WO1999048916A2 - Hypoxia-inducible human genes, proteins, and uses thereof - Google Patents
Hypoxia-inducible human genes, proteins, and uses thereof Download PDFInfo
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- WO1999048916A2 WO1999048916A2 PCT/US1999/006860 US9906860W WO9948916A2 WO 1999048916 A2 WO1999048916 A2 WO 1999048916A2 US 9906860 W US9906860 W US 9906860W WO 9948916 A2 WO9948916 A2 WO 9948916A2
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- 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
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the present invention relates to hypoxia-inducible genes, and fragments thereof, and to the use of these sequences in the diagnosis and treatment of disease conditions involving hypoxia, including stroke, heart attack, and cancer.
- Hypoxia is responsible for regulating a number of cellular and systemic processes, including angiogenesis, erythropoiesis, and glycolysis. Hypoxic insult and hypoxia- induced gene expression also play a role in a variety of severe pathological conditions including ischemia, retinopathy, neonatal distress, and cancer.
- hypoxia-induced gene expression is associated with ischemia (and reperfusion) in many tissues including the liver, heart, eyes, and brain. Many of the hypoxia-induced genes are believed to be involved in the protection or repair ofthe cells exposed to hypoxia. Enhancement ofthe body's protective expression of some stress-induced genes is therefore likely to be beneficial in many ischemia/reperfusion-related conditions such as liver transplantation, bypass operations, cardiac arrest, and stroke. For instance, in the brain, the response to brain ischemia includes the enhanced expression of growth factors and anti- apoptosis genes (Koistinaho et al. (1997) Neuroreport 20:i-viii). However, the ischemic induction of gene expression is not always favorable.
- brain ischemia can also result in the expression of apoptosis genes or other genes which promote degeneration ofthe neuronal cells.
- Ischemia can also induce an extreme inflammatory reaction in the injured brain via the upregulation of proinflammatory cytokines, chemokines, and endothelial- leukocyte adhesion molecules (Feuerstein et al (1997) Ann. N. Y. Acad.Sci. 15:179- 93). There is some evidence that this hypoxia-induced inflammatory response is a major cause of brain damage.
- Eye diseases associated with neovascularization also involve hypoxia. These eye diseases include diabetic retinopathy, retinopathy of prematurity, and sickle cell retinopathy. All can be serious enough to lead to blindness.
- VEGF vascular endothelial growth factor
- the feasibility of treatment of retinopathy of prematurity by antisense inhibition of a hypoxia-induced gene, vascular endothelial growth factor (VEGF), has been demonstrated (Robinson, Patent No. 5,661,135).
- the process of wound healing also involves the induction of gene expression by hypoxia (Anderson et al., Patent No. 5,681,706). TNF- ⁇ (tumor necrosis factor- ⁇ ) expression and secretion by macrophages is one response involved in wound healing that is induced by low oxygen.
- hypoxia-induced effects include the formation of scar tissue.
- tissue hypoxia is responsible for regulating expression of genes in the developing embryo, particularly with regard to angiogenesis and vasoformation (Iyer et al. (1998) Genes and Development 12: 149-162; Maltepe et al. (1997) Nature 386:403-407).
- Hypoxia also plays a role in neonatal stress and pregnancy-related diseases. For instance, oxygen tension appears to regulate cytotrophoblast proliferation and differentiation within the uterus (Genbacev et al. (1997) Science 277:1669-1672).
- IGFBP- 1 insulin-like Growth Binding Protein
- hypoxia has also been established to play a key role in neoplastic tissues.
- the progression of human tumors to malignancy is an evolutionary process involving the differential expression of multiple genes in response to unique microenvironments.
- Low oxygen conditions create a dominant tumor microenvironment which directly favors processes driving malignant progression, such as angiogenesis or elimination of p53 tumor suppressor activity.
- the connection between tumor hypoxia and the treatment of cancer is further exemplified by a study of cervical cancer that showed that the oxygen level of a tumor was an independent prognostic factor (Hoeckel et al. (1996) Semin. Radiat. Oncol. 6:1-8).
- the prognostic value ofthe oxygen level of a tumor was found to be more significant than all other indicators such as the age ofthe patient, clinical stage, or tumor size.
- hypoxia inducible factor-1 HIF-1
- HRE hypoxia-responsive element
- HIF-1 has been cloned and found to not be activated by stressors such as heat shock and ionizing radiation.
- Differential-display polymerase chain reaction PCR has been used to identify additional genes induced by hypoxia (O'Rourke et al. (1996) Eur. J. Biochem. 241 :403-410). Six hypoxia-induced genes were identified, three of which were of known function.
- the present invention relates to genes whose expression is induced under hypoxic conditions.
- One aspect ofthe present invention provides the isolated polynucleotide having the sequence shown as SEQ ID NO: 1 (Fig. 1 A), comprising the cDNA of the hypoxia-induced human gene HIG1, and encoding the polypeptide sequence of SEQ ID NO:2 (HIG1; Fig. IB).
- Polynucleotides with sequences complementary to SEQ ID NO:l, fragments of SEQ ID NO:l which are at least twelve nucleotides in length, and sequences which hybridize to SEQ ID NO: l are also contemplated by the present invention.
- one aspect ofthe invention concerns the fragment ofthe sequence set forth in SEQ ID NO: l comprising nucleotides 62- 343, the nucleotides representing the coding sequence of human HIG1.
- the complements to the coding sequence, at least twelve nucleotide-long fragments of the coding sequence, and sequences which hybridize to the coding sequence of HIG1 are also provided by the invention.
- Another aspect ofthe present invention provides the isolated polynucleotide having the sequence shown as SEQ ID NO: 3 (Fig. 2 A), comprising the cDNA ofthe hypoxia induced gene HIG2, and encoding the polypeptide sequence of SEQ ID NO:4 (HIG2; Fig. 2B).
- the complements to SEQ ID NO:3, as well as at least twelve nucleotide-long fragments thereof and sequences which hybridize thereto are also provided.
- the invention refers in particular to a polynucleotide having a sequence corresponding to nucleotides 274-465 ofthe sequence set forth in SEQ ID NO:3, or complements thereof, or at least twelve nucleotide-long fragments thereof, or sequences which hybridize thereto. Nucleotides 274-465 represent the coding sequence of human HIG2.
- the present invention also encompasses expression vectors and delivery vehicles which contain polynucleotides ofthe present invention and host cells that are genetically engineered with polynucleotides ofthe present invention.
- the invention provides for an oligonucleotide probe comprising fragments, preferably at least about 15 nucleotides long, ofthe polynucleotides of SEQ ID NO: l or SEQ ID NO:3, or the complement thereto.
- Polypeptides ofthe sequences set forth in SEQ ID NO:2 ( ⁇ IG1) and SEQ ID NO:4 ( ⁇ IG2), or biochemically equivalent fragments ofthe polypeptides of either sequence, are further contemplated by the present invention.
- Antibodies that are specifically immunoreactive to the hypoxia-induced polypeptides HIG1 or HIG2 ofthe present invention are also provided.
- the present invention provides for arrays of polynucleotides or polypeptides corresponding to at least two different hypoxia- inducible genes, hypoxia-induced polypeptides, or antibodies immunoreactive with hypoxia-induced polypeptides.
- Hypoxia-inducible genes suitable for use in the arrays, diagnostic methods, and treatment methods ofthe invention described herein are not limited to HIG and HIGl, or derivatives thereof, but also include a number of known genes now determined to be hypoxia-inducible.
- Additional hypoxia-induced genes useful in the methods and arrays ofthe present invention include, but are not limited to, the genes of annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, f ⁇ roblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor- 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1 , quiescin, growth arrest DNA damage-inducible protein 45, DEC1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lac
- the present invention provides diagnostic and prognostic tools for assaying for the expression of hypoxia-inducible genes in a tissue of an animal, for determining the presence of hypoxia in a tissue in an animal, and for evaluating a hypoxia-related condition in an animal particularly in order to tailor therapy to a known hypoxic state.
- mRNA transcripts or proteins ofthe hypoxia-inducible genes of HIGl, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor- 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein,
- a polynucleotide array or antibody array ofthe invention may be contacted with polynucleotides or polypeptides, respectively, either from or derived from a sample of body fluid or tissue obtained from the animal.
- the amount and position of polynucleotide or polypeptide from the animal's sample which binds to the sites of the array is determined.
- the gene expression pattern observed may be correlated with an appropriate treatment.
- aspects ofthe invention concern treating a tissue which is a tumor by first determining the presence of hypoxia in the tumor and, second, treating the tumor with an established form of therapy for cancers such as radiation therapy, chemotherapy, and surgery.
- the invention provides for methods of attenuating the hypoxic response of a tissue by blocking expression of a hypoxia-inducible gene HIGl, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor- 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creat
- Methods for enhancing the response of tissue to hypoxia involve administering expression vectors comprising the hypoxia-inducible genes ofthe present invention or administering polypeptide expression products of hypoxia- inducible genes to the tissue.
- Figure 1 shows the human HIGl cDNA and protein sequences.
- the nucleotide sequence for the human HIGl gene is shown in Figure 1A from 5' to 3 '(SEQ ID NO:l).
- the coding sequence is underlined.
- the other regions are untranslated regions (5' and 3' UTR) ofthe gene.
- the protein sequence of human HIGl is shown in Figure IB (SEQ ID NO:2).
- Figure 2 shows the human HIG2 cDNA and protein sequences.
- the nucleotide sequence for the human HIG2 gene is shown in Figure 2A from 5 ' to 3 ' (SEQ ID NO:3).
- the coding sequence is underlined.
- the other regions are untranslated regions (5' and 3' UTR) ofthe gene.
- the protein sequence of human HIG2 is shown in Figure 2B (SEQ ID NO:4).
- Figure 3 shows the murine HIGl cDNA and protein sequences.
- the nucleotide sequence for the murine HIGl gene is shown in Figure 3 A from 5' to 3' (SEQ ID NO:5).
- the coding sequence is underlined.
- the other regions are untranslated regions (5' and 3' UTR) ofthe gene.
- the protein sequence of murine HIGl is shown in Figure 3B (SEQ ID NO:6).
- Figure 4 shows the HIGl cDNA and protein sequences of seriola quinqueradiata.
- the nucleotide sequence for this fish HIGl is shown in Figure 4A from 5' to 3' (SEQ ID NO:7).
- the coding sequence is underlined.
- the other regions are untranslated regions (5' and 3' UTR) ofthe gene.
- the protein sequence of fish HIGl is shown in Figure 4B (SEQ ID NO:8).
- Figure 5 shows the murine HIG2 cDNA and protein sequences.
- the nucleotide sequence for the murine HIG2 gene is shown in Figure 5 A from 5' to 3' (SEQ ID NO: 9).
- the coding sequence is underlined.
- the other regions are untranslated regions ofthe gene (5' and 3' UTR).
- the protein sequence of murine HIG2 is shown in Figure 5B (SEQ ID NO: 10).
- FIG 6 shows the alignment of human HIGl and HIG2 protein sequences with the HIGl and HIG2 sequences of other species.
- the HIGl homologues from humans (hHIGl), mice (mHIGl), and fish (seriola quinqueradiate) (fHIGl or GHL1) are aligned in Figure 6A; the HIG2 homologues from humans (hHIG2) and mice (mHIG2) are aligned in figure 6B.
- Figure 7 schematically illustrates the addition of linkers to cDNA library fragments. The linker addition is followed by PCR amplification.
- Figure 8 illustrates how the subtraction protocol is used to enrich the tester cDNA library with sequences unique to the tester cDNAs.
- hypoxic tissue will have an oxygen content that is less than or equal to about 2%.
- Normoxic or oxic conditions are conditions comprising a normal level of oxygen for that particular environment. Normoxic or oxic tissue typically has an oxygen content above about 5%.
- hypoxia-induced or “hypoxia-inducible” when referring to a gene means that the gene is expressed at a higher level when the host cell is exposed to hypoxic conditions than when exposed to normoxic conditions.
- the number of mRNA transcripts of a hypoxia-induced gene would is at least about 20% higher in a hypoxic cell versus a normoxic cell.
- expression ofthe hypoxia-induced gene is at least about 2-fold higher in hypoxic versus normoxic cells.
- expression ofthe hypoxia-inducible gene is at least about 5-fold higher in hypoxic cells versus normoxic cells.
- hypoxia-related condition in an animal is a condition where hypoxia or altered (typically, enhanced) levels of expression of hypoxia-inducible genes in a tissue ofthe animal is involved.
- the hypoxia or altered expression of hypoxia- inducible genes may either be a symptom or play a role in the cause, development, progression, amelioration, or cure ofthe condition.
- a hypoxia-related condition may optionally be a disease or pathological condition.
- Hypoxia-related conditions include, but are not limited to, cancer, ischemia, reperfusion, retinopathy, neonatal distress, preeclampsia, cardiac arrest, stroke, and wound healing.
- hypoxia hypoxia
- isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
- naturally-occurring polynucleotides or polypeptides present in a living animal are not isolated, but the same polynucleotides or polypeptides could be part of a vector or composition, and be isolated in that such vector or composition is not part of its natural environment.
- sample obtained from a patient or a “sample obtained from an animal” may be a sample of tissue or a sample of body fluid.
- tissue is used herein to refer to any biological matter made up of one cell, multiple cells, an agglomeration of cells, or an entire organ.
- tissue as used herein, encompasses a cell or cells which can be either normal or abnormal (i.e. a tumor).
- body fluid may be any liquid substance extracted, excreted, or secreted from an organism or a tissue of an organism. The body fluid need not necessarily contain cells. Body fluids of relevance to the present invention include, but are not limited to, whole blood, serum, plasma, urine, cerebral spinal fluid, tears, and amniotic fluid.
- biochemically equivalent variations means protein or nucleic acid sequences which differ in some respect from the specific sequences disclosed herein, but nonetheless exhibit the same, or substantially the same, functionality.
- cDNA for example, this means that modified sequences which contain other nucleic acids than those specifically disclosed are encompassed, provided that the alternate cDNA encodes mRNA which in turn encodes a protein of this invention. Such modifications may involve the substitution of only a few bases, or many. The modifications may involve substitution of degenerate coding sequences or replacement of one coding sequence with another; introduction of non-natural nucleic acids is contemplated. It is not necessary for the alternate DNA to hybridize with that disclosed herein provided that the functional criterion is met.
- the modified nucleic acid sequence hybridizes to and is at least 95% complementary to the sequence of interest.
- polynucleotide includes, but is not limited to, mRNA, cDNA, genomic DNA, and synthetic DNA and RNA sequences, comprising the natural nucleoside bases adenine, guanine, cytosine, thymine, and uracil.
- sequences having one or more modified nucleosides also encompasses sequences having one or more modified nucleosides.
- polynucleotide and oligonucleotide are used interchangeably herein. No limitation as to length or to synthetic origin are suggested by the use of either of these terms herein.
- polypeptide means a poly(amino acid) comprising at least two amino acids linked by peptide bonds.
- a "protein” is a polypeptide which is encoded by a gene.
- Negtralizing a polypeptide or protein means inhibiting, partially or wholly, the bioactivity ofthe polypeptide or protein. This inhibition of activity may mean inhibition of catalytic activity, prevention of binding to a receptor or ligand, blockage or dimer formation, or the like.
- sequences which hybridize thereto means polynucleotide sequences which are capable of forming Watson-Crick hydrogen bonds with another polynucleotide sequence under normal hybridization conditions, such as in buffered (pH.
- aqueous, saline solutions for instance, 1 to 500 mM NaCI
- normal hybridization conditions will depend on the length ofthe polynucleotides involved, typically they include the presence of at least one cation such as Na + , K + , Mg2+, or Ca2+, a near neutral pH, and temperatures less than 55°C.
- sequences which hybridize to a polynucleotide may be about 90%- 100% complementary to the polynucleotide, if the sequences are of sufficient length, in solutions with high salt concentrations, and/or under low temperature conditions, polynucleotides with complementarity of 70% or above, or even just 50% or above, may hybridize to the polynucleotide. Sequences which hybridize thereto typically comprise at least 12 nucleotides, and preferably at least about 15 nucleotides, which are complementary to the target polynucleotide .
- a "coding sequence” is a polynucleotide or nucleic acid sequence which is transcribed and translated (in the case of DNA) or translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
- the boundaries ofthe coding sequence are determined by a translation start codon at the 5 ' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
- a transcription termination sequence will usually be located 3' to the coding sequence.
- control sequences refer to translational start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, as necessary and sufficient for the transcription and translation of a given coding sequence in a defined host cell.
- control sequences suitable for eucaryotic cells are promoters, polyadenylation signals, and enhancers. All of these control sequences need not be present in a recombinant vector so long as those necessary and sufficient for the transcription and translation ofthe desired gene are present.
- operably or operatively linked refers to the configuration ofthe coding and control sequences so as to perform the desired function.
- control sequences operably linked to a coding sequence are capable of effecting the expression ofthe coding sequence.
- a coding sequence is operably linked to or under the control of transcriptional regulatory regions in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA that can be translated into the encoded protein.
- the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
- intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
- the expression products described herein may consist of proteinaceous material having a defined chemical structure. However, the precise structure depends on a number of factors, particularly chemical modifications common to proteins. For example, since all proteins contain ionizable amino and carboxyl groups, the protein may be obtained in acidic or basic salt form, or in neutral form.
- the primary amino acid sequence may be derivatized using sugar molecules (glycosylation) or by other chemical derivatizations involving covalent or ionic attachment with, for example, lipids, phosphate, acetyl groups and the like, often occurring through association with saccharides. These modifications may occur in vitro, or in vivo, the latter being performed by a host cell through posttranslational processing systems.
- Vector means a polynucleotide comprised of single strand, double strand, or circular DNA or RNA.
- An "expression vector” is comprised ofthe following elements operatively linked at appropriate distances for allowing functional gene expression: replication origin, promoter, enhancer, 5' mRNA leader sequence, ribosomal binding site, nucleic acid cassette, termination and polyadenylation sites, and selectable marker sequences. One or more of these elements may be omitted in specific applications.
- the nucleic acid cassette can include a restriction site for insertion ofthe nucleic acid sequence to be expressed.
- the nucleic acid cassette contains the nucleic acid sequence to be expressed including translation initiation and termination sites.
- An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation ofthe coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" ofthe control sequences. Modification ofthe sequences encoding the particular protein of interest may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; or to maintain the reading frame.
- the control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector.
- the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site which is in reading frame with and under regulatory control ofthe control sequences.
- a “regulatory element” is a segment of DNA to which a transcription factor(s) binds and alters the activity of a gene's promoter either positively (induction) or negatively (repression).
- Stress-responsive element or “stress-responsive regulatory element” is a regulatory element which binds transcription factors activated by the cell in response to environmental stress.
- Environmental stressors may include one or more ofthe following: oxygen depletion; radiation; heat shock; pH change; hypothermia; or glucose starvation.
- a “delivery vehicle”, as used herein, refers to a means of delivering a polypeptide or a polynucleotide to a cell.
- the delivery vehicle is preferably used to deliver an expression vector to a cell or a cell in an organism.
- a delivery vehicle may be a virus, such as a retrovirus, an adenovirus, an adeno-associated virus, a herpes simplex virus, or a vaccinia virus.
- Liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. They have internal aqueous space useful for entrapping water soluble compounds such as polynucleotides. Recognition molecules can be attached to their surface for the targeting ofthe delivery vehicles to specific tissues.
- an "antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Antibodies may exist as intact immunoglobulins or as a number of fragments, including those well-characterized fragments produced by digestion with various peptidases. While various antibody fragments are defined in terms ofthe digestion of an intact antibody, one of skill will appreciate that antibody fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies.
- Antibody fragments encompassed by the use ofthe term “antibodies” include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments.
- the phrase "specifically binds to a polypeptide” or “specifically immunoreactive with”, when referring to an antibody refers to a binding reaction which is determinative ofthe presence ofthe polypeptide (or protein) in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
- Specific binding to a protein under such conditions may require an antibody that is selected for its specificity for a particular protein or polypeptide.
- a variety of immunoassay formats may be used to select anitbodies specifically immunoreactive with a particular protein.
- solid-phase ELISA immunoassays are rountinely used to select monoclonal antibodies specifically immunoreactive with a protein.
- HIGl Hypoxia-inducible Genes and Expression Products
- Polynucleotides with sequences complementary to SEQ ID NO:l polynucleotides that are fragments of SEQ ID NO:l of at least twelve nucleotides in length and polynucleotides which hybridize to SEQ ID NO: 1 are also within the scope ofthe present invention.
- the fragments of SEQ ID NO:l are preferably at least 15 nucleotides long.
- HIG2 a second, novel human gene, herein referred to as HIG2, whose expression is induced by cellular response to hypoxia.
- HIG2 a second, novel human gene, herein referred to as HIG2, whose expression is induced by cellular response to hypoxia.
- HIG2 a second, novel human gene, herein referred to as HIG2, whose expression is induced by cellular response to hypoxia.
- HIG2 a second, novel human gene, herein referred to as HIG2
- the cDNA sequence ofthe HIG2 gene is shown in Fig. 2A (SEQ ID NO:3).
- the coding sequence of HIG2 comprises nucleotides 274-465 of SEQ ID NO:3.
- Fragments ofthe HIG2 sequence, and of the HIG2 coding sequence in particular, of at least twelve, and preferably fifteen, nucleotides in length are provided by the present invention as well.
- Polynucleotides of sequence which is complementary to SEQ ID NO: 3 (especially to nucleotides 274-465) or polynucleotides which hybridize to polynucleotides of the sequence set forth in SEQ ID NO:3 (especially to nucleotides 274-465), are also contemplated.
- Polypeptides encoded by the polynucleotides of HIGl (SEQ ID NO:2; Fig. IB) and HIG2 (SEQ ID NO:4; Fig. 2B), or biochemically equivalent variations of either protein, are also provided by the present invention. Fragments of these polypeptides which consist of at least eight amino acids are provided as well. Preferably, the fragments are at least 15 amino acids in length.
- mouse and fish ⁇ IG1 polynucleotide and polypeptide sequences can be considered biochemically equivalent variations ofthe human ⁇ IG1.
- the mouse ⁇ IG2 polynucleotide and polypeptide sequences are likewise understood to be biochemically equivalent variations ofthe human HIGl.
- polynucleotides of this invention may readily be incorporated within expression vectors by one of ordinary skill in the art.
- the polynucleotide sequence comprising nucleotides 62-343 of SEQ ID NO:l (the coding sequence of HIGl) or nucleotides 274-465 of SEQ ID NO:2 (the coding sequence of HIG2) is operably linked with appropriate control sequences, such as a promoter.
- larger fragments ofthe polynucleotides of SEQ ID NO:l or SEQ ID NO:2 which comprise portions ofthe untranslated regions ofthe genes may be used in an expression vector instead. This may be particularly useful when hypoxia-induciblity is desired, since the untranslated regions may contain critical regulatory regions such as hypoxia-responsive elements.
- the polynucleotides of this invention may also be incorporated within a host cell.
- transfection may be used to introduce an expression vector containing one ofthe polynucleotides ofthe invention into the cell.
- the polynucleotide ofthe transfected vector may also be operably linked with control sequences including regulatory elements to effect the expression within the cell of exogenous protein or polypeptide sequences encoded by the polynucleotides ofthe present invention.
- a HIGl or HIG2 polynucleotide may be introduced into an animal either by first incorporating the vector into a cell and then transferring the cell to the animal (ex vivo) or by incorporating the vector into a cell within an animal directly (in vivo).
- the introduction of a HIGl or HIG2 polynucleotide into a cell may be achieved by directly injecting the nucleic acid into the cell or by first mixing the nucleic acid with polylysine or cationic lipids which will help facilitate passage across the cell membrane.
- introduction ofthe polynucleotide into the cell is preferably achieved through the use of a delivery vehicle such as a liposome or a virus.
- Viruses which may be used to introduce a HIGl or HIG2 polynucleotide or expression vector into a cell include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and vaccinia viruses.
- Antisense oligonucleotides complementary to HIGl and HIG2, particularly those which are capable of blocking expression of HIGl or HIG2 are provided by the present invention.
- the antisense oligonucleotide is preferably an oligonucleotide having a sequence complementary to at least a portion (preferably at least about 12 nucleotides in length) of SEQ ID NO:l or SEQ ID NO:3.
- the antisense oligonucleotide is preferably between about 15 and about 22 nucleotides in length. Modifications ofthe sequence or bases ofthe antisense oligonucleotide may be desirable to facilitate transfer into a cell, stability, or tight binding to the
- HIGl or HIG2 mRNA are examples of HIGl or HIG2 mRNA.
- An oligonucleotide probe is provided by another embodiment ofthe invention.
- the probe consists of one ofthe polynucleotides of this invention, or an at least 12 nucleotide-long fragment thereof.
- the probe may be used to assay for, and if the probe is properly labeled, quantitate, the hypoxia-induced expression of HIGl or HIG2 in a cell.
- the probe is at least about 15 nucleotides in length. In a particularly preferred embodiment, the probe is between 15 and 22 nucleotides in length.
- Antibodies specifically immunoreactive with the HIGl or HIG2 polypeptides represent still another embodiment ofthe invention. These antibodies may be monoclonal or polyclonal. The antibodies may optionally be recombinant or purely synthetic. The antibody may be an intact antibody or fragment. The preparation of antibodies specific to the HIGl and HIG2 polypeptides would be routine for those skilled in the art. In addition to the identification ofthe new genes HIGl and HIG2 which were found to be hypoxia-inducible, we have also established for the first time that several previously known genes are hypoxia-inducible in humans (see the specific examples, Examples 2 and 9, below).
- genes include annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC- like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1 , fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogenase, Bcl-2-interacting killer, Nip3L/Nix, and Pim-1
- GPDH glyceraldehyde-3 -phosphate dehydrogenase
- Another aspect ofthe invention involves the presentation of multiple (at least two, and preferably more than four) hypoxia-inducible gene sequences, polynucleotide probes complementary to the hypoxia-inducible gene sequences, hypoxia-induced polypeptides, or antibodies (immunoreactive with hypoxia- induced polypeptides) on an array.
- more than about 10 different polynucleotides, polypeptides, or antibodies are presented on the array.
- the number of different polynucleotides, proteins, or antibodies on the array is greater than about 25, or even greater than about 100.
- One aspect ofthe invention provides an array of polynucleotides which comprises at least two different hypoxia-inducible genes, or complements thereto, or at least twelve nucleotide-long fragments thereof, or sequences which hybridize thereto.
- the hypoxia-inducible genes or their fragments may optionally be selected from HIGl, HIG2, any ofthe hypoxia-inducible genes listed in Table 1 (below), Table 3 (Example 8, below), and Table 5 (Example 9, below). However, it is understood that all ofthe hypoxia-inducible gene sequences on the array need not be derived only from those hypoxia-inducible listed herein.
- the polynucleotides on the array are typically single-stranded.
- the polynucleotide array on of the multiple polynucleotides on the array is derived from either the HIGl or HIG2 gene sequences.
- the polynucleotides ofthe array may comprise the entire sequence of one strand ofthe gene, or may comprise at least 12 nucleotide long fragments thereof, or sequences which hybridized thereto.
- one ofthe polynucleotides ofthe array comprises a polynucleotide corresponding to nucleotides 62-343 of SEQ ID NO: l (HIGl) or nucleotides 274- 465 of SEQ ID NO:2 (HIG2), or complements to one ofthe coding sequences, or at least twelve nucleotide-long fragments of one ofthe coding sequences, or sequences which hybridize to one ofthe coding sequences.
- the second polynucleotide sequence may be selected from HIGl, HIG2, any ofthe hypoxia-inducible genes represented in Table 1, shown below, any ofthe expressed sequence tags of hypoxia-inducible genes shown in Table 3 (see Example 8), or any other hypoxia-inducible gene or expressed sequence tag from a hypoxia-inducible gene. It is understood that regardless of which genes are represented on the array, the gene sequences do not have to be represented in their entirety.
- the polynucleotide sequences that are immobilized on the array are most preferably, single-stranded and complementary to the mRNA transcripts ofthe relevant hypoxia-inducible genes.
- the immobilized polynucleotides may be fragments or complementary sequences ofthe gene or EST sequence that contain at least twelve nucleotides and preferably at least fifteen nucleotides.
- longer gene fragments including EST fragments of at least 50 or at least 100 nucleotides may be used.
- the array is made up of many different gene sequences.
- polynucleotide array only polynucleotides correlating to hypoxia-inducible genes expressing gene products of a similar function are included on the array. At least two, but preferentially more than two, different hypoxia-induced genes encoding proteins from a single functional category are represented on the array. Examples of seven functional categories of hypoxia-inducible proteins are as follows: (1) glycolytic enzymes/proteins; (2) angiogenesis/tissue remodeling proteins; (3) erythropoiesis/vascular regulatory proteins; (4) metabolic/homeostatic proteins; (5) apoptosis proteins; (6) DNA repair proteins; and (7) cell-cycle proteins. These categories are shown in Table 1, below, along with some representative members of each ofthe categories.
- a preferred embodiment of this array comprises polynucleotide sequences complementary to the mRNA transcripts ofthe relevant hypoxia inducible genes.
- a particularly preferred embodiment of an array displays multiple polynucleotide sequences, each of which is complementary to a different gene which encodes a protein involved in angiogenesis and/or tissue remodeling.
- PGK phosphoglycerate kinase
- glucose transporter isoform 3 (Glut-3)
- GPDH glyceraldehyde-3 -phosphate dehydrogenase
- AK-3 adenylate kinase isoenzyme 3
- VEGF vascular endothelial growth factor
- PDGF ⁇ platelet-derived growth factor ⁇
- TGF ⁇ transforming growth factor ⁇
- TNF ⁇ tumor necrosis factor ⁇
- IL-6 interleukin-6
- IL-2 interleukin-2
- FGF-3 fibroblast growth factor
- PAI-1 plasminogen activator inhibitor- 1
- MIF macrophage migration inhibitory factor
- EPO erythropoietin
- alpha-fetoprotein AFP
- IGFBP- 1 insulin-like growth factor binding protein- 1
- t-NOS-1 epidermal growth factor receptor (EGFR)
- HAP-1 huntingtin-associated protein 1
- GRP78 glucose-regulated protein 78
- GRP90 glucose-regulated protein 90
- GPDH glyceraldehyde-3 -phosphate dehydrogenase
- heterogeneous nuclear ribonucleoprotein Al hnRNP Al
- glucose transporter isoform 3 (Glut-3)
- AK-3 adenylate kinase isoenzyme 3
- IGFBP-3 insulin-like growth factor binding protein-3
- Bcl-2-interacting killer (BIK) 19 kDa-interacting protein 3 long/Nip3-like protein X (NipP3L/Nix)
- B-cell translocation gene-1 (BTG-1)
- RTP tunicamycin responsive protein
- GADD45 growth arrest DNA damage-inducible protein 45
- polynucleotides correlating to the gene sequences encoding proteins belonging to at least two different functional categories of hypoxia-inducible genes are displayed on a single array. Although at least two different polynucleotide sequences are required to form the array, in a preferred embodiment many more than two are used. Again, a preferred embodiment of this array comprises polynucleotide sequences complementary to the mRNA transcripts ofthe relevant hypoxia inducible genes of at least 12 nucleotides in length, and preferably fifteen.
- the present invention also provides for polypeptide arrays analogous to the polynucleotide arrays discussed above, except that the polypeptide sequences of the hypoxia-inducible genes, or fragments thereof, are displayed in an array.
- the polypeptide array comprises the polypeptide expression products of at least two hypoxia-inducible genes, or biochemically equivalent fragments thereof.
- the polypeptide array my comprise the protein HIGl or HIG2 and at least one other protein which is a hypoxia induced gene product.
- the polypeptide array may instead comprise at least one protein selected from the group consisting of HIGl, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor- 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydr
- Another aspect ofthe invention concerns a polypeptide array comprising at least two different hypoxia-induced proteins, or biochemically equivalent fragments thereof, wherein each hypoxia-induced protein belongs to a different functional category.
- the polypeptide array comprises at least two different hypoxia-induced proteins or biochemically equivalent fragments thereof, wherein said hypoxia-induced proteins are all proteins belonging to a single functional category.
- the functional category may be selected from the group consisting of glycolytic enzymes/proteins, metabolic/homeostatic proteins, apoptosis proteins, DNA repair proteins, angiogenesis/tissue remodeling proteins, cell-cycle proteins, and erythropoiesis/vascular regulatory proteins. (See Table 1, above).
- Yet another alternative embodiment ofthe invention is an array analogous to a polypeptide array described above, except that antibodies immunoreactive with the hypoxia-induced polypeptides are immobilized to form the array, rather than the polypeptide sequences themselves.
- Each array comprises at least two different antibodies, each of which is immunoreactive with a different hypoxia- induced protein.
- Each ofthe two antibodies is specifically immunoreactive with the polypeptide expression products of hypoxia-inducible genes, such as, but not limited to, HIGl or HIG2.
- the antibody array comprises at least one antibody immunoreactive with a protein selected from the group consisting of HIGl, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor- 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC1, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid
- the antibody array further comprises at least one of a second antibody, wherein said second antibody specifically binds a second hypoxia-induced gene product or a biochemically equivalent fragment thereof.
- the antibodies on the array may be monoclonal or polyclonal. They may be intact antibodies or fragments of antibodies that are capable of specifically binding the polypeptides ofthe present invention.
- the antibody array preferably comprises at least four different antibodies, and preferably more than about 10 different antibodies.
- the material to which the polynucleotides or polypeptides are immobilized in the array may vary.
- Possible substrates for construction of a biomolecule array include, but are not limited to, cellulose, glass, silicon, silicon oxide, silicon nitride, polystyrene, germanium,(poly)tetrafluorethylene, and gallium phosphide.
- the animal is preferably a mammal. Most preferably, the mammal is a human.
- hypoxia-inducible genes such as HIGl or HIG2, or combinations thereof
- detection of abnormal levels ofthe transcripts of hypoxia-inducible genes such as HIGl or HIG2, or combinations thereof, in the tissues or body fluids of an animal can be used in both a diagnostic and prognostic manner for hypoxia-related conditions.
- the abnormal levels may be characterized by either increased levels or decreased levels, depending upon the hypoxia-related condition being analyzed. In other cases, either the complete absence or any presence of a hypoxia-inducible gene transcript may be indicative of an abnormal condition.
- hypoxia-induced polypeptides can be used in either a diagnostic or prognostic manner for hypoxia-related conditions.
- the presence of hypoxia in a tissue can be evaluated by testing for the presence or absence ofthe transcripts or polypeptides encoded by the polynucleotides ofthe invention in either the tissue or in the body fluids ofthe animal. Detection ofthe transcripts or polypeptides can be either qualitative or quantitative.
- One aspect ofthe invention provides a method of determining the presence of hypoxia in a tissue in an animal or evaluating a hypoxia-related condition in a tissue in an animal.
- These methods comprise assaying for either the messenger RNA (mRNA) transcripts or the polypeptide expression product of at least one gene selected from the group consisting of HIGl, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor- 1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC- like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC
- This method determining the presence of hypoxia in a tissue may be used to diagnose a hypoxia-related condition in a animal.
- the presence of hypoxia in a tissue or the degree of expression of hypoxia- inducible genes determined by these methods may be used to select an appropriate treatment for the animal.
- the hypoxia-related condition being evaluated may be cancer and the tissue which is the target ofthe evaluation may optionally be a tumor.
- the degree to which the tumor is showing gene expression patterns characteristic of hypoxia or the activation of genes involved in angiogenesis can be usefully correlated with appropriate treatment of tumors of that particular type.
- the hypoxia-related condition need not necessarily be cancer.
- the hypoxia-related condition may instead be any condition in which hypoxic conditions play a role (favorable or detrimental to the animal).
- hypoxic conditions include, but are not limited to, ischemia, reperfusion, retinopathy, neonatal 5 distress, preeclampsia, cardiac arrest, stroke and wound healing.
- the transcripts of hypoxia-inducible genes may be detected by any of several means known to those skilled in the art.
- diagnostic detection involves annealing to the transcript, in vivo or in vitro, a labeled nucleic acid probe complementary to the transcript sequence.
- the labeled probe can be l o fluorescent, radioactive, immunoreactive, colormetric or otherwise marked for detection.
- amplification ofthe transcript in a tissue or fluid sample from the animal may first be performed to aid subsequent detection ofthe transcript. Amplification ofthe hypoxically-induced transcripts can be readily achieved using the polynucleotides ofthe present
- the antibodies can be applied to the tissue in vivo, or to tissue or body fluid samples removed from the animal.
- tissue or body fluid samples removed from the animal Various forms of typical immunoassays known to those skilled in the art would be applicable here. These assays include both competitive and non-competitive assays. For instance, in one
- immobilized antibodies that specifically react with HIG2 polypeptide are contacted with the biological tissue or fluid sample. Presence ofthe immobilized HIG2-antibody complex could then be achieved by application of a second, labeled antibody immunoreactive with either the HIG2 polypeptide or the HIG2-antibody complex.
- a Western blot type of assay could also be used in an alternative embodiment of the present invention.
- a removed tissue is to be analyzed in vitro, typically, degradation ofthe tissue is preferred prior to testing for the presence of either an mRNA transcript or a gene product. For instance, if detection of polynucleotides is desired, proteolytic degradation is useful (Temsamani et al., Patent No. 5,693,466). Extraction or isolation of proteins or nucleic acids in the sample is also preferred prior to carrying out a diagnostic screen. Numerous methods for the isolation of proteins or nucleic acids from cells or biological fluids are well established in the art.
- a diagnostic evaluation of hypoxia-induced gene expression involves assaying the expression levels of more than one hypoxia-inducible inducible genes at a time.
- the arrays ofthe invention are particularly useful for assaying the expression of multiple hypoxia-inducible genes in parallel.
- the diagnostic detection methods mentioned above in regard to in vitro detection would also apply as methods for detecting the presence of polynucleotides and polypeptides in a tissue or a body fluid upon administration of a sample ofthe tissue or fluid to one ofthe arrays ofthe present invention.
- hypoxia-inducible or hypoxia-repressible
- the pattern of expression of hypoxia-inducible genes can therefore be used in a diagnostic or prognostic manner to aid in the treatment of a hypoxia-related condition in an animal.
- the polypeptide arrays ofthe present invention also can be used to screen for drugs useful in the treatment of hypoxia-related conditions. These drugs may be drugs which are capable of inhibiting the hypoxic response of a tissue.
- methods of assaying for expression of hypoxia-inducible genes in a tissue in an animal, determining the presence of hypoxia in a tissue in an animal, or evaluating a hypoxia-related condition in a tissue in an animal comprise first contacting the proteins or messenger RNA of a sample of body fluid or tissue obtained from the animal with an antibody array or polynucleotide array, respectively, ofthe invention. Tissue or fluid samples from an animal may be contacted directly with an array, and binding ofthe proteins or mRNA transcripts on the array detected. (The cells in a tissue to be assayed would preferably be lysed prior to application to the array.) Alternatively, the tissue or fluid sample may be purified to isolate the proteins or mRNA transcripts prior to application to the array.
- cDNA is first prepared from the messenger RNA ofthe sample by reverse transcription and then the cDNA is applied to a polynucleotide array.
- the method comprises detecting the amount and position of the protein, mRNA or cDNA which remains bound to the array after removal of excess or non-bound protein, mRNA, or cDNA.
- a method of diagnosing a hypoxia-related condition in an animal may optionally comprise the additional step of correlating the result ofthe evaluation ofthe hypoxia-related condition in the tissue in the animal with an appropriate treatment for the animal.
- the hypoxia-related condition which may be evaluated, diagnosed or treated by any ofthe above methods may a condition such as cancer, ischemia, reperfusion, retinopathy, neonatal distress, preeclampsia, cardiac arrest, or stroke.
- Another aspect ofthe invention provides for a method of treating a tumor.
- This method involves first determining the presence of hypoxia in a tumor by any ofthe methods described above (with or without arrays). The method further comprises treating said tumor with any combination of an established form of therapy for cancer such as radiation therapy, chemotherapy, or surgery.
- HIGl or HIG2 polynucleotides or the polynucleotides corresponding to the gene sequences of other hypoxia-inducible gene sequences, such as those listed in Table 1 may be used to attenuate the response of a tissue to hypoxia.
- hypoxia-inducible sequences can be targeted within a tissue by the introduction of antisense oligonucleotides, triple-helix probes, catalytic nucleic acids or the like in a manner which inhibits expression ofthe HIG genes or other hypoxia-inducible genes within the tissue.
- the method of attenuating the hypoxic response of tissue comprises inhibiting the expression of a gene selected from the group consisting of HIGl, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DECl, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase,
- This inhibition of expression of a hypoxia-inducible gene may optionally be achieved by introducing into the cells of said tissue a nucleic acid molecule such as an antisense oligonucleotide, a triple-helix probe, a deoxyribozyme, or a ribozyme which is specific to the hypoxia-inducible gene.
- a nucleic acid molecule such as an antisense oligonucleotide, a triple-helix probe, a deoxyribozyme, or a ribozyme which is specific to the hypoxia-inducible gene.
- the HIGl or HIG2 proteins or other expression products of hypoxia-inducible genes may instead be targeted to attenuate the hypoxic response of a tissue.
- antibodies, antagonists, inhibitors, or proteases that are specific to the expression products of hypoxia-induced genes may be introduced to the tissue.
- a method of attenuating the hypoxic response of a tissue comprises neutralizing a protein selected from the group consisting of HIG 1 , HIG2, annexin V, lipocortin 2, hnRNP A 1 , Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin- responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage- inducible protein 45, DECl, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase
- the methods described above for attenuating the hypoxic response of a tissue may be used to treat a hypoxia-related condition in an animal.
- the treatment of a hypoxia-related condition in an animal may be effected by targeting the hypoxia-induced gene sequences ofthe hypoxic (or potentially hypoxic) tissue via one or more ofthe techniques known to those skilled in the art. These techniques include, but are not limited, to introduction of antisense oligonucleotides, triple-helix probes, deoxyribozymes, or ribozymes into the subject's tissue of concern.
- the animal to be treated is a human.
- hypoxia-related condition towards which this treatment may be directed is ischemia, stroke, heart attack, neonatal distress, retinopathy, or any other disease condition in which hypoxia plays a significant role.
- the hypoxia-related condition to be treated is cancer and the tissue is a tumor.
- the disclosed treatment ofthe tumor may be coupled with any combination of other cancer therapies such as radiation therapy, chemotherapy, or surgery.
- treatment ofthe hypoxia-related conditions may also be achieved by neutralizing the protein expression products of hypoxia-inducible genes, as described above.
- antibodies, antagonists, inhibitors, proteases, or the like which target and neutralize HIGl and HIG2 polypeptides may be introduced into the animal, preferably human, containing the tissue to be treated.
- the protein expression products ofthe genes which have been newly identified as being hypoxia-inducible may be used to identify or screen for drugs, such as inhibitors, useful in the treatment of hypoxia-related conditions.
- small molecule drug candidates or peptides may be tested against the any ofthe proteins of HIGl, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC-like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DEC 1 , low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lac
- Combinatorial libraries of small molecules or libraries of peptides such as those produced by phage display may alternatively be screen against one ofthe hypoxia-induced proteins described herein.
- the expression of some gene products induced by hypoxia can be helpful in protecting cells from damage or death.
- this invention also provides for methods of enhancing the hypoxic response of a tissue and thereby and treating hypoxic tissue (or potentially hypoxic tissue).
- the method comprises introducing an expression vector into the tissue and allowing for expression ofthe coding sequence on the vector to take place.
- the coding sequence ofthe expression vector comprises the sequence of at least one ofthe genes HIGl, HIG2, annexin V, lipocortin 2, hnRNP Al, Ku autoantigen, phosphoribosylpyrophosphate synthetase, acetoacetylCoA thiolase, ribosomal L7, fibroblast growth factor-3, EPH receptor ligand, plasminogen activator inhibitor-1, macrophage migration inhibitory factor, fibronectin receptor, lysyl hydroxylase-2, endothelin-2, B-cell translocation gene-1, reducing agent and tunicamycin-responsive protein, CDC- like kinase-1, quiescin, growth arrest DNA damage-inducible protein 45, DECl, low density lipoprotein receptor related protein, hamster hairy gene homologue, adipophilin, cyclooxygenase-1, fructose bisphosphatase, creatine transporter, fatty acid binding protein, lactate dehydrogena
- Nip3L/Nix, o ⁇ Pim-1 Expression ofthe vector's hypoxia-inducible gene within the tissue should occur at a level which is higher than would occur in the absence ofthe expression vector.
- the coding sequence of the expression vector may be operably linked to its native promoter, another hypoxia-inducible promoter, or a constitutive promoter.
- the proteins ofthe hypoxia-inducible genes may be introduced into the tissue directly to enhance the hypoxic response ofthe tissue and for treatment of hypoxia. Delivery ofthe proteins may be achieved through the use of liposomes, hydrogels, controlled-release polymers, or any ofthe other vehicles known in the art to be useful for the delivery of polypeptides as drugs, e) Methods for Identifying Stress-Inducible Genes
- RDA Representational Difference Analysis
- the present invention provides for methods of identifying both stress- inducible and stress-repressible genes.
- the methods identify differences between 5 mRNA from cell populations exposed to different stress conditions.
- a representative protocol for the identification of stress-inducible genes is outlined in detail in a specific example below (Example 1).
- the method for identifying stress-inducible or stress-repressible genes and fragments of genes involves first subjecting one of two populations of cells to o stress prior to preparation of two cDNA libraries from the mRNA libraries ofthe two populations. Protocols for the generation of cDNA libraries through reverse transcription of mRNA sequences are well known in the art and kits for doing so are commercially available (from Gibco BRL, for instance).
- the cDNAs are synthesized by using a mixture of 5 oligo-dT primers containing equal proportions of oligomers having a G, A, or C residue at the 3 '-end ("indexed" or "registered” primers).
- oligo-dT primers also have a defined DNA sequence (20 to 24 base pairs in length) that is incorporated into each cDNA fragment. This tag permits the use of two PCR primers to specifically amplify the 3 '-end of each cDNA.
- the two cDNA libraries are digested separately with restriction enzymes and then linker sequences are ligated to the ends ofthe digested cDNA fragments, as shown in Fig. 7. Restriction digests and ligation of linkers may be performed in any manner known to those skilled in the art. Some examples of such methods may be found in Sambrook et al.
- the cDNA library from one ofthe two cell populations is amplified with tagged oligonucleotide primers by means ofthe polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- the "tag" on the oligonucleotide primers is biotin.
- any chemical or biological moiety which provides a means of selection or isolation ofthe tagged entity is suitable as a tag.
- use of biotin as a tag allows for removal ofthe tagged sequences on a streptavidin resin.
- oligonucleotides bearing a thiol group may instead be used as the tagged primer, since oligonucleotides with attached thiol groups can be retained on a variety of affinity resins, such as thiopropyl sepharose columns or mercurial resins.
- affinity resins such as thiopropyl sepharose columns or mercurial resins.
- the cDNA library from the stressed cells is amplified with normal, non- tagged, oligonucleotide primers in a separate polymerase chain reaction.
- the cDNA PCR-amplified in this manner is referred to herein as "tester" cDNA.
- the non-tagged, amplified, tester cDNA is heated and then reannealed in the presence of a large excess (typically about 5- to about 100-fold) ofthe tagged, amplified, driver cDNA. See Fig. 8.
- those DNA strands which either are themselves tagged or are duplexed with tagged DNA are removed from the mixture. This removal is typically done via exposure ofthe mixture of DNA strands to a resin or matrix which has affinity for the tag used on the primers earlier.
- magnetic beads coated with streptavidin are used.
- Other resins, such as streptavidin agarose could be used in conjunction with a biotin tag.
- Tagged single-stranded or duplex cDNA will be retained on the affinity resin, and the non-tagged species, which are not retained, can be found in the flowthrough or supernatant.
- the cDNA from the non- stressed cell population is "subtracted” from the cDNA ofthe stressed cell population.
- the remaining, non-tagged cDNA library is said to be "enriched”.
- the remaining, non-tagged cDNA sequences are then again amplified by means of the polymerase chain reaction with non-tagged primers.
- the non- tagged cDNA library is again heated and reannealed in the presence of a large excess (typically about 5- to about 100-fold) ofthe original tagged cDNA library. Removal of all tagged DNA molecules and reamplification of remaining tagged sequences again follows. The combination of steps involving heating and reannealing, removed tagged molecules, and reamplifying remaining, non-tagged molecules constitutes one round.
- the methods ofthe present invention involve repeating the rounds from zero to many times. In a preferred embodiment, the method involves a total of approximately 3 to 5 rounds.
- the method involves performing the steps as described above in parallel with a second set of steps in which the cDNA library from the stressed population of cells is instead subtracted from the cDNA library from the non-stressed population.
- the cDNA library from the stressed cell population is amplified with tagged primers and the cDNA library from the non-stressed cell population is amplified with non-tagged primers.
- the original cDNA ofthe stressed cell population is repeatedly subtracted from the cDNA ofthe non-stressed cell population, and separately, the original cDNA ofthe non- stressed cell population is repeatedly subtracted from the stressed cell population.
- one ofthe two enriched cDNA libraries obtained from the two sets of steps is subtracted from the other enriched cDNA library. Which enriched library is subtracted from which is entirely dependent upon whether stress-inducible or stress-repressible sequences are sought. If stress-inducible sequences are sought, the enriched, non- stressed cDNA library is subtracted from the enriched, stressed, cDNA library. If stress-repressible sequences are sought, the enriched, stressed-cell cDNA library is subtracted from the enriched non-stressed-cell cDNA library.
- the final subtraction step of one enriched library against another is beneficial since the initial subtraction rounds ofthe procedure tend to remove only the cDNAs that are in common and present at high frequency in the two populations, because cDNA fragments derived from rare messages will initially be present at such low concentrations that they might not find a complementary strand during the hybridization step.
- the rare sequences will begin to increase in concentration so that they can then be effectively subtracted.
- the rarest sequences from both conditions are enriched in the libraries, and subtraction of one enriched library from another yields an effective isolation of either stress-inducible or stress-repressible genes.
- the enriched cDNA library may be cloned and sequenced using any one ofthe multitude of techniques known to those skilled in the art.
- a particularly convenient method of inserting PCR-amplified DNA strands into vectors suitable for cloning and sequencing, known as "T-A cloning", is commercially available from companies such as Invitrogen and Novagen.
- T-A cloning is commercially available from companies such as Invitrogen and Novagen.
- Other alternative methods can be found in Molecular Cloning: A Laboratory Manual, 2nd. ed, Vol. 1-3, eds. Sambrook et al., Cold Spring Harbor Laboratory Press (1989).
- the stress to which one ofthe two cell populations is exposed is hypoxia.
- the method may also be applied to the investigation of responses to other stresses, such as ionizing radiation, heat, glucose starvation, hypothermia, or pH change.
- the response to a stress such as a toxin or a drug may be investigated by employment ofthe disclosed method.
- HCE.E6.E7 Normal human cervical epithelial cells stably immortalized with the human papillomavirus E6 and E7 oncoproteins (HCE.E6.E7) served as the starting material for the construction of a cDNA library enriched by representational difference analysis (RDA). HCE.E6E7 were cultured in synthetic medium PFMR-
- the linker pair of SEQ ID NO: 13 and SEQ ID NO: 14 was used for the hypoxically incubated cell cDNAs.
- the two separate linker strands were dissolved in 10 mM Tris-HCl (pH 7.6), 10 mM MgCl2 buffer ( 10 ⁇ M of each oligomer), then heat-denatured and slowly cooled to room temperature before use in a ligation reaction.
- the excess linkers were removed by gel filtration through a spin- column containing Sephacryl S-300HR.
- the linker-ligated cDNA fragments were collected in the microfuge tube while the excess unligated linkers were trapped in the Sephacryl with other low molecular- weight components.
- the gel-filtered, linker-ligated cDNA fragments were then lyophilized to dryness.
- the linker-ligated cDNA fragments were amplified by a single-primer PCR technique. Again, if the preparation was to be used as the driver cDNA, it was amplified by using PCR primers with a biotin residue at the 5 '-end. If the preparation was to be used as the test cDNA from which the driver is used to subtract sequences, then it was amplified by using untagged primers.
- the ligated cDNA (0.1 ⁇ g aliquot) was amplified in a standard PCR buffer containing 1 ⁇ M primer, 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl 2 , and 0.01% gelatin.
- the nicked PCR template had to be repaired by TAQ polymerase during a 5-min extension reaction at 72 ° C.
- a standard PCR reaction of 35 cycles (94 °C, 30s; 56 °C, 30s; 72 °C, 60s) was performed in a Perkin Elmer DNA Thermal Cycler.
- the oligonucleotide primers used in the amplification step were as follows:
- the first round of subtraction was performed by mixing 3 ⁇ g ofthe biotinylated driver cDNA with 0.1 ⁇ g ofthe test cDNA.
- the mixture was lyophilized in a 0.5 mL microfuge tube and carefully redissolved in 2 ⁇ L of 50 mM HEPES (pH 7.5), 10 mM EDTA, 1.5 mM NaCI, and 2% sodium dodecyl sulfate (SDS). This very small amount of solution was overlaid with 50 ⁇ L of mineral oil to prevent evaporation, and the tube was place in the thermal cycler and heated at 95 °C for 10 min.
- the biotinlyated cDNAs and any hybridized sequences were removed by mixing the diluted solution with a 100 ⁇ L slurry containing 1 mg of M-280 Streptavidin Dynabeads (Dynal) in the same incubation buffer. The incubation was continued at room temperature for 30 min with slow tumbling. The beads were then pelleted to the bottom ofthe tube by using a magnet and the supernatant was removed and desalted by passing through a 1 mL Sephacryl spin column as described above. The cDNA solution was then lyophilized and redissolved in 10 ⁇ L of water.
- the small amount of cDNA remaining after subtraction was reamplified by PCR using the same primers.
- a single-stranded binding protein was added to the PCR reaction mixture used to reamplify the subtracted cDNA fragments: 1 ⁇ L (one-tenth volume) ofthe subtracted cDNA preparation was placed in 100 ⁇ L of PCR buffer containing 1 ⁇ g of Escherchia Coli single-stranded binding protein (Perfect MatchTM, Stratagene).
- the cDNA was amplified during 25 PCR cycles (94 °C, 30 s; 54 °C, 30 s; 72 °C, 60 s), and the product was analyzed by ethidium agarose gel electrophoresis. The appearance of this reamplified cDNA was similar to that ofthe initial material described above.
- the subtraction libraries were prepared in parallel, so that the library enriched for sequences expressed under hypoxic conditions was prepared at the same time as the library enriched for sequences expressed under normoxic conditions.
- the driver used for the initial rounds of subtraction was the original set of cDNA fragments.
- the enriched library prepared in parallel was used as the driver for the fourth round. In this way, the rarest sequences from both conditions were enriched in the final library. For instance, to obtain hypoxically induced sequences in this final round, the cDNA library enriched for sequences expressed under normoxic conditions served as the driver library and the cDNA library enriched for sequences expressed under hypoxic conditions served as the test library.
- cDNA fragments were sequenced from each ofthe two enrichment libraries produced by the subtraction protocol of Example 1 from HCE.E6E7 cells cultured under hypoxic and aerobic conditions.
- Four rounds of RDA subtraction ofthe oxic cDNAs from the hypoxic cDNAs generated a population of fragments in one ofthe enrichment libraries representing genes that theoretically are induced by hypoxic treatment.
- Five hundred randomly chosen clones from the cDNA library were partially sequenced. The obtained sequences were analyzed by NCBI-blast to determine the frequency of each ofthe genes/ESTs in the enriched population and to identify whether the isolated, hypoxia-induced ESTs corresponded to previously identified genes or ESTs.
- the northern blot assays were used to confirm that, ⁇ -tubulin mRNA, detected in the HCE.E6E7 aerobic enrichment library, decreased in response to hypoxia in HCE.E6E7 cells, whereas mRNA corresponding to the HIG2 EST, found in the hypoxic enrichment library, strongly increased under the same hypoxic conditions.
- Hybridization was carried out in 0.5 M Na2HPOzi, 7% SDS, 1 mM EDTA at 56°C for HIGl and 65 «C for HIG2, washed to 0.2-0.5 x SSC at 56°C or 65°C, exposed to a phosphorimager plate, and visualized on a Storm 860 phosphoimager (Molecular Dynamics).
- the hypoxia-inducibility of ESTs as determined by Northern blot is summarized in Table 2, above.
- the HIGl and HIG2 sequences both demonstrated hypoxia-inducibility in the Northern blot assay.
- HCE.E6E7s SiHa cervical squamous carcinoma, MCF-7 breast carcinoma, HI 299 lung carcinoma, Hctl 16 colonic carcinoma cells; human cervical fibroblasts (HCFs) and HCF.E6E7s] probed for HIG2 expression demonstrated the following: (1) the gene is expressed as a single 1.5 kb transcript (the original EST cross-hybridizes with unknown 1.6- and 4-kb transcripts in HCE.E6E7s); (2)
- HIG2 mRNA increases from undetectable in 21% O2 (air) to abundant in 0.02% O2 in HCE.E6E7, SiHa, and MCF-7 cells after 6 h of hypoxia; (3) HIG2 is moderately expressed in HI 299 and Hctl 16 cells after 6 h of hypoxia; (4) there is no detectable HIG2 mRNA in HCFs and HCF.E6E7s; (5) in SiHa cells, HIG2 remains elevated for 48 h of hypoxia but decreases moderately by 72 h of exposure; and (6) no HIG2 induction is found in SiHa cells 6 h and 24 h after treatment with UV-C (20 J/m 2 ), ⁇ -irradiation (6 Gy), MMS (100 ⁇ g/mL for 1 h), serum deprivation (0.1%), or glucose starvation (4%, ⁇ 1 mM); (7) HIG2 expression is extinguished after exposure of hypoxic cells to 2 hours of reoxygenation.
- hypoxia inducibility of HIGl has been found to range between about 2-fold and about 5-fold across a variety of different human cell lines studied.
- the hypoxia-inducibility of HIG2 ranges between about 10- and about 20-fold across the various human cell lines studied. (See also Example 4, below).
- HIGl and HIG2 are also known genes identified by the subtraction method in Example 1 and confirmed by Northern blots to be hypoxia inducible. These genes are also listed in Table 2. ESTs corresponding to the genes of annexin V, lipocortin 2, hnRNP Al , Ku (70) autoantigen, glyceraldehyde-3 -phosphate dehydrogenase, ribosomal L7, acetoacetylCoA thiolase, and PRPP synthetase were identified by multiple hits in the hypoxia screen. All of these previously known genes were confirmed to be hypoxia-inducible by Northern blot.
- acetoacetyl CoA thiolase sequence tag is listed as induced, the reported, major RNA (1.8 kb) for the gene does not change. However, there is a larger, hybridizing, RNA species (4.2 kb) that is induced after 24-48 h hypoxia (data not shown).
- ESTs corresponding to glyceraldehyde 3 -phosphate dehydrogenase were especially prevalent amongst the cDNA clones.
- the hypoxia- induced expression of glyceraldehyde-3 -phosphate dehydrogenase had been previously identified only in normal, non-transformed cells.
- the HIG2 EST (142 bp) was used to probe a conventional cDNA library constructed from mRNA isolated from SiHa cells exposed to 16 h hypoxia to obtain the full-length cDNA clone HIG2.
- This library was probed with radiolabelled HIG2 tag using conventional methods.
- Full length HIGl was isolated by first identifying overlapping ESTs from the NCBI human EST database, until a full length sequence was generated (1.35 kb). PCR primers were then synthesized corresponding 5 ' and 3 ' UTRs in order to amplify the complete sequence using RT-PCR of SiHa RNA isolated after a 16 h hypoxia treatment. The full-length HIGl cDNA was then cloned and sequenced to confirm the predicted sequence.
- the full-length cDNA sequence of HIGl is shown in Figure 1 A.
- the full- length cDNA sequence of HIG2 is shown in Figure 2A.
- the translations ofthe putative open reading frames from HIGl and HIG2 are listed in Figure IB and 2B, respectively, and both encode small peptides (95 and 64 aa residues respectively) without obvious functional motifs.
- Example 4 Hypoxic induction of HIGl and HIG2 in cervical cancer cell lines. Because HIGl and HIG2 represent two novel genes whose functions are unknown, these genes were investigated in more detail.
- the expression of HIGl and HIG2 was examined in a series of human cervical cancer cell lines (SiHa, CaSki and C33a) under oxic and hypoxic conditions in vitro.
- the cell lines SiHa, CaSki and C33a were obtained from the ATCC and were cultured in Dulbecco's modified Eagle's medium (DMEM) or RPMI1640 supplemented with 10% fetal bovine serum.
- DMEM Dulbecco's modified Eagle's medium
- RPMI1640 supplemented with 10% fetal bovine serum.
- HIG2 is more consistently induced from low basal levels in all the cervical cancer cells tested.
- the major HIG2 mRNA species is 1.4 kb in length, but there are two other mRNA species of minor abundance (8.0 and 9.0 kb) that are induced with identical kinetics to the major species.
- Example 5 Hypoxic induction of HIGl and HIG2 in tumor xenografts.
- hypoxic induction of HIGl and HIG 2 in vivo was also tested in tumor xenografts generated from the C33a cell line by Northern blot analysis of total tumor RNA.
- Gene expression in untreated xenografts was compared to that in xenografts that were made hypoxic by treatment ofthe host animal with flavone acetic acid (FAA) 24 hours prior to explantation and RNA isolation.
- FFAA flavone acetic acid
- To generate tumor xenografts 2.5-5 x 10 ⁇ cells were injected subcutaneously into the flank of scid mice and allowed to grow into tumors that reached 1-2 cm in diameter before harvest.
- FAA Lipha Chemical, NY
- FAA treatment resulted in increased tumor hypoxia as measured by ependorff electrode and increased HIGl and HIG2 expression by 1.2 and 2.4 fold respectively.
- the moderate level of HIGl induction in vivo is not unexpected, due to the in vitro data.
- the portion ofthe human gene used for a probe in these experiments has low homology with mouse RNA and under the conditions used, did not cross- hybridize.
- Example 6 Specificity ofthe induction of HIGl and HIG2.
- HIGl and HIG2 induction is unique to hypoxic stress, or if it is elicited by other tumor microenvironment stresses such as glucose deprivation, serum starvation, or by genotoxic stresses such as UV or ionizing radiation.
- hypoxia-mimetic, iron-chelating compound desferoxamine that has been shown to induce expression from HIF-1 responsive genes.
- cells were plated overnight and then treated the next day with either 256 nm UV at 1.2 J/m ⁇ /sec, or gamma irradiation from 137£ s source at 3.8 Gy/min.
- Glucose and serum deprivation experiments were performed by washing the cells three times in phosphate-buffered saline (PBS) and replacing the indicated media (glucose free RPMI with dialyzed serum, or 0.1% FBS RPMI).
- PBS phosphate-buffered saline
- HIGl was poorly responsive to hypoxic stress over this timecourse, but strongly induced by glucose deprivation.
- HIG2 was induced strongly by hypoxia, the hypoxia-mimetic stress desferoxamine (DFO), and glucose deprivation.
- UV light seemed to have little effect upon either HIGl or HIG2 expression.
- ionizing radiation did not change HIGl expression levels, it did result in a moderate 2.5 fold induction of HIG2 by 24 hours.
- HIF-1 may be important in HIG2 expression.
- Example 7 Identification of HIGl and HIG2 sequences from non-human species.
- a search ofthe NCBI-dbEST database for fragments of genes from other species that might represent evolutionarily conserved orthologues identified overlapping mouse EST fragments that encode for similar peptides to the human version of HIGl and HIG2.
- the murine HIGl and HIG2 orthologues are shown in Figures 3 A and 5A, respectively. These mouse genes code for predicted peptides ( Figures 3B amd 5B, respectively) with 84% and 76% identity to the human peptides respectively.
- a sequence comparison ofthe HIGl homologues is shown in Figure 6 A.
- a sequence comparison of the HIG2 homologues is shown in Figure 6B.
- RNA isolated from SCCVII cells Both mHIGl (murine HIGl) and mH/G2 (murine HIG2) have hypoxia-inducible species of RNA by this analysis.
- Murine HIGl has two major RNA species that strongly hybridize to the probe, at approximately 1.2-1.4 kb in length. The larger message is modestly induced, while the smaller message is strongly induced to approximately 5 fold by a 12h exposure to hypoxia.
- Murine H/G2 also has two RNA species at approximately 1.4 and 2.2 kb. Both the murine HIG2 rnRNAs seem to be mildly hypoxia- inducible with 2-3 fold induction by 6-12 hours. For comparison, the same blot was probed with vascular endothelial growth factor (VEGF) and this message shows an approximately 5-fold induction by 6h.
- VEGF vascular endothelial growth factor
- Nylon filters containing GDA arrays were purchased from Genome Systems (St Louis, MO) that have affixed to them nucleic acids that were originally characterized by the I.M.A.G.E. consortium (LLNL). This array represents 18,394 cDNA clones that have been categorized as either known genes or ESTs (expressed sequence tags) isolated by the consortium. This filter was used to quantitatively determine the mRNA expression levels of all these arrayed cDNAs in SIHA tumor cells both under oxic conditions and hypoxic conditions (18 hrs, ⁇ 0.2 %). Messenger RNA was isolated from control and hypoxic SIHA cells and cDNA probe was generated using MoML reverse transcriptase.
- Example 9 Analysis of Gene Expression under Hypoxia using GEM ⁇ M microarrays
- the hypoxic induction of genes in FaDu cells was analyzed by comparing the expression of genes in FaDu cells exposed to hypoxic conditions (5% C ⁇ 2/5% H2/90% N2 for 16 hours at 37 °C) to those exposed to normal, oxic conditions. This differential expression was analyzed using GEM ⁇ M technology provided by Genome Systems Inc. Messenger RNA (mRNA) was extracted from hypoxic FaDu cells, and separately from oxic FaDu cells.
- mRNA Messenger RNA
- the poly A+ RNA was isolated from total RNA essentially according to the standard Genome Systems Inc. protocol, as follows. To purify polyA RNA, the total RNA sample was passed twice over OligoTex mRNA isolation columns from Qiagen. After the elution ofthe polyA RNA, the polyA RNA was ethanol precipitated, and the final product was brought up in DEPC H2O or TE. For 50 ⁇ l of elution from the OligoTex column, 40 ⁇ l of IX TE and 1 ⁇ l of glycogen (5 mg/ml) was added. Then 120 ⁇ l of 100% EtOH was added and the sample was frozen at -80°C for 10 minutes. The sample was then spun at 12,000 x g for 10 minutes at 4°C.
- the supernatant was removed and 250 ⁇ l of 75% EtOH was added.
- the pellet was spun at 12,000 x g for 5 minutes at 4°C.
- the supernatant was again removed and the pellet dried for 10 minutes at room temperature.
- the pellet was then dissolved in DEPC H20 to a concentration of 50 ng/ ⁇ l.
- RNA samples were sent to Genome Systems Inc. to perform a GEM mieroarray analysis.
- fluorescent labeled cDNA probes were prepared by Genome Systems Inc. using standard methodologies familiar to those skilled in the art.
- the cDNA probes corresponding to the mRNA sample from the oxic FaDu cells were labeled with a different, distinguishable fluorescent label than the cDNA probes corresponding to the mRNA sample from the hypoxic FaDu cells.
- the two fluorescent probe samples were then simultaneously applied by Genome Systems Inc. to their Human UniGEM V mieroarray for hybridization to the arrayed cDNA molecules.
- the Human UniGEM V mieroarray contains sequence verified Genome Systems Inc. proprietary cDNA clones representing more than 4,000 known human genes and up to 3,000 ESTs mapped to the UniGene database. (All ofthe genes on the mieroarray were selected for criteria such as known functions, homologies, and presence on the human transcript map.)
- the genes or gene fragments ofthe GEM microrarray (each 500-5000 base pairs in length) are arrayed on glass surface to which they have been chemically bonded.
- the mieroarray was washed free of probe molecules which had not hybridized.
- the different gene/EST sites ofthe GEM mieroarray are then scanned for the each ofthe two fluorescent labels. Presence ofthe fluorescent label at a particular gene site indicates the expression of that gene in the cell corresponding to that fluorescent label.
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EP1064378A2 (en) | 2001-01-03 |
WO1999048916A8 (en) | 2008-05-08 |
WO1999048916A3 (en) | 2000-01-20 |
CA2322843A1 (en) | 1999-09-30 |
JP2002507405A (en) | 2002-03-12 |
AU3455599A (en) | 1999-10-18 |
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