroups were analyzed using the log-rank test. Univariate and multivariate analyses were performed with the Cox proportional-hazard regression model to determine associations between clinicopathological variables and cancer-related mortality. First, associations between death and possible prognostic factors including age, gender, pT-classification, and pN-classification were analyzed taking into consideration one factor at a time. Second, multivariate Cox analysis was applied on backward (stepwise) procedures that always forced strong CDC45L expression into the model, along with any and all variables that satisfied an entry level of a P-value less than 0.05. As the model continued to add factors, independent factors did not exceed an exit level of P < 0.05.
Identification of CDC45L-interacting proteins. Cell extracts were precleared by incubation at 4 degrees C for 1 hour with 100 microliter of protein G-agarose beads, in final volumes of 2 ml of immunoprecipitation buffer (0.5% NP40, 50 mmol/L Tris-Hcl, 150 mmol/L NaCl) in the presence of proteinase inhibitor. After centrifugation at 1,000 rpm for 1 minute at 4 degrees C, the supernatants were incubated at 4 degrees C with a rabbit polyclonal anti-CDC45L antibody or control normal rabbit IgG for 4 hours. In addition, 30 microliter of protein G-agarose beads were added into each supernatant and incubated another 2 hours. After the beads were collected from each sample by centrifugation at 5,000 rpm for 2 minutes and washed six times with 1 ml of immunoprecipitation buffer, they were resuspended in 50 microliter of Laemmli sample buffer and boiled for 5 minutes before the proteins were separated on 5% to 10% SDS-PAGE gels (BioRad). After electrophoresis, the gels were stained with silver. Protein bands found specifically in extracts immunoprecipitated with anti-CDC45L antibody were excised to serve for analysis by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS; AXIMA-CFR plus, SHIMAZU BIOTECH). To confirm the interaction between CDC45L and PIF1, the immunoprecipitation experiment was carried out, as described elsewhere (NPL18, 23).
Immunocytochemical analysis. Cells were plated on glass coverslips (Becton Dickinson Labware), fixed with 4% paraformaldehyde, and permeablilized with 0.1% Triton X-100 in PBS for 3 minutes at room temperature. Non-specific binding was blocked by CASBLOCK (ZYMED) for 10 minutes at room temperature. Cells were then incubated for 60 minutes at room temperature with primary antibodies diluted in PBS containing 3% BSA. After being washed with PBS, the cells were stained by FITC-conjugated secondary antibody (Santa Cruz) for 60 minutes at room temperature. After another wash with PBS, each specimen was mounted with Vectashield (Vector Laboratories, Inc.) containing 4',6'-diamidine-2'-phenylindolendihydrochrolide (DAPI) and visualized with Spectral Confocal Scanning Systems (TSC SP2 AOBS: Leica Microsystems).
RNAi assay. Small interfering RNA (siRNA) duplexes (100 nM) were transfected into lung cancer cell lines, A549 and SBC-3, using 24 microliter of Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. The transfected cells were cultured for 7 days, and the number of colonies was counted by Giemsa staining, and viability of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (cell counting kit-8 solution; Dojindo Laboratories), at 7 days after transfection. To confirm suppression of CDC45L or PIF1 expression, semiquantitative RT-PCR was carried out with synthesized primers specific for CDC45L or PIF1 described above. The target sequences of the synthetic oligonucleotides for RNAi were as follows: control-1 (LUC: luciferase gene from Photinus pyralis), 5'-CGUACGCGGAAUACUUCGA-3' (SEQ ID NO: 7); control-2 (EGFP: enhanced green fluorescent protein (GFP) gene, a mutant of Aequorea victoria GFP), 5'-GAAGCAGCACGACUUCUUC-3' (SEQ ID NO: 8);
si-CDC45L#1, 5'-GCAAACACCUGCUCAAGUC-3' (SEQ ID NO: 9);
si-CDC45L#2, 5'-GGACGUGGAUGCUCUGUGU-3' (SEQ ID NO: 10);
si-PIF1#1, 5'-GAAAGGCCAGAGCAUCUUC-3' (SEQ ID NO: 11);
si-PIF1#2, 5'-GGCAUGACCCUGGAUUGUG-3' (SEQ ID NO: 12).
Flow cytometry. A549 cells were plated at densities of 3.5 X 105 cells/100 mm dish and transfected with siRNA oligonucleotide. Cells were trypsinized 48 hours after transfection, collected in PBS, and fixed in 70% cold ethanol for 30 minutes. After treatment with 100 microgram/mL RNase (Sigma-Aldrich), the cells were stained with 50 microgram/mL propidium iodide (Sigma-Aldrich) in PBS. Flow cytometry was done on a Becton Dickinson FACScan and analyzed by ModFit software (Verity Software House, Inc.). The cells selected from at least 10,000 ungated cells were analyzed for DNA content.
Results
CDC45L expression in lung cancer and normal tissues. To identify novel molecules that can be applicable to detect presence of cancer at an early stage and to develop novel treatments based on the biological characteristics of cancer cells, genome-wide expression profile analysis of clinical lung carcinoma was performed using a cDNA microarray (NPL7-12). Among 27,648 genes or ESTs screened, elevated expression (3-fold or higher) of CDC45L transcript in the great majority of the clinical lung cancer samples examined was identified. It was confirmed its overexpression by means of semi-quantitative RT-PCR experiments in 12 of 15 lung cancer tissues and in all of 15 lung-cancer cell lines (Figs. 1A and 1B).
Subsequently, overexpression of CDC45L protein was confirmed in all of 6 cancer cell lines, but no band was detected in normal bronchial epithelia derived cells (SAEC) by western-blot analysis using anti-CDC45L antibody (Fig. 1C). Subsequently, immunofluorescent analysis was performed to examine the subcellular localization of endogenous CDC45L in lung cancer cell line A549, and its strong staining in nucleus and weak staining in cytoplasm was found (Fig. 1D). Northern blotting using CDC45L cDNA as a probe identified a 2.2-kb band only in testis among 16 normal tissues examined (data not shown). Furthermore, by immunohistochemistry using anti-CDC45L polyclonal antibody, CDC45L protein expression levels in 5 normal tissues (heart, lung, liver, kidney, and testis) were compared with those in lung cancers. CDC45L expressed abundantly in the nucleus and cytoplasm of testis and lung cancer cells, but its expression was hardly detectable in the remaining four normal tissues (Fig. 2A).
Association of CDC45L expression with poor prognosis for NSCLC patients. To investigate the biological and clinicopathological significance of CDC45L in pulmonary carcinogenesis, the present inventors carried out immunohistochemical staining on tissue microarray containing tissue sections from 267 NSCLC patients who had surgical resection. CDC45L staining with polyclonal antibody specific to CDC45L was mainly observed at nucleus and cytoplasm of tumor cells, but not detected in normal lung cells (Fig. 2B). Of the 267 NSCLCs, CDC45L was stained positively in 171 cases (64.0%) and negatively in 96 cases (36.0%) (details are shown in Table 1A). The present inventors then compared the status of CDC45L positivity with clinicopathological factors, and found that the CDC45L expression in NSCLCs was significantly associated with older age (>= 65; P = 0.0417, Fisher's exact test; Table 1A), male gender (P = 0.0008), non-ADC histology (P < 0.0001), and larger tumor size (pT2-3; P = 0.0066). The median survival time of NSCLC patients with CDC45L-positive tumor was significantly shorter than those with CDC45L-negative tumor (P = 0.0045 by log-rank test; Fig. 2C). The present inventors also applied univariate analysis to evaluate associations between patient prognosis and several factors including age (<65 versus >= 65), gender (female versus male), histological type (ADC versus non-ADC), pT stage (tumor size; T1 versus T2+T3), pN stage (node status; N0 versus N1+N2), and CDC45L status (negative versus positive)(Table 1B). All of the parameters were significantly associated with poor prognosis. In multivariate analysis, CDC45L status did not reach the statistically significant level as an independent prognostic factor for surgically treated lung cancer patients enrolled in this study (P = 0.4332), while pT and pN stages as well as the age did so, suggesting the relevance of CDC45L expression to these clinicopathological factors in lung cancer.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Effects of CDC45L on growth of cancer cells. To assess whether CDC45L is essential for growth and/or survival of lung-cancer cells, siRNA oligonucleotides specific to CDC45L sequences were constructed and transfected into lung cancer cell lines, A549 (adenocarcinoma) and SBC-3 (small cell lung cancer), that endogenously expressed CDC45L at high levels. A knockdown effect was confirmed by semi-quantitative RT-PCR when si-CDC45L#1 and si-CDC45L#2 constructs were used (Fig. 3A). Subsequent MTT and colony-formation assays (Figs. 3B and 3C) revealed a drastic reduction in the number of living cells and colonies in the cells transfected with si-CDC45L#1 and si-CDC45L#2. To clarify the mechanisms of this effect further, we performed flow cytometrical analysis using A549 cells that had been transfected with si-CDC45L#2, and found that the sub-G1 proportion of the cells transfected with si-CDC45L#2 was significantly higher than those treated with control siRNA (si-LUC) (Fig. 3D). The data suggested that CDC45L knockdown induced apoptosis of lung cancer cells.
Identification of PIF1 as a protein interacting with CDC45L. To elucidate the biological mechanism of CDC45L overexpression in lung carcinogenesis, the present inventors attempted to identify proteins that would interact with CDC45L in lung cancer cells. Cell extracts from SBC-3 cells were immunoprecipitated with a rabbit polyclonal anti-CDC45L antibody or rabbit IgG (control). Following separation by SDS-PAGE, protein complexes were silver-stained. A protein band, which was seen in immunoprecipitates by anti-CDC45L antibody, but not in those by rabbit IgG, was excised, trypsin-digested, and subjected to mass spectrometry analysis. Peptides from the extracted protein band matched to those of PIF1. The present inventors subsequently re-examined primary NSCLC tissues and lung-cancer cell lines by semi-quantitative RT-PCR experiments, and found increased PIF1 expression in 9 of 15 NSCLC clinical samples as well as in all of 15 lung-cancer cell lines examined, while its expression was scarcely detected in normal lung tissues and normal bronchial epithelia derived cells, SAEC (Fig. 4A). The expression pattern of the CDC45L gene showed good concordance with that of PIF1 gene, suggesting that these two genes were likely to be co-activated in lung cancer cells. To confirm the possible interaction of CDC45L with PIF1 in lung cancer cells, plasmids designed to express FLAG-tagged PIF1 protein (pCAGGSn3FC-PIF1-FLAG) were constructed, and transfected into SBC-3 cells, and then immunoprecipitated the proteins with anti-FLAG antibody. Western blot analysis of the precipitates using anti-CDC45L antibody indicated that endogenous CDC45L was co-precipitated with exogenous PIF1 (Fig. 4B). Immunocytochemical analysis confirmed the co-localization of endogenous CDC45L and exogenous PIF1 in nucleus and cytoplasm (Fig. 4C).
Effects of PIF1 on growth of cancer cells. To further assess whether expression of PIF1 plays a role in growth and/or survival of lung-cancer cells, the present inventers examined the biological significance of the PIF1 function in pulmonary carcinogenesis using siRNAs against PIF1. Transfection of siRNA oligonucleotides against PIF1 (si-PIF1#1 and -#2) into A549 and SBC-3 cells effectively suppressed the expression of the endogenous PIF1 protein, while no effect was observed by the control siRNAs (Fig. 5A). In accordance with the reduced expression of PIF1, A549 and SBC-3 cells showed significant decreases in cell viability and numbers of colonies (Figs. 5B and 5C). These results strongly supported the possibility that PIF1 might also play a significant role in growth and/or survival of lung cancer cells.
Discussion
The development of new molecular-targeting anti-cancer drugs that provide good survival and few serious adverse effects is extremely important. Therefore, this invention has established an effective system to identify therapeutic targets for developing small-molecule compounds that have more efficient anti-cancer effect with fewer adverse reaction than current therapies (Kikuchi T. et al. Oncogene 2003;22:2192-205., Kakiuchi S. et al. Mol Cancer Res 2003;1:485-99., Kakiuchi S. et al. Hum Mol Genet 2004;13:3029-43., Kikuchi T. et al. Int J Oncol 2006;28:799-805., Taniwaki M. et al. Int J Oncol 2006;29:567-75., Yamabuki T. et al. Int J Oncol 2006;28:1375-84.).
Although CDC45L is known to be a replication-initiation protein, its involvement in clinical cancer has not been indicated. Recently advanced understanding of the regulation of replication factors has provided new sources for possible proliferation markers as well as therapeutic targets. For instance, serine-threonine kinase CDC7 is activated to phosphorylate the MCM proteins and thereby recruits additional factors to promote unwinding of the DNA as well as binding of DNA polymerase, essential for initiation of DNA replication. Treatment with potent CDC7 kinase inhibitor, PHA-767491 resulted in induction of apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models (Montagnoli A, et al. Nat Chem Biol 2008;4:357-65.). Another selective CDC7 kinase inhibitor, BMS-863233, which restricts initiation of DNA replication and shows antitumor activity, is under preparation of clinical tirals in patients with refractory hematologic cancer (//clinicaltrials.gov/show/NCT00838890). Moreover, in terms of biomarkers, MCM proteins, CDC6, and geminin (GMNN) were immunohistochemically examined as prognostic biomarkers for patients with various types of cancers including breast, prostate, lung, brain, uterus, and kidney cancers (Gonzalez MA et al., Nat Rev Cancer 2005;5:135-41.). The treatment of lung cancer cells with specific siRNA shown in the Examples above reduced expression of CDC45L and then led to apoptosis. Moreover, clinicopathological evidence through tissue microarray experiments in the Examoles demonstrated that NSCLC patients with tumors expressing CDC45L revealed shorter cancer-specific survival periods than those with negative CDC45L expression. The results obtained by in vitro and in vivo assays demonstrate that overexpressed CDC45L is an important molecule that induces a highly malignant phenotype of lung-cancer cells. To our best knowledge, this is the first study to show the prognostic value of CDC45L expression as a cancer biomarker.
The present invention also identified the interaction between CDC45L and PIF1 proteins in lung cancers. PIF1 is classified as a member of SFI 5'-to-3' DNA helicase conserved from yeast to human, which is mainly reported as a factor for DNA replication in yeast (NPL 42, 43). In the Examoles, PIF1 was also highly transactivated in the great majority of lung cancers and treatment of lung cancer cells with specific siRNA resulted in suppression of the growth activity. These data indicated that PIF1 also plays an important role in the pulmonary carcinogenesis.
Saccharomyces cerevisiae PIF1 helicase functions in DNA replication with the DNa2 helicase/nuclease and DNA polymerase delta (Budd ME et al., Mol Cell Biol 2006;26:2490-500.). On the other hand, CDC45L interacts with the MCM2-7 complex, the GINS complex, and DNA polymerases delta, and plays an important role in elongation of DNA replication by bridging the processive DNA polymerases delta and epsilon with the replicative helicase in the elongating machinery (NPL39). Based on the data provided here, targeting the CDC45L-PIF1 complex as well as CDC45L expression is a new approache to suppress the cancer cell proliferation and/or survival.
The gene-expression analysis of cancers described herein, using the combination of laser-capture dissection and genome-wide cDNA microarray, has identified a specific gene as a target for cancer prevention and therapy. Based on the expression of this differentially expressed gene, i.e., CDC45L,, the present invention provides a novel molecular diagnostic marker for identifying and detecting cancers as well as assessing the prognosis. Further, PIF1, identified as the gene that its translation product was interacted with CDC45L, was confirmed overexpression in cancers. Therefore, the present invention also provides a novel diagnostic strategy using CDC45L or PIF1.
Furthermore, as described herein, CDC45L and PIF1 are involved in cancer cell survival. Therefore, the present invention also provides novel molecular targets for treating and preventing cancer. They may be useful for developing novel therapeutic drugs and preventative agents without adverse effects.
The methods described herein are also useful for the identification of additional molecular targets for prevention, diagnosis, and treatment of cancers. The data provided herein add to a comprehensive understanding of cancers, facilitate development of novel diagnostic strategies, and provide clues for identification of molecular targets for therapeutic drugs and preventative agents. Such information contributes to a more profound understanding of tumorigenesis, and provides indicators for developing novel strategies for diagnosis, treatment, and ultimately prevention of cancers.
All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.
Furthermore, while the invention has been described in detail and with reference to specific embodiments thereof, it is to be understood that the foregoing description is exemplary and explanatory in nature and is intended to illustrate the invention and its preferred embodiments. Through routine experimentation, one skilled in the art will readily recognize that various changes and modifications can be made therein without departing from the spirit and scope of the invention. Thus, the invention is intended to be defined not by the above description, but by the following claims and their equivalents.

Claims (33)

  1. A method of detecting or diagnosing cancer in a subject, comprising determining a expression level of CDC45L and/or PIF1 in a subject-derived biological sample, wherein an increase of said level compared to a normal control level of said gene indicates that said subject suffers from or is at risk of developing cancer, or the presence of cancer in said subject, wherein the expression level is determined by a method selected from a group consisting of:
    (a) detecting an mRNA of CDC45L gene and/or an mRNA of PIF1 gene,
    (b) detecting a protein encoded by CDC45L gene and/or a protein encoded by PIF1 gene, and
    (c) detecting (a) biological activity(activities) of a protein encoded by CDC45L gene and/or a protein encoded by PIF1 gene.
  2. The method of claim 1, wherein said increase is at least 10% greater than said normal control level.
  3. The method of claim 1, wherein the subject-derived biological sample is a biopsy sample.
  4. The method of claim 1, wherein the cancer is lung cancer.
  5. A kit for use in diagnosis or detection of cancer, wherein the kit comprises a reagent which binds to a transcription or translation product of the CDC45L gene and/or PIF1 gene.
  6. A method for assessing prognosis of a subject with lung cancer, wherein the method comprises steps of:
    (a) detecting an expression level of CDC45L in a subject-derived biological sample;
    (b) comparing the detected expression level to a control level; and
    (c) determining prognosis of the patient based on the comparison of (b).
  7. The method of claim 6, wherein the control level is a good prognosis control level and an increase of the expression level compared to the control level indicates poor prognosis.
  8. The method of claim 7, wherein the increase is at least 10% greater than said control level.
  9. The method of claim 6, wherein said expression level is determined by a method selected from a group consisting of:
    (a) detecting an mRNA of CDC45L gene;
    (b) detecting a protein encoded by CDC45L gene; and
    (c) detecting a biological activity of the protein encoded by CDC45L gene.
  10. A method of screening for a candidate compound for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
    a) contacting a test compound with a CDC45L or PIF1 polypeptide, or fragment thereof;
    b) detecting binding activity between the polypeptide or fragment thereof, and the test compound; and
    c) selecting the test compound that binds to the polypeptide or fragment thereof.
  11. A method of screening for a candidate compound for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
    a) contacting a test compound with a cell expressing CDC45L gene and/or PIF1 gene;
    b) detecting (an) expression level(s) of CDC45L gene and/or PIF gene; and
    c) selecting the test compound that reduces the expression level(s) of CDC45L gene and/or PIF1 gene in comparison with the expression level(s) detected in absence of the test compound.
  12. A method of screening for a candidate compound for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
    a) contacting a test compound with a CDC45L or PIF1 polypeptide or fragment thereof;
    b) detecting a biological activity of the polypeptide or fragment thereof of step (a); and
    c) selecting the test compound that suppresses a biological activity of the polypeptide or fragment thereof in comparison with a biological activity detected in the absence of the test compound.
  13. The method of claim 12, wherein the biological activity is cell proliferative activity.
  14. A method of screening for a candidate compound for treating or preventing cancer, or inhibiting cancer cell growth, said method comprising steps of:
    a) contacting a test compound with a cell into which a vector comprising a transcriptional regulatory region of CDC45L or PIF1 genes and a reporter gene that is expressed under control of transcriptional regulatory region has been introduced,
    b) measuring expression or activity of said reporter gene; and
    c) selecting the test compound that reduces an expression or activity level of said reporter gene, in comparison with the level detected in the absence of the test compound.
  15. A method of screening for a candidate compound inhibits a binding between a CDC45L polypeptide and a PIF1 polypeptide, said method comprising steps of:
    (a) contacting CDC45L polypeptide or functional equivalent thereof with a PIF1 polypeptide or functional equivalent thereof in the presence of a test compound;
    (b) detecting a binding between the polypeptides;
    (c) comparing the binding level detected in the step (b) with the level detected in the absence of the test compound; and
    (d) selecting the test compound that reduces or inhibits the binding level in comparison with the level detected in the absence of the test compound.
  16. The method of claim 15, wherein the functional equivalent of CDC45L polypeptide comprises a PIF1-binding domain.
  17. The method of claim 15, wherein the functional equivalent of PIF1 polypeptide comprises a CDC45L-binding domain.
  18. The method of any one of claims 10 to 17, wherein the cancer is lung cancer.
  19. A double-stranded molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12, and the antisense strand comprises a nucleotide sequence complementary to said target sequence, wherein said sense strand and said antisense strand hybridize to each other to form the double-stranded molecule, wherein said double-stranded molecule, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits the expression of said gene.
  20. The double-stranded molecule of claim 19, wherein the sense strand hybridizes with the antisense strand at the target sequence to form the double-stranded molecule having between 19 and 25 nucleotide pair in length.
  21. The double-stranded molecule of claim 19 or 20, wherein said double-stranded molecule is a single polynucleotide comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
  22. The double-stranded molecule of claim 21, wherein said polynucleotide has a general formula
    5'-[A]-[B]-[A']-3'
    wherein [A] is a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotides; and [A'] is a nucleotide sequence complementary to [A].
  23. A vector comprising each or both of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to SEQ ID NOs: 9, 10, 11 or 12, and wherein the antisense strand comprises a nucleotide sequence which is complementary to said sense strand, wherein transcripts of said sense strand and said antisense strand hybridize to each other to form said double-stranded molecule, and wherein said vector, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits expression of said gene.
  24. Vectors comprising each of a combination of polynucleotide comprising a sense strand nucleic acid and an antisense strand nucleic acid, wherein said sense strand nucleic acid comprises nucleotide sequence corresponding to SEQ ID NOs: 9, 10, 11 or 12 and said antisense strand nucleic acid consists of a sequence complementary to the sense strand, wherein the transcripts of said sense strand and said antisense strand hybridize to each other to form a double-stranded molecule, and wherein said vectors, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits the cell proliferation.
  25. The vector of claim 23, wherein the polynucleotide is an oligonucleotide of between about 19 and about 25 nucleotides in length.
  26. The vector of claim 23 or 25, wherein said double-stranded molecule is a single nucleotide transcript comprising the sense strand and the antisense strand linked via a single-stranded nucleotide sequence.
  27. The vector of claim 26, wherein said polynucleotide has a general formula
    5'-[A]-[B]-[A']-3'
    wherein [A] is a nucleotide sequence corresponding to a target sequence selected from the group consisting of SEQ ID NO: 9, 10, 11 and 12; [B] is a nucleotide sequence consisting of about 3 to about 23 nucleotides; and [A'] is a nucleotide sequence complementary to [A].
  28. A method of treating or preventing cancer in a subject, comprising administering to said subject a pharmaceutically effective amount of a double-stranded molecule against CDC45L gene or PIF1 gene, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits cell proliferation as well as the expression of the gene.
  29. A method of claim 28, wherein the double stranded molecule is that of any one of claims 19 to 22.
  30. A method of claim 28, wherein the vector is that of any one of claims 23 to 27.
  31. A composition for treating or preventing cancer, which comprises a pharmaceutically effective amount of a double-stranded molecule against a CDC45L or PIF1, or a vector encoding said double-stranded molecule, wherein the double stranded molecule, when introduced into a cell expressing the CDC45L gene or PIF1 gene, inhibits cell proliferation as well as the expression of the gene, and a pharmaceutically acceptable carrier.
  32. The composition of claim 31, wherein the double stranded molecule is that of any one of claims 19 to 22.
  33. The composition of claim 31, wherein the vector is that of any one of claims 23 to 27.
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