WO2001010199A1 - A knockout mouse for the tumor suppressor gene anx7 - Google Patents
A knockout mouse for the tumor suppressor gene anx7 Download PDFInfo
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- WO2001010199A1 WO2001010199A1 PCT/US2000/021409 US0021409W WO0110199A1 WO 2001010199 A1 WO2001010199 A1 WO 2001010199A1 US 0021409 W US0021409 W US 0021409W WO 0110199 A1 WO0110199 A1 WO 0110199A1
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
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- A01K67/0276—Knockout animals
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
Definitions
- the invention is directed to tumor-susceptible non-human animals.
- the invention further pertains to the use of such animals in the development of anti-cancer agents and therapies.
- Oncogenes are mutated, dominant forms of cellular proto-oncogenes that stimulate cell proliferation, while tumor suppressor genes are recessive and normally inhibit cell proliferation (Cooper, 1995). The loss or inactivation of tumor suppressor genes is widely thought to be one of the contributors to unregulated cancer cell growth. While the discovery and identification of oncogenes has been relatively straightforward, identifying tumor suppressor genes has been much less so (Fearon, The Genetic Basis of Human Cancer (B. Vogelstein et al., eds.) pp. 229-236 (1998)).
- oncogenes and tumor-suppressing genes have a basic distinguishing feature.
- the oncogenes identified thus far have arisen only in somatic cells and thus have been incapable of transmitting their effects to the germ line of the host animal.
- mutations in tumor-suppressing genes can be identified in germ line cells and are thus transmissible to an animal's progeny. About a dozen such tumor suppressor genes have been identified, with the hope that knowledge of their mechanism(s) might yield therapeutically relevant insights.
- Tumor suppressor gene action depends on either mutation or deletion of both tumor suppressor alleles or on a reduction in the absolute level of expressed tumor suppressor protein. In their natural state, tumor suppressor genes act to suppress cell proliferation. Damage in such genes leads to a loss of this suppression, and thereby results in tumorigenesis. Knudson's "two-mutation hypothesis" is a well studied statistical model for tumor suppressor gene action which is based on the epidemiological analysis of retinoblastoma. (Knudson, A.G., Proc. Nat. Acad. Sci. USA. 68:820-823
- Tumor suppressor genes are principally known for control of cell proliferation by their action on the cell cycle.
- Well-studied examples include
- p27 KIP1 Another example of a tumor suppressor gene acting on the cell cycle is the p27 KIP1 gene, also known simply as p27, which physiologically inhibits cyclin-dependent kinases, and thereby blocks cell proliferation (Fero, M. L, et al., Nature 396:177-180 (1998)).
- cyclin dependent kinases that consist of an activating cyclin subunit and a catalytic Cdk subunit (Polyak, K., et al., Cell 78:59-66 (1994)); Hartwell, L., Cell 71:543-546, (1992)); Nurse, P., Nature 344:503-508,(1990)).
- the functions of the respective cyclins and Cdk's in mammalian cells correspond to the different phases of the cell cycle. For example, during the G1 phase, cyclin D-Cdk4/6 and cyclin E-Cdk2 are catalytically active and rate limiting for cell cycle progression.
- D-type cyclins induce the synthesis of D-type cyclins to initiate the G1 phase.
- the D-type cyclins then associate with Cdk4/Cdk6, and the active Cdk's then hyperphosphorylate Rb to drive the cell past the restriction point (Buchkovich, K., et al., Cell 58:1097-1105 (1989)); see Weinberg, R.A., Cell 81:323-330 (1996)).
- Tumor suppressor genes have been found to affect the function of both of these types of subunits.
- tumor suppressor genes can also control cellular differentiation by acting as transcription factors and/or by modulating specific downstream DNA repair targets involved in maintaining genomic integrity.
- the tumor suppressor gene, inactivation of the tumor suppressor gene, p53 is the most common, resulting in a somatic mutation that causes malignancy (Nigro, J.M., et al., Nature 342:705-708 (1989); cf., review by Nguyen and Jameson, 1998).
- a germline mutation for p53 is the basis for a familial cancer, the Li- Fraumeni syndrome (Srivastava, S., et al., Nature 348:747-749 (1990)).
- Li- Fraumeni syndrome Srivastava, S., et al., Nature 348:747-749 (1990)
- p53 works in the following manner: When DNA is damaged, a resulting signal causes stabilization of p53, which in turn causes transcriptional deregulation of p21 , resulting in cell cycle arrest in the G1 phase (Hunter, T., Cell 75:839-841 (1993)).
- tumor suppressor genes have also been implicated in controlling apoptotic cell death (Graeber, T. G., et al., Nature 379:88 (1996)). Again, p53 figures prominently in this process as well (Basu, A., et al., Mol. Hum. Reprod. 4:1099-1109 (1998)).
- the clear message from this brief summary is that the individual tumor suppressor genes cannot be viewed from a single perspective. ln order to study these tumor suppressor genes, model systems must be developed. Recent advances in recombinant DNA and genetic technologies have made it possible to discover and assess new tumor suppressor genes.
- One of the key model systems available is the transgenic animal. Such animals have been engineered to contain gene sequences that are not normally or naturally present in an unaltered animal. The techniques have also been used to produce animals which exhibit altered expression of naturally present gene sequences.
- the present invention provides a transgenic knockout mammal having somatic and germline cells comprising a chromosomally incorporated transgene. At least one allele of a genomic tumor suppressing annexin gene is disrupted by the transgene such that the expression of a tumor suppressing annexin gene is inhibited. This inhibition of the endogenous tumor suppressing annexin gene results in an increased susceptibility to formation of tumors as compared to a wild type mammal.
- the transgenic mammal may be heterozygous for this disruption.
- the genomic tumor suppressing annexin gene is annexin VII.
- the preferred transgenic mammal is a transgenic rodent, and the more preferred transgenic mammal is a mouse.
- Another embodiment of this method is the generation of transgenic embryonic stem cells.
- the method involves the steps of: (a) constructing a transgene construct containing
- Another embodiment of the present invention comprises a method for generating a transgenic mammal having a functionally disrupted endogenous tumor suppressing annexin gene. The method involves the steps of: (a) constructing a transgene construct containing
- a recombination region having all or a portion of the endogenous tumor suppressing annexin gene and (ii) a marker sequence which provides a detectable signal for identifying the presence of the transgene in a cell;
- step (d) transferring the cells of step (c) into a blastocyst and implanting the resulting chimeric blastocyst into a female mammal
- the preferred transgenic mammal for this method is a transgenic rodent, and the more preferred transgenic mammal is a transgenic mouse.
- the most preferred transgenic stem cell is a transgenic mouse stem cell.
- the most preferred tumor suppressing annexin gene is an annexin VII gene.
- Another embodiment of the invention comprises a method for evaluating the carcinogenic potential of a test agent by contacting a transgenic mammal containing a disrupted tumor suppressing annexin gene with a test agent, and comparing the number of transformed cells in a sample of the treated transgenic mammal with the number of transformed cells in a sample from an untreated transgenic mammal. Alternatively, one can compare the number of transformed cells in a sample of the treated transgenic mammal with a control agent. The difference in the number of transformed cells in the treated transgenic mammal, compared to the number of transformed cells in the absence of treatment or in the presence of a control agent, indicates the carcinogenic potential of the test agent.
- Another embodiment comprises a method of treating mammalian cancer cells lacking endogenous wild-type annexin protein, which comprises introducing a wild-type annexin tumor suppressor gene into the mammalian cancer cells, whereby the phenotype of abnormal proliferation of these mammalian cancer cells' is suppressed by the expressed annexin protein.
- the mammalian cancer cell lacks at least one allele of the wild-type annexin tumor suppressor gene.
- the mammalian cancer cell is an osteosarcoma cell, lung carcinoma cell, lymphoma cell, leukemia cell, soft-tissue sarcoma cell, breast carcinoma cell, bladder carcinoma cell, or prostate carcinoma cell. More preferably, the mammalian cancer cell has a mutated annexin tumor suppressor gene.
- Another embodiment comprises a method for treating a patient having a neoplasm characterized by abnormally proliferating cells in a mammal comprising administering an effective dose of a recombinant replication deficient virus comprised of a DNA segment that expresses a protein having the cell growth inhibition activity of the annexin tumor suppressor gene product.
- the patient has a neoplasm comprised of cells that substantially lack a functional annexin tumor suppressor gene product.
- the neoplasm is comprised of cells that substantially lack a functional annexin VII gene product.
- the replication-deficient virus is selected from the group consisting of a retrovirus, an adenovirus, a herpes simplex virus, a vaccinia virus, a papillomavirus, and an adeno-associated virus.
- the virus is a recombinant replication deficient adenovirus expression vector.
- Another embodiment comprises a composition for therapy of a neoplastic disease characterized by the lack a functional annexin tumor suppressor gene product.
- the treatment comprises administering a therapeutically effective dose of a recombinant replication deficient adenovirus in a pharmaceutically deliverable form.
- Another embodiment comprises a method of treating a disease characterized by abnormally proliferating cells in a mammal, by:
- Another embodiment is a DNA construct containing a recombination region having all or a portion of the endogenous tumor suppressing annexin gene and a marker sequence which provides a detectable signal for identifying the presence of the transgene in a cell.
- the construct is KSBX.pPNT, as described below.
- Another embodiment is an expression vector comprising an isolated polynucleotide sequence, which hybridizes to an annexin sequence under standard hybridization conditions and encodes a protein having the cell growth inhibition activity of an annexin protein.
- the expression vector is selected from the group consisting of a retrovirus, an adenovirus, a herpes simplex virus, a vaccinia virus, a papillomavirus, and an adeno-associated virus. More preferably, the expression vector is a recombinant replication deficient adenovirus, and the polynucleotide sequence corresponds to the annexin VII gene.
- Another embodiment comprises a cell transformed by the expression vector mentioned above.
- Another embodiment comprises a method for identifying a polymorphism or a mutation in an exon of a human or animal tumor suppressor annexin VII gene. This method involves: (a) incubating, under amplification conditions, a sample of genomic DNA
- DNA comprising an exon of a human or animal tumor suppressor annexin gene with a primer pair comprising:
- a first primer which hybridizes to a promoter region or to an intron upstream of the exon
- a second primer which hybridizes to the 3'-noncoding region or to an intron downstream of the exon, such that at least one primer of the primer pair hybridizes to an intron
- step (d) comparing the sequence of the exon obtained in step (b) to the sequence of a corresponding wild type exon.
- the exon is selected from the group consisting of exon 4, exon 5, exon 6, exon 7, and exon 8.
- Another embodiment comprises a pharmaceutical preparation comprising an expression vector comprising an isolated polynucleotide sequence, which hybridizes to an annexin sequence under standard hybridization conditions and that encodes a protein having the cell growth inhibition activity of annexin VII, and a physiologically tolerable diluent.
- Figure 1 A depicts the targeting vector, KSBX.pPNT.
- Figure 1B depicts the restriction map of the mouse anx7 gene.
- Figure 2 depicts the southern blot analysis of the targeting construct.
- Figure 3A depicts the PCR analysis of ES cell clones transfected with
- Figure 3B depicts the southern blot analysis of genomic DNA digested with
- Figure 4 illustrates a schematic sketch of the generation of chimera.
- Figure 5A illustrates a recipient nonagouti (a/a) (black female) C57B1/6 blastocyst.
- Figure 5B illustrates the synexin transgenic chimeric mouse generated by injecting ES cells (with targeting construct) derived from an agouti (A/A) mouse (brown).
- Figure 5C illustrates the all agouti progeny from this chimeric mouse, when bred to a C57B1/6 black male or C57B1/6 black female.
- Figure 6 illustrates the PCR analysis of genomic DNA from yolk sac of anx7(+/+), anx7(+/-), and anx7(-/-) embryos.
- Figure 7A illustrates the increased growth of an anx7(+/-) mouse compared with the control mouse.
- Figure 7B Representative growth curve of thirty anx7(+/+) and anx7(+/-) littermates as a function of age.
- Figure 8. The percent increase in organ weights of control mice compared with anx7 (+/-) transgenic mice.
- Figure 9A Metastatic Lymphosarcoma of the thymus stained with hematoxylin and eosin (H and E) from a control littermate of an anx7 (+/-) mouse.
- FIG 9B Metastatic Lymphosarcoma of the thymus stained with hematoxylin and eosin (H and E) from an anx7 (+/-) mouse.
- Figure 10A Lymphosarcoma of the thymus metastatic to the lung from a control littermate of an anx7 (+/-) mouse.
- Figure 10B Lymphosarcoma of the thymus metastatic to the lung from anx7
- FIG 11 A Hepatocellular carcinoma in liver tissue from a control littermate of an anx7 (+/-) mouse.
- Figure 11B Hepatocellular carcinoma in liver tissue from an anx7 (+/-) mouse.
- Figure 12A Growth suppression of tumor cells by anx7 and p53 in DU145, a prostate tumor cell line, transfected with pcDNA3.1 alone (vector) or vector expressing anx7 (+anx7) or p53 (+ p53).
- Figure 12B Growth suppression of tumor cells by anx7 and p53 in LNCaP, a prostate tumor cell line, transfected with pcDNA3.1 alone (vector) or vector expressing anx7 (+anx7) or p53 (+ p53).
- Figure 12C Growth suppression of tumor cells by anx7 and p53 in MCF-7, a breast cancer cell line, transfected with pcDNA3.1 alone (vector) or vector expressing anx7 (+anx7) or p53 (+ p53).
- Figure 12D Growth suppression of tumor cells by anx7 and p53 in Saos, an osteosarcoma cell line, transfected with pcDNA3.1 alone (vector) or vector expressing anx7 (+ anxT) or p53 (+ p53).
- Figure 13 depicts EM pictures showing pancreatic ⁇ -cells from anx7 (+/-) and control (+/+) littermates.
- Figure 14 depicts the dominant negative activity of ANX7J mutant when mixed with wild typ ANX7 in an in vitro membrane fusion assay.
- Figure 15 depicts the phoshorylation of ANX7 by different protein kinase subunits.
- Figure 16A depicts the frequency of ANX7 expression in a stage specific manner in a prostate tissue microarray containing 301 specimens from all stages of human prostate tumor progression.
- Figure 16B depicts H&E stained sections (left side) and brown diaminobenzidine (DAB) stain from an anti-ANX-7 monoclonal antibody immunostaining (right side) of typical examples taken from samples of the human tumor microarray shown in Figure 16A.
- BPH benign prostatic hypertrophy
- PIN primary intraepithelial neoplasms.
- Figure 17A depicts the immunostaining of human prostate cancer cells by Ki67 antibody.
- Figure 17B depicts example histological images stained by antibody Ki67 (left column) or consecutive sections stained for ANX7 protein (right column).
- Ki67 benign prostatic hypertrophy
- PIN primary intraepithelial neoplasms
- Figure 18 depicts data from a breast cancer tissue microarray; normal breast tissue expresses virtually no ANX7 (see Figure 26). The percent of samples that are positive for ANX7 become progressively greater as the diagnostic category gets "worse”. Fractions are number of cases that are positive/ total cases in this diagnostic category. Abbreviations: dcis (ductal carcinoma in situ).
- FIG. 19 Survival curves for patients in the pathological stage pT:1.
- Figure 20 Survival curves for patients in the pathological stage pT:2.
- Figure 21 Survival curves for patients in the pathological stage pT:3.
- FIG. 23 Survival curves for patients in the clinical stage BRE:1.
- Figure 24 Survival curves for patients in the clinical stage BRE:2.
- Figure 25 Survival curves for patients in the clinical stage BRE:3.
- ANX7 3,
- FIG 26 Tumor types in which the normal tissue is low in ANX7, and where some of the tumors tend to be higher. Data are given as percent of tumor cells positive for ANX7 protein. Upper left panel: Breast cancer; see Figure 18 for these data without control. Upper right panel: Sarcoma's. Lower left panel: lung cancer; note that normal adult lung is virtually deficient in ANX7, while fetal lung is 25% positive. Carcinoid, small, and large cell lung cancers are profoundly distinct from ANX7 levels found in normal tissue. Lower right panel: testes; ledig tumor seems to be the most distinct from normal tissue.
- Figure 27 Tumor types in which the normal tissue is high in ANX7, and where some of the tumors tend to be low.
- Upper left panel skin; melanomas appear to be the most distinct.
- Upper right panel lymphoid tissue; the three types of tumors studied appear to be distinct from normal lymph node tissue.
- Lower left panel prostate; see earlier parts of this description for detailed studies on the prostate.
- Lower right panel nerve. Another type of tumor with as aspect of this pattern of behavior is gynecological (see Figure 28).
- FIG. 28 Tumor types in which the normal levels of ANX7 protein can be ca. 50%, and where tumors also vary in the same range.
- Upper left panel salivary gland tumors; note that adenocarcinoma is completely positive.
- Upper right panel renal.
- Lower left panel gynecological; while the normal uterine cervix is completely positive, the normal placenta is intermediate.
- Figure 29 Other tumors for which controls are not necessarily obvious.
- Figure 30 Brain; levels of ANX7 are generally low in this tissue, and in derived tumors.
- Figure 31 Gl tumors vary in level of ANX7. Normal exocrine pancreas is
- a cell's "genome” consists of all of its heritable DNA (either chromosomal or non-chromosomal (i.e. episomal, viral, etc.).
- One of the two alleles of a gene is provided by the animal's or cell's maternal parent; the other set is provided by its paternal parent.
- the diploid nature of human and animal cells is described by Lewin, B. (Genes V, Oxford Univ. Press, New York (1994)), and in other similar treatises.
- An allele may be capable of being expressed by the natural processes operating in a cell. The expression of an allele results in the production of a gene product.
- the term "allele” as used herein is intended to denote any nucleotide sequence that affects the expression of a particular gene. It thus is intended to refer to any enhancer, promoter, processing, intervening, coding or termination sequence or region of the gene, or any sequence that stabilizes the gene product, or its mRNA, etc.
- An allele of a gene is said to be "mutated” if (1 ) it is not expressed in a cell or animal, (2) the expression of the allele is altered with respect to the expression of the normal allele of the gene, or (3) the allele expresses a gene product, but that gene product has altered structure, activity, or characteristics relative to the gene product of a normal allele of that gene.
- mutation or “mutated” as used herein are intended to denote an alteration in the "normal” or “wild-type” nucleotide sequence of any nucleotide sequence or region of the allele.
- wild-type are intended to be synonymous, and to denote any nucleotide sequence typically found in nature.
- mutated and “normal” are thus defined relative to one another; where a cell has two chromosomal alleles of a gene that differ in nucleotide sequence, at least one of these alleles is a "mutant" allele as that term is used herein.
- an "endogenous tumor suppressing gene” is the "wild-type” tumor suppressing gene that exists normally in a cell, and a “mutated annexin tumor suppressor gene” defines a gene that differs in nucleotide sequence from the wild-type gene.
- Mutations may have one of three effects.
- One effect is that a mutation may detectably alter the expression of an allele. This denotes any change in nucleotide sequence affecting the extent to which the allele is transcribed, processed or translated. Such alterations may be, for example, in (1 ) an enhancer, (2) a promoter, (3) a coding or termination region of the allele, (4) a mutation which stabilizes the gene product, or its mRNA, etc.
- a second effect is that a mutation may detectably alter the activity of an allele.
- This denotes any change in nucleotide sequence that alters the capacity of the expressed gene product to mediate a function of the gene product.
- Such mutations include changes that diminish or inactivate one or more functions of the expressed product.
- such mutations also include changes that result in an increase in the capacity of the gene product to mediate any function (for example, a catalytic or binding activity) of that gene product.
- a mutation may detectably alter the function of an allele. This denotes any change in nucleotide sequence that alters the capacity of a binding molecule (such as a binding protein) to specifically bind to the allele.
- the mutations that cause these effects in a tumor suppressing annexin gene can be readily identified by sequencing, tumorigenicity, resilience to tumorigenicity, binding activity, etc. (see, for example, Eliyahu et al., Nature 312:646-649 (1984); Finlay et al., Molec. Cell. Biol. 8:531-539 (1988); Nigro,
- An allele is said to be "chromosomal" if it either is, or replaces, one of the two alleles of a gene which a cell inherits from its ancestors, or which an animal inherits from its parents.
- An allele is not "chromosomal,” as that term is used herein, if the allele increases the copy number of the total number of alleles of a particular gene which are present in a cell.
- the cells that can be produced in accordance with the present invention include both “germ-line” and “somatic” cells.
- a “germ-line” cell is a sperm cell or egg cell, or a precursor or progenitor of either; such cells have the potential of transmitting their genome (including the altered tumor-suppressor allele) in the formation of progeny animals.
- a “somatic” cell is a cell that is not a germ-line cell.
- transgene refers to a nucleic acid sequence which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
- a transgene can be operably linked to one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
- exemplary transgenes of the present invention encode, for instance an annexin polypeptide, preferably an ANX7-polypeptide.
- Other exemplary transgenes are directed to disrupting one or more genomic annexin genes by homologous recombination with genomic sequences of an annexin gene, preferably an anx7 gene.
- transgenic animals of the present invention all include within a plurality of their cells a transgene of the present invention, which transgene alters the phenotype of the "host cell” with respect to regulation of cell growth, death and/or differentiation. Since it is possible to produce transgenic organisms of the invention utilizing one or more of the transgene constructs described herein, a general description will be given of the production of transgenic organisms by referring generally to exogenous genetic material. This general description can be adapted by those skilled in the art in order to incorporate specific transgene sequences into organisms utilizing the methods and materials described below.
- the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal.
- Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell.
- the specific line(s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.
- the haplotype is a significant factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, Me.).
- the line(s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i.e., obtained from animals which have one or more genes partially or completely suppressed).
- the transgene construct may be introduced into a single stage embryo.
- the zygote is the best target for micro-injection.
- the use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). As a consequence, all cells of the transgenic animal will carry the incorporated transgene.
- the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below. In some species such as mice, the male pronucleus is preferred. It is most preferred that the exogenous genetic material be added to the male DNA complement of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus.
- the exogenous genetic material should be added to the male complement of DNA or any other complement of DNA prior to its being affected by the female pronucleus.
- the exogenous genetic material is added to the early male pronucleus, as soon as possible after the formation of the male pronucleus, which is when the male and female pronuclei are well separated and both are located close to the cell membrane.
- the exogenous genetic material could be added to the nucleus of the sperm after it has been induced to undergo decondensation.sperm containing the exogenous genetic material can then be added to the ovum or the decondensed sperm could be added to the ovum with the transgene constructs being added as soon as possible thereafter. Any technique which allows for the addition of the exogenous genetic material into nucleic genetic material can be utilized so long as it is not destructive to the cell, nuclear membrane, or other existing cellular or genetic structures. Introduction of the transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
- the exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection.
- Microinjection of cells and cellular structures is known and is used in the art.
- the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1 -2pl of DNA solution.
- the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention.
- the number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1 ,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. As regards the present invention, there will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences.
- Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.
- DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene.
- the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
- Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
- suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like.
- Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
- Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal.
- the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.
- the partner may be a parental line.
- in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
- the transgenic animals produced in accordance with the present invention will include exogenous genetic material.
- the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of an
- ANX7 protein (either agonistic or antagonistic), the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.
- Retroviral infection can also be used to introduce transgene into a non-human animal. The developing non-human embryo can be cultured in vitro to the blastocyst stage. During this time, the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds.
- the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al. (1985) PNAS 82:6927-6931 ; Van der Putten et al. (1985) PNAS 82:6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388). Alternatively, infection can be performed at a later stage. Virus or virus-producing cells can be injected into the blastocoele (Jahner et al.
- transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
- ES embryonal stem cell
- ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981 ) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83:9065-9069; and Robertson et al. (1986) Nature 322:445-448).
- Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
- the present invention relates to the production of non-human transgenic and chimeric animals and cells which contain at least one mutated chromosomal allele of a tumor suppressor gene.
- the present invention encompasses the formation of such cells and non-human animals for any annexin tumor suppressor gene.
- the invention is illustrated below with reference to the annexin VII tumor suppressor gene, but this example is not meant to limit the scope of the invention.
- the ability to manipulate this gene and to produce non-human transgenic animals which carry such mutated alleles is illustrated with respect to a particular disrupted allele. It is to be understood, however, that the invention and the methods disclosed herein can be used to produce any possible mutation in the anx7 gene.
- the invention includes the production of animal cells and non-human transgenic or chimeric animals which carry the particular mutations of the anx7 gene that are responsible for the lethal nullizygous state discussed below.
- the nullizygous anx7 (-/-) transgenic mouse mutant has a lethal phenotype during early embryogenesis.
- transgenic mouse exhibits a phenotype of gender dimorphic gigantism, generalized organomegaly, focal hyperplasia and dysplasia, and increased incidence of disparate spontaneous tumors.
- the combination of dysplasia and increased incidence of tumors was the first hint that the anx7 gene may be a tumor suppressor gene.
- the wild type human anx7 gene was transfected into two human prostate tumor cell lines, a breast cancer cell line, and an osteosarcoma cell line. The experiments with the anx7 gene systematically result in tumor cell growth arrest, as did the positive controls with the wild type p53 gene. It was therefore concluded that the anx7 is a tumor suppressor gene.
- the loss (either by mutation or deletion) of both anx7 alleles has been found to be an embryonic lethal event.
- Annexin Genes and Specifically, the Annexin VII Gene (anx 7)
- the present invention concerns a non-human animal or an animal (including human) cell in which one of the two naturally present copies of an annexin gene, preferably the anx7 gene, of such non-human animal or animal cell has been rendered non-functional through a mutation (such as a deletion, insertion, or substitution in the naturally occurring annexin gene sequence).
- a mutation such as a deletion, insertion, or substitution in the naturally occurring annexin gene sequence.
- Annexins are a family of structurally related proteins that all have the ability to bind Ca 2+ and phospholipid. These genes have been described in many organisms from mammals to molds to plants. (Raynal and Pollard, BBA 1197:63-93 (1994)). In the presence of Ca 2+ , the annexins bind to acidic phospholipids with very high affinity (K d in the nM range for annexin V.) The Ca 2+ binding similarities of all the annexins is due to their common primary structure, a unique N-terminal domain (the 'tail') and a conserved C-terminal domain (the 'core').
- annexin VI the conserved C-terminal domain is always composed of 4 repeats (annexin VI having 8) of ⁇ 70 amino acids containing an increased homology region called the "endonexin fold". Due to this conserved primary structure, all annexins have a high degree of identity with each other. Within mammals, annexins have between 40% and 60% identity with any other member of the family. (Hauptmann, R. et al. Eur. J. Biochem. 185:63-71 (1989)).
- annexin genes may derive from a common ancestor gene, but that a precursor underwent divergent remodeling during its evolution towards annexins I, II, and III, on the one hand, and anx7 on the other. There is no apparent relationship between the exon-intron organization of annexin genes and the primary structure of the their respective proteins.
- Annexins as Tumor Suppressor Genes Recently attention has been directed towards the family of annexin genes, particularly the anx7 gene (a.k.a. synexin), as tumor suppressor gene candidates. Early work on the anx7 gene has shown that it is expressed in small amounts in nearly every cell (Creutz, E.C., et al., J. Biol. Chem. 254:553-558 (1978); ibid, 1979; Raynal and Pollard, BBA Biomembranes 1197:63-93 (1994)).
- anx7 is found throughout phylogeny as a single copy gene in organisms as diverse as man (Shirvan, A., et al., Biochemistry 33:6888-6901 (1994)), mouse (Zhang-Keck, Z-Y., et al., Biochem. J. 289: 735-741 (1993), Zhang-Keck, Z-Y., et al., Biochemical J. 301 :835-845 (1994), Xenopus (Srivastava, S., et al., Biochemical J. 316:729-736 (1996)), and Dictyostelium (Greenwood, M., et al., Biochim Biophys Acta 1088(3):429-
- the anx7 gene is found on chromosome 10q21.
- Other potential tumor suppressor genes have been hypothesized to exist on chromosome 10q in the same vicinity as the anx7 gene. Examples include myxoid chondrosarcoma at 10q21.1 (Shen, W.P., et al., Cancer Genet. Cytogenet. 45:207-215 (1990)); sporadic nonmedullary thyroid carcinoma at 10q21.1 (Jenkins, R.B., et al., Cancer 66: 1213-1220 (1990)); renal cell carcinoma at 10q21-23 (Morita, R., et al., Cancer Res.
- gliobiastoma two independent regions at 10pter-q11 and 10q24-q26 (Steck, P.A., et al., Genes Chromosomes Cancer 12:255-261 (1995)); colonic denocarcinoma, an inverted, non-ret duplication of 10q11 to 10q21 (Solic, N., et al., Int. J. Cancer 62:48-57, (1995)); lung carcinoma at 10q21-10qter (Petersen, S., et al., Br. J. Cancer 77:270-276 (1998)); hepatocellular carcinoma at 10q (Piao, Z., et al., Int.
- ANX7 protein has Ca 2+ -dependent membrane fusion activity (Creutz, C.E., et al., J. Biol. Chem. 253:2858-2866
- ANX7 GTPase activity is sensitive to such critical modulators of conventional G-proteins as AI 2 F 6 and mastoparan (Caohuy, H., et al., Secretory Systems and Toxins (eds., Linial, M., et al.) 2:439-449 (1998)).
- ANX7 can be shown to bind and hydrolyze GTP.
- ANX7 protein also forms Ca 2+ channels in membranes (Pollard, H.B., et al., Proc. Natl. Acad. Sci. (USA) 85:2974-2978 (1988)), which can be stabilized in long open states by GTP.
- Protein kinase C phosphorylates ANX7 with a 2:1 P/Protein molar ratio, both in vitro and in vivo. This is of possible relevance to ANX7 function in the cell cycle since many isoforms of PKC have been directly implicated in activating intracellular signaling (Nishizuka, Y., Science 258:607-614 (1992), and in specifically activating mitosis (Kolch, W., et al., Nature 364:426-428
- EGF epidermal growth factor
- PGDF platelet derived growth factor
- the simplest model to explain the mechanism of action of a tumor-suppressing gene is that malignancy requires two separate genetic events (e.g., loss by deletion or mutation of both functional anx7 alleles in a cell). Inactivation of only one of the two natural anx7 alleles causes the animal to be more susceptible to cancerous growths.
- Transgenic animals may be used to investigate the biological implications of tumor-suppressing genes (Capecchi, M. R., Science
- Lavrequisite, A. et al. constructed a transgenic mouse which had a single added mutant p53 gene in addition to the endogenous two wild-type p53 alleles. The mouse and its progeny overexpressed the added p53 gene. The mice were found to have a high incidence of lung, bone, and lymphoid tumors (Lavrequisite, A. et al., Molec. Cell. Biol. 9:3982-3991 (1989)).
- this invention provides a transgenic animal whose genome possesses one normal and functional anx7 allele and one non-functional (mutant) anx7 allele. Such animals could be used to study the consequences resulting from the loss of one anx7 allele, and thus would more clearly aid in elucidating the processes of oncogenesis and tumorigenesis. Such animals would also be useful in screening potential carcinogens, in developing novel antineoplastic therapeutics, and in gene therapy.
- the present invention uses the process of homologous recombination to introduce a specific mutation into the naturally present anx7 sequence of an animal cell, most preferably an embryonic stem (ES) cell.
- the mutated ES cells of non-human animals can then be either cultured in suitable cell culture medium or introduced into the uterus of a suitable recipient and permitted to develop into a non-human animal.
- the methods of the present invention may be used to alter the somatic cells of a non-human animal to produce a chimeric non-human animal.
- homologous recombination is a well-studied natural cellular process which results in the scission of two nucleic acid molecules having identical or substantially similar sequences (i.e., "homologous"), and the ligation of the two molecules such that one region of each initially present molecule is now ligated to a region of the other initially present molecule (Sedivy, J. M., Bio-Technol. 6:1192-1196 (1988)).
- homologous recombination is, thus, a sequence specific process by which cells can transfer a "region" of DNA from one DNA molecule to another.
- a "region" of DNA is intended to generally refer to any nucleic acid molecule.
- the region may be of any length from a single base to a substantial fragment of a chromosome.
- the molecules For homologous recombination to occur between two DNA molecules, the molecules must possess a "region of homology" with respect to one another. Such a region of homology must be at least two base pairs long and having a substantially similar nucleic acid sequence.
- Recombination is catalyzed by enzymes which are naturally present in both prokaryotic and eukaryotic cells.
- the transfer of a region of DNA may be envisioned as occurring through a multi-step process.
- either of the two participant molecules is a circular molecule
- the recombination event results in the integration of the circular molecule into the other participant.
- regions of homology which may be the same, but are preferably different
- two recombinational events may occur, and result in the exchange of a region of
- DNA between two DNA molecules may be "reciprocal,” and thus results in an exchange of DNA regions between two recombining DNA molecules.
- it may be "non-reciprocal,” (also referred to as “gene conversion”) and result in both recombining nucleic acid molecules having the same nucleotide sequence.
- the frequency of recombination between two DNA molecules may be enhanced by treating the introduced DNA with agents which stimulate recombination.
- agents which stimulate recombination include trimethylpsoralen, UV light, etc.
- ES embryo-derived stem
- Gene targeting has been used to produce chimeric and transgenic mice in which an nptll gene has been inserted into the ⁇ 2 -microglobulin locus (Koller, B. H. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:8932-8935 (1989);
- the gene of interest In order to utilize the "gene targeting" method, the gene of interest must have been previously cloned, and the intron-exon boundaries determined. The method results in the insertion of a marker gene (i.e. the nptll gene) into a translated region of a particular gene of interest. Thus, use of the gene targeting method results in the gross destruction of the gene of interest.
- a marker gene i.e. the nptll gene
- the chimeric or transgenic animal cells of the present invention are prepared by introducing one or more DNA molecules into a precursor pluripotent cell, most preferably an ES cell, or equivalent (Robertson, E. J., In: Current Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), pp. 39-44, which reference is incorporated herein by reference).
- a precursor pluripotent cell most preferably an ES cell, or equivalent
- the pluripotent (precursor or transfected) cell may be cultured in vivo, in a manner known in the art (Evans, M. J. et al., Nature 292:154-156 (1981)) to form a chimeric or transgenic animal. Any ES cell may be used in accordance with the present invention. It is, however, preferred to use primary isolates of ES cells. Such isolates may be obtained directly from embryos such as the CCE cell line disclosed by Robertson, E. J., In: Current Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), pp. 39-44), or from the clonal isolation of ES cells from the CCE cell line
- ES cell lines which have been clonally derived from embryos are the ES cell lines, AB1 (hprt + ) or AB2.1 (hprt).
- the ES cells are preferably cultured on stromal cells (such as STO cells (especially SNC4 STO cells) and/or primary embryonic fibroblast cells) as described by E. J. Robertson (In: Teratocarcinomas and Embryonic Stem Cells: A Practical
- ES cell lines may be derived or isolated from any species (for example, chicken, etc.), although cells derived or isolated from mammals such as rodents (i.e. mouse, rat, hamster, etc.), rabbits, sheep, goats, fish, pigs, cattle, primates and humans are preferred.
- rodents i.e. mouse, rat, hamster, etc.
- rabbits sheep, goats, fish, pigs, cattle, primates and humans are preferred.
- the present invention provides human or animal cells which contain a desired gene sequence in one of the two annexin gene alleles of the cell's genome.
- the invention also provides a means for producing non-human chimeric or transgenic animals whose cells contain such a sequence.
- the animals which may be produced through application of the described method include chicken, non-human mammals (especially, rodents (i.e. mouse, rat, hamster, etc.), rabbits, sheep, goats, fish, pigs, cattle and non-human primates).
- the cells and non-human animals of the present invention have both diagnostic and therapeutic utility.
- A. Diagnostic Utility i.e. mouse, rat, hamster, etc.
- the present invention can be used to identify an agent that is capable of affecting a characteristic of an animal cell that is attributable to the presence or expression of a tumor-suppressing gene.
- a characteristic of an animal cell is said to be "attributable to the presence or expression of a tumor-suppressing gene," if the characteristic is altered by the absence or lack of expression of the tumor-suppressing gene. Examples of such characteristics include tumorigenesis, resilience to tumorigenesis, the extent, distribution, incidence, location, grade, etc. of tumors, etc.
- such agents can decrease the tumorigenic (or neoplastic) potential of the cells or animals. Such agents are discussed below with regard to the therapeutic potential of the invention.
- such agent can increase the tumorigenic (or neoplastic) potential of the cells or animals.
- the cells and non-human animals of the present invention have utility in testing potential or suspected carcinogens for tumorigenic activity. They may be used to identify and assess the tumorigenic effects of agents that may be present, for example, in the environment (such as environmental pollutants in air, water or soil), or resulting from environmental exposures to chemicals, radioisotopes, etc. They may also be used to facilitate studies of the effects of diet on oncogenesis. They may be used to determine whether potential or present food additives, chemical waste products, chemical process by-products, water sources, proposed or presently used pharmaceuticals, cosmetics, etc., have tumorigenic activity. They may also be used to determine the tumorigenic potential of various energy forms (such as UV rays, X-rays, ionizing radiation, gamma rays of elemental isotopes, etc.).
- energy forms such as UV rays, X-rays, ionizing radiation,
- the frequency at which a mutational event occurs is dependent upon the concentration of a mutagenic chemical agent, or the intensity of a mutagenic radiation.
- the frequencies of a single cell receiving two mutational events is the square of the frequency at which a single mutational event will occur, the cells and non-human animals of the present invention shall be able to identify neoplastic (mutagenic) agents at concentrations far below those needed to induce neoplastic changes in natural cells or animals. This is because one allele of the tumor suppressing gene anx7 has already been mutated in the transgenic mouse of the present invention.
- One especially preferred cell is a non-human cell in which one of the natural anx7 alleles has been replaced with a functional human anxl allele and the other of the natural anx7 alleles has been mutated to a non-functional form.
- Such cells may be used, in accordance with the methods described above, to assess the neoplastic potential of agents in cells containing the human anxl allele. More preferably, such cells are used to produce non-human animals which do not contain any natural functional anx7 alleles, but which contain only one functional human anxl allele. Such non-human animals can be used to assess the tumorigenicity of an agent in a non-human animal expressing the human anx7 gene product. 1. In Vitro Assays
- This embodiment therefore comprises an in vitro assay of tumorigenic activity.
- the present invention permits the identification of such "latent” carcinogenic and "co-carcinogenic” agents.
- the presence of a "latent” carcinogen can be identified by merely maintaining cell or animal exposure to a candidate agent.
- the cells of the present invention can be incubated in
- conditioned culture medium i.e. medium containing the candidate agent that was used to culture other cells before being used to culture the cells of the present invention.
- the present invention permits the identification of co-carcinogenic factors capable of inducing neoplastic effects in the presence of a second agent.
- factors can be identified by culturing the cells of the present invention in the presence of two or more candidate agents simultaneously, and then assaying for neoplasia.
- the transformation of the cells to a neoplastic state would be indicative of tumorigenic (or neoplastic) activity of the assayed agent.
- a neoplastic state may be evidenced by a change in cellular morphology, by a loss of contact inhibition, by the acquisition of the capacity to grow in soft agar, or most preferably, by the initiation of expression of tumor antigens.
- tumor antigens as a means of detecting neoplastic activity is preferred since such antigens may be readily detected.
- antibodies, or fragments of antibodies may be used to quantitatively or qualitatively detect the presence tumor of antigens on cell surfaces. Since any cell type (i.e. lung, kidney, colon, etc.) may be employed to form the a ⁇ x7-mutated cells of the present invention, it is possible to determine whether an agent has a tissue specific tumorigenic potential. To accomplish this goal, one would incubate a candidate agent in the presence of anx7-mutated cells derived from any of a variety of tissue types.
- tumors have tumor-specific antigens, and since antibodies capable of binding to such antigens have been isolated, it is possible to use such antibodies to characterize any tumor antigens which may be expressed by the anx7-mutated cells.
- detection may be accomplished using any of a variety of immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect the antigen through the use of radioimmune assays.
- RIA radioimmune assay
- radioisotopic labels include 3 H, 111 ln, 125 l, 131 l, 32 P, 35 S, 14 C, 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 Eu, 90 Y, 67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, etc.
- enzyme labels non-radioactive isotopic labels, fluorescent labels, chemiluminescent labels or other suitable labels can be employed.
- suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholine esterase, etc.
- suitable non-radioactive isotopic labels include
- fluorescent labels include an 152 Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, a fluorescamine label, etc.
- chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, an aequorin label, etc.
- chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, an aequorin label, etc.
- suitable labels which may be employed in accordance with the present invention.
- the binding of these labels to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J. H., et al. (Clin. Chim. Ada 7_0:
- the above-described in vitro assay has the advantageous features of potentially lower cost than presently used assays, and the capacity to readily screen large numbers of agents.
- Use of this embodiment facilitates comparisons of test results obtained at different times and conditions.
- it is possible to use very large numbers of cells in such assays it is possible to detect the tumorigenic activity of tumorigenic agents even at very low concentrations.
- this embodiment can be performed using human cells, it provides a means for determining the tumorigenic (or neoplastic) potential of a test compound on human cells.
- the formation of tumors in such animals would be indicative of tumorigenic activity of the assayed agent.
- the use of the non-human animals of the present invention is preferred over naturally occurring non-human animals because natural animals contain two functional anxl alleles and thus require two mutational events in order to lead to loss of functional anxl activity.
- the non-human animals of the present invention have only one functional anxl allele, only one mutational event is needed to cause total loss of anxl function.
- the detection of tumors in such animals can be accomplished by biopsy, imaging, or by assaying the animals for the presence of cells which express tumor antigens.
- detection may be accomplished by removing a sample of tissue from a subject and then treating the isolated sample with any suitably labeled antibodies (or antibody fragments) as discussed above.
- in situ detection is accomplished by removing a histological specimen from the subject, and providing the labeled antibody to such specimen.
- the antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a sample of tissue.
- the detection of tumor cells may be accomplished by in vivo imaging techniques, in which the labeled antibodies (or fragments thereof) are provided to the subject, and the presence of the tumor is detected without the prior removal of any tissue sample.
- in vivo detection procedures have the advantage of being less invasive than other detection methods, and are, moreover, capable of detecting the presence of antigen-expressing cells in tissue which cannot be easily removed from the patient.
- body fluids such as blood, lymph, etc.
- stools such assay for the presence of tumor antigens in body fluids (such as blood, lymph, etc.), stools, or cellular extracts.
- the antibodies (or antibody fragments) may be utilized in liquid phase or bound to a solid-phase carrier, as described below.
- the use of an in vivo assay has several advantageous features.
- the in vivo assay permits one not only to identify tumorigenic agents, but also to assess the kind(s) of tumors induced by the agent, the number and location (i.e. whether organ or tissue specific) of any elicited tumors, and the grade (clinical significance) of such elicited tumors. It further permits an assessment of tumorigenicity which inherently considers the possible natural metabolism of the introduced agent, the possibility that the introduced agent (or its metabolic by-products) might selectively accumulate in specific tissues or organs of the recipient animal, the possibility that the recipient animal might recognize and repair or prevent tumor formation.
- such an assay provides a true biological model for studying and evaluating the tumorigenic potential of an agent in a living non-human animal.
- Immunoassays of Tumor Antigens The in vitro, in situ, or in vivo detection of tumor antigens using antibodies (or fragments of antibodies) can be improved through the use of carriers.
- Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
- the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
- the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen.
- the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
- the surface may be flat such as a sheet, test strip, etc.
- the binding molecules of the present invention may also be adapted for utilization in an immunometric assay, also known as a "two-site” or “sandwich” assay.
- an immunometric assay also known as a "two-site” or “sandwich” assay.
- a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support that is insoluble in the fluid being tested (i.e., blood, lymph, liquified stools, tissue homogenate, etc.) and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
- Typical immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody.
- This type of forward sandwich assay may be a simple "yes/no” assay to determine whether antigen is present or may be made quantitative by comparing the measure of labeled antibody with that obtained for a standard sample containing known quantities of antigen.
- Such "two-site” or “sandwich” assays are described by Wide at pages 199-206 of
- Radioimmune Assay Method edited by Kirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970.
- a simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
- stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.
- the immunometric assays for antigen require that the particular binding molecule be labeled with a "reporter molecule.”
- reporter molecules or labels are conventional and well-known to the art.
- enzyme labels are a preferred embodiment. No single enzyme is ideal for use as a label in every conceivable immunometric assay. Instead, one must determine which enzyme is suitable for a particular assay system.
- Criteria important for the choice of enzymes are turnover number of the pure enzyme (the number of substrate molecules converted to product per enzyme site per unit of time), purity of the enzyme preparation, sensitivity of detection of its product, ease and speed of detection of the enzyme reaction, absence of interfering factors or of enzyme-like activity in the test fluid, stability of the enzyme and its conjugate, availability and cost of the enzyme and its conjugate, and the like.
- Included among the enzymes used as preferred labels in the immunometric assays of the present invention are peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, glycoamylase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase.
- Urease is among the more preferred enzyme labels, particularly because of chromogenic pH indicators which make its activity readily visible to the naked eye.
- the cells and animals of the present invention can be used to identify agents that decrease the tumorigenic (or neoplastic) potential of the cells or animals.
- agents can be "anti-tumor agents” and/or “chemopreventative agents.”
- Anti-tumor agents act to decrease the proliferation of the cells (or the growth, dissemination, or metastasis of tumors in the chimeric or transgenic animals).
- Chemopreventative agents act to inhibit the formation of new tumors. Such agents may have general activity
- the present invention permits the identification of novel antineoplastic therapeutics. Any of the assays in section A. above may be used for determining tumor-suppressing activity.
- the transgenic cells and non-human animals of the present invention can be used to study human gene regulation of the anxl gene.
- such cells and animals can be used to investigate the interactions of the anxl gene with oncogenes or other tumor suppressor genes.
- they may be used to identify therapeutic agents which have the ability to impair or prevent neoplastic or tumorigenic development.
- therapeutic agents have utility in the treatment and cure of cancer in humans and animals.
- potential therapeutic agents are frequently found to induce toxic effects in one animal model but not in another animal model. To resolve the potential of such agents, it is often necessary to determine the metabolic patterns in various species, and to then determine the toxicities of the metabolites.
- the present invention permits one to produce transgenic cells or animals which could facilitate such determinations.
- pharmaceutically acceptable carriers i.e. liposomes, etc.
- Such agents can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle.
- Suitable vehicles and their formulation are described, for example, in Nicolau, C. et al. (Crit. Rev. Ther. Drug Carrier Syst. 6:239-271 (1989)), which reference is incorporated herein by reference.
- compositions suitable for effective administration, such compositions will contain an effective amount of the desired gene sequence together with a suitable amount of carrier vehicle. Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the desired gene sequence (either with or without any associated carrier). The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
- macromolecules for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate
- Another possible method to control the duration of action by controlled release preparations is to incorporate the agent into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
- a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
- colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsion
- the cells and non-human animals of the present invention may be used to investigate gene regulation, expression and organization in animals.
- the methods of the present invention may be used to produce alterations in a regulatory region of the native anx7 gene sequence.
- the invention provides a means for altering the nature or control of transcription or translation of the anxl gene, and of altering the anxl gene itself.
- the invention enables one to introduce mutations which result in increased or decreased gene expression.
- it enables one to impair or enhance the transcriptional capacity of the natural anx7 gene in order to decrease or increase its expression.
- the present invention permits the manipulation and dissection of the anxl gene. Such abilities are especially valuable in gene therapy protocols and in the development of improved animal models of cancer.
- the principles of gene therapy are disclosed by Oldham, R. K. (In:
- DNA encoding either a functional anx7 gene, variants of that gene, or other genes which influence the activity of the anx7 gene may be introduced into the somatic cells of an animal (particularly mammals including humans) in order to provide a treatment for cancer (i.e. "gene therapy").
- gene therapy i.e. "gene therapy”
- viral or retroviral vectors are employed for this purpose.
- Retroviral vectors are a common mode of delivery and in this context are retroviruses from which all viral genes have been removed or altered so that no viral proteins are made in cells infected with the vector. Viral replication functions are provided by the use of retrovirus "packaging" cells that produce all of the viral proteins but that do not produce infectious virus. Introduction of the retroviral vector DNA into packaging cells results in production of virions that carry vector RNA and can infect target cells, but such that no further virus spread occurs after infection. To distinguish this process from a natural virus infection where the virus continues to replicate and spread, the term transduction rather than infection is often used.
- Non-retroviral vectors have been used in genetic therapy.
- One such alternative is the adenovirus (Rosenfeld, M. A., et al., Cell 68:143155 (1992); Jaffe, H. A. et al., Nature Genetics 1:372-378 (1992); Lemarchand, P. et al.,
- adenovirus vectors Major advantages of adenovirus vectors are their potential to carry large segments of DNA (36 Kb genome), a very high titre (10 11 /ml), ability to infect non-replicating cells, and suitability for infecting tissues in situ, especially in the lung.
- the most striking use of this vector so far is to deliver a human cystic fibrosis transmembrane conductance regulator (CFTR) gene by intratracheal instillation to airway epithelium in cotton rats (Rosenfeld, M. A., et al., Cell 63: 143-155 (1992)).
- CFTR cystic fibrosis transmembrane conductance regulator
- herpes viruses may also prove valuable for human gene therapy (Wolfe, J. H. et al., Nature Genetics 1:379-384 (1992)).
- any other suitable viral vector may be used for genetic therapy with the present invention.
- Plasmid DNA should be easy to certify for use in human gene therapy because, unlike retroviral vectors, it can be purified to homogeneity.
- liposome-mediated DNA transfer several other physical DNA transfer methods, such as those targeting the DNA to receptors on cells by conjugating the plasmid DNA to proteins, have shown promise in human gene therapy (Wu, G. Y., et al., J. Biol. Chem. 266:14338-14342 (1991); Curiel, D.
- such gene therapy can be provided to a recipient in order to treat (i.e. suppress, attenuate, or cause regression) an existing neoplastic state
- the principles of the present invention can also be used to provide a prophylactic gene therapy to individuals who, due to inherited genetic mutations, or somatic cell mutation, contain cells having impaired anxl gene expression (for example, only a single functional allele of the anxl gene).
- Such therapy could be administered in advance of the detection of cancer in order to lessen the individual's predisposition to the disease.
- transgenic mice Before beginning the actual production of transgenic mice, the genomic locus for annexin VII (anxl) was characterized. Then, a transgene construct ("targeting vector”) was prepared based on this characterization. This construct carries the necessary elements to facilitate the transgenic animal construction.
- the anx7 genomic locus from a 129SV/CPJ mouse genomic library (Stratagene), which contained 14 exons of the anx7 gene spanning about 34 kb, was screened with mouse anxl cDNA probe (Zhang-Keck, et al., Biochem. J. 301 :835-845.)
- mouse anxl cDNA probe Zhang-Keck, et al., Biochem. J. 301 :835-845.
- several restriction fragments including three Xba I fragments (1.9, 3.6 and 3.1 kb) and one Xho I fragment (2.0 kb) encompassing this region were subcloned, labeled with 32 [P], and tested for the presence of repetitive sequences.
- the 2.0 kb Xho I genomic DNA fragment (containing exons 4 and 5) and the 3.1 kb Xba I genomic DNA fragment (containing exons 7 and 8) were inserted into the Xho I site and the Xbal site of pPNT, respectively, to generate the replacement type targeting vector termed KSBX.pPNT (See Figure 1A).
- the vector, pPNT (obtained from Dr. Heiner Westphal's laboratory at NICHD, NIH) contained PGKneo and PGKtk cassettes, separated and flanked by a number of unique cloning sites. The neo gene was determined to be in the same orientation as the anx7 gene.
- TK herpes simplex virus thymidine kinase
- Pluripotent embryonic stem cells are cells which may be obtained from embryos until the early post-implantation stage of embryogenesis. The cells may be propagated in culture, and are able to differentiate either in vitro or in vivo upon implantation into a mouse as a tumor.
- ES cells have a normal karyotype (Evans, M. J. et al., Nature 292:154-156 (1981 ); Martin, G. R. et al., Proc. Natl. Acad. Sci. (U.S.A.)
- ES cells Upon injection into a blastocyst of a developing embryo, ES cells will proliferate and differentiate, thus resulting in the production of a chimeric animal. ES cells are capable of colonizing both the somatic and germ-line lineages of such a chimeric animal (Robertson, E. et al., Cold Spring Harb.
- ES cells may be propagated in vitro, it is possible to manipulate such cells using the techniques of somatic cell genetics.
- it is possible to select ES cells which carry mutations such as in the hprt gene (encoding hypoxanthine phosphoribosyl transferase) (Hooper, M. et al., Nature 326:292-295 (1987); Kuehn, M. R. et al., Nature 326:295-298 (1987)).
- Such selected cells can then be used to produce chimeric or transgenic mice which fail to express an active enzyme, and thus provide animal models for diseases.
- the ES cells used here were derived from mouse strain 129SvJ and maintained in culture on primary mouse embryo fibroblast (PMEF) feeder cells carrying a neomycin gene.
- the culture medium was supplemented with leukemia inhibitory factor (1500 units/ml).
- the targeting vector was linearized by the restriction endonuclease Not I and transfected into the J1 cell line (Li et al. 1992) by electroporation of 3 X 10 6 ES cells.
- the genetically altered ES cells containing the targeted allele were selected with G418 (Gibco) at 350 ⁇ g/ml and Gancyclovir (Bristol Myers) at 0.2 ⁇ M.
- Genomic DNA was isolated from those clones exhibiting dual resistance (selected against neomycin and Gancyclovir). Genomic DNA was isolated from cultured cells by digestion overnight at 55°C in lysis buffer (10 mM Tris-HCI, pH 7.5/100 mM NaCI/1 mM EDTA/100 ⁇ g/ml proteinase K) followed by precipitation with iso- propanol. The pellet was washed with 70% ethanol and dissolved in 100 ⁇ l of
- FIG. 3A depicts the PCR analysis of ES cell clones transfected with KSBX.pPNT.
- DNA from PCR-positive ES clones was digested with Xba I and hybridized with a genomic DNA probe, KXX, which is external to the 5'- flank introduced into the targeting vector (See Figure 3B).
- the probe detected the predicted 3.6 kb wild-type and 4.5 kb mutant fragments representing the normal and altered alleles of anx7, respectively.
- the data indicated that the targeting vector had been successfully generated and that genetically altered heterozygous ES cells had been isolated which had undergone a single targeted integration event at the anx7 locus.
- C Preparation of Chimeras
- the strategy for generating the chimeras containing the desired targeted mutation is shown in Figure 4.
- the altered ES cells were microinjected into the blastocoel cavity of a 4.5 day preimplantation mouse embryo from a C57B1/6J mouse. Then, the embryos were transferred surgically into the uterine horn of a pseudopregnant mouse and development was allowed to progress to birth.
- mice Resulting chimeric animals were backcrossed to C57BL6/J mice, and germline transmission was scored by coat color. All agouti (A/A) mice (i.e., brown) offspring were tested for the presence of the mutated anx7 allele by PCR amplification using the same conditions described above for the detection of homologous recombination events in the ES cells. Normally, the agouti (A/A) mice (i.e., brown) offspring were tested for the presence of the mutated anx7 allele by PCR amplification using the same conditions described above for the detection of homologous recombination events in the ES cells. Normally, the
- ES cells are derived from mice with distinguishable coat color alleles (brown, shown in Figure 4 as black) compared to recipient blastocyst (black, shown in Figure 4 as white).
- ES cells (with targeting construct) derived from an agouti (A/A) mouse (brown) were injected into a recipient nonagouti (a/a)
- mice from embryonic days E8 to E17 were genotyped. Of the viable 120 embryos analyzed, 25% were anx7 (-/-) at E10, but by E11 none of them had survived.
- the Yolk sac DNA of these embryos was used as a template for PCR analyses as described above. Polymerase chain reaction analysis showed the absence of anxl transcripts in anx7 (-/-) mutants. (See Figure 6.)
- mice Male anx7 (+/-) mice begin to grow at a greater rate than normal littermate controls by about the fourth week after birth. By contrast, female anx7 (+/-) mice do not vary from their controls. Male mutant growth thereafter does not appear to abate.
- the data in Figure 7 show growth up to six months of age. However, when these same animals were weighed at 13 months of age, evidence of continued growth over this subsequent time period was noted. Weights as high as 60 grams were noted for some of these heterozygous animals. Postmortem examination has systematically shown that the animals are not fat, but merely large. Organ weight studies performed at 6 months of age showed that many internal organs in anx7 (+/-) males weighed much more than normal, but were of grossly normal structure. It is remarkable that, in light of our subsequent focus on the hyperplastic Islets of Langerhans, the pancreas was one of several organs that were not larger than normal. However, the islets do make up less than 2% of the islet by volume.
- the growth phenotype of gender-specific gigantism and organomegaly of the anxl (+/-) mouse is fundamentally different from that of other reported mouse knockouts.
- the reported instances of mutation-based gigantism are mostly endocrine in origin, and are due to increases of either growth hormone (Palmiter, R., et al., Nature 300:611-615 (1982)), IGF-1 (Mathews, L, et al., Endocrinology 123:2827-2833 (1988)), or
- IGF-2 (Wolf, E., et al., Endocrinology 135:1877-1886 (1994)).
- the levels of serum IGF-1 in the anx7 (+/-) mouse are within normal limits.
- the levels of IGF-1 integrate the pulsatile levels of growth hormone, it was concluded that average GH levels were probably normal as well in anxl (+/-) mice.
- GH levels measured in overnight-fasted animals showed no change in males.
- One qualitative parallel between the growth kinetics of male anx7 (+/-) mice and those mice transgenic for GH or IGF-1 is a postpartum delay in the onset of enhanced growth.
- mice and mice transgenic for GH begin to grow at 3-4 weeks, while those transgenic for IGF-1 begin to grow only after 6-8 weeks.
- Mice overproducing IGF-2 are heavier than control mice at birth, but do not sustain the increase in weight into adulthood.
- pituitary gland histology in male and female anx7 (+/-) mutants cannot be distinguished from wildtype histologies (data not shown).
- the selective distribution of organomegaly noted for the anx7 (+/-) male mutant is distinct from that associated with high levels of GH, IGF-1 and IGF-2 (Palmiter, R., et al., Science 222:809-814 (1983); Mathews,
- (+/-) mice were not profoundly different from levels in control animals, indicating that hyperinsulinism is not a viable explanation either. Together, these data thus further validate the conclusion that the documented growth anomalies in the anx7 (+/-) mouse are probably not related to pituitary hyperfunction. The fact that unique growth anomalies in the anx7 (+/-) mouse are gender-specific constitute a further distinct internal genetic control for the anxl (+/-) mouse mutation.
- Lymphosarcoma of the thymus is a frequently occurring tumor in these mutants.
- This tumor was found as an unencapsulated, 1-cm 3 tan fleshy mass occupying the anterior thoracic cavity, which surrounded the heart and compressed the lungs.
- the tumor mass is composed of sheets of monomorphic cells supported by a fine fibrovascular stroma.
- the cells are small, round, and non-adherent, with well-demarcated borders, scant lightly basophilic cytoplasm, single central round deeply basophilic nuclei, and, generally, a single central prominent nucleolus.
- the hepatocellular carcinoma shown in Figure 11 is an unencapsulated mass (1 X 0.5 X 0.5 cm) composed of large polygonal cells arranged in cords and trabeculae.
- the mitotic rate is less than 1/10 high power fields.
- Cells have discrete cytoplasmic borders, abundant granular to finely vacuolated eosinophilic cytoplasm and a large centralized round vesicular nucleus. In most cells there is a single prominent magenta nucleolus, although occasional nuclei contain multiple nucleoli. Neoplastic cells are observed to infiltrate adjacent hepatic parenchyma.
- FIG. 9B A section from a lymphosarcoma of the thymus, taken at 50-X magnification, is shown in Figure 9B, with a sample of normal thymus shown in Figure 9A for comparison.
- the board certified veterinary pathologist's description is as follows:
- thymic mass in mouse MS9801634: There is a 1 cm 3 tan fleshy mass occupying the anterior thoracic cavity, surrounding the heart and compressing the lungs.
- the mass is composed of sheets of monomorphic cells supported by a fine fibrovascular stroma.
- the cells are small, round, and non-adherent, with well demarcated borders, scant lightly basophilic cytoplasm, single central round deeply basophilic nuclei and generally a single central prominent nucleolus.
- the mass is unencapsulated.
- Neoplastic cells infiltrate and expand the mediastinum, extend into the lung along branches of the pulmonary artery, efface the bronchial lymph nodes, and disseminate to the kidneys.
- Cell morphology is consistent with lymphosarcoma.
- a section is shown of tumor cell infiltration into the lung in Figure 10B, in which extensions along branches of the pulmonary artery are prominent.
- FIG. 11 B A section from a hepatocellular carcinoma, taken at 100-X magnification, is shown in Figure 11 B.
- Figure 11A For comparison a sample of normal liver from an anx7(+/+) mouse shown in Figure 11A.
- the board certified veterinary pathologist's description is as follows.
- the wild type human anx7 gene suppresses growth of a variety of human
- (+/-) mouse exhibit organomegaly. Tissues from anx7 (+/-) and control mice were
- ANX7 from heart has a tissue-
- ANX7 levels appear to be much lower in mutant than in control pancreas
- the adenovirus Compared with the chemically based gene transfer systems, the adenovirus
- Adenovirus recombinants of anx7 sense, anti-sense, and mutations are constructed
- cDNA sequence are formed by homologous recombination.
- Wild type and mutant anx7 genes are engineered into replication-deficient
- adenoviral vectors prepared, purified, titered, and
- mutant anx7 genes have been prepared in the adenovirus vector, and many have
- Example V is directed against GTP binding sites; and an antisense anx7 construct.
- anx7 constructs with dominant negative activity to suppress the function of
- the combinations were single mutations (e.g., 1 , 2, 3 & 4);
- mutations at two sites e.g., 1 & 2, 1 & 3, etc.
- mutations at three sites e.g., 1&2&3,
- ANX7 proteins behaves as a dominant negative mutant in the in vitro
- Threonine/serine protein kinases such as Protein Kinase C (PKC) and tyrosine
- kinases are known to phosphorylate tumor suppressor genes such as p53 or BRCA1 ,
- receptors are mixed with cells, and endogenous ANX7 labeling is detected.
- PKA protein Kinase
- PKG cGMP-dependent protein kinase
- Casein kinase I and
- EGFR-kinase epidermal growth factor receptor kinase
- casein kinase I casein kinase II
- casein kinase II casein kinase II
- Ca 2 7calmodulin kinase II Ca 2 7calmodulin kinase II
- ANX7 was detected by immunoprecipitation, which was blocked by specific PKC
- ANX7 might be labeled when human adenocarcinoma A431 cells were exposed to
- EGF epidermal growth factor
- PDGF platelet derived growth factor
- ANX7 can be labeled by a broad spectrum of protein kinases, both in
- casein kinases I and II serve to distinguish ANX7
- ANX7 is a Ca 2+ -activated GTPase, which contains the five putative RAS-type
- G-2 QinT
- G-4 N-RNsN
- G-5 EiSG
- Binding of 8-azido-GTP could then be used to assess GTP binding.
- the wild type and mutant ANX7 proteins were expressed in the pTrc99A
- Tumor cell lines can be grown in vitro, and this growth is suppressed when
- wild type tumor suppressor genes are transfected into the tumor cells (e.g.,
- certain human prostate tumor cell lines can be suppressed when a
- mutated Rb gene is supplanted by a wild type Rb gene (Huang, H.J-S., et al.,
- susceptible tumor cells contain p53 mutations, it is even possible to suppress the growth of cancer cells with transfected p53 which contain endogenous wild type p53
- Prostate cancer cell line DU145 was cultured in Eagle's Minimum Essential Medium with 2 mM L-glutamine and Earle's BSS, adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate (Sigma Chemical Co., St. Louis, MO ), and 10% fetal bovine serum (Intergen Co., Purchase, NY).
- Prostate cancer cell line LNCaP was cultured in RPMI 1640 medium with 2mM L-glutamine adjusted to contain 1.5% sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM pyruvate (Sigma Chemical Co.), and10% fetal bovine serum (Intergen Co. , Purchase, NY).
- Osteosarcoma cell line Saos-2 was cultured in McCoy's 5a medium with 1.5 mM L-glutamine (Sigma Chemical Co.), and 15% fetal bovine serum (Intergen Co. , Purchase, NY).
- Breast cancer cell line MCF7 was cultured in Eagle's Minimum Essential Medium with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate (Sigma Chemical Co., St. Louis, MO ), 0.01 mg/ml bovine insulin (Life Technologies Inc.), and 10% fetal bovine serum (Intergen Co., Purchase, NY). Cells were plated in 6-well plates (35 mm wells) and grown in appropriate
- pcDNA3 the cells were rinsed with phosphate-buffered saline (PBS), fixed with 2%
- prostate tumor cell lines DU145 ( Figure 12A) and LNCaP ( Figure 12B), a breast
- Tumor suppressor genes are principally known for control of cell proliferation
- anx7 mRNA and ANX7 protein in human IMR-90 fibroblasts can be studied by the
- IMR-90 cells obtained from the ATCC, were cultured and synchronized by
- RNA transcripts Matched tumor and adjacent normal tissues from mice and human are obtained and immediately embedded in OCT (Miles Inc. Diagnostics Division Elkhart, IN) and frozen at -70°C. With LCM (laser-gene capture microdissection) of tumor and normal cells are obtained from a heterozygous mouse and human specimens.
- a cryotome 1.0-micron sections are cut from frozen tissues and stained by hematoxylin (H) and eosin (E).
- H hematoxylin
- E eosin
- the H & E slides are read by the pathologist to ensure the presence of >70% tumor cells.
- the neoplastic area are outlined on each slide.
- the unstained frozen sections on the slides are stored at -70°C until DNA was extracted.
- the H&E stained slides are used as a template and corresponding frozen sections will be superimposed on it.
- the normal and tumor cells dissected by LCM are used for RNA extraction and purification by Ransom B reagent (Tel-Test, Inc., Friendwood, Texas).
- Tumor and normal tissue RNAs are reverse-transcribed using random hexamers and Superscript (Life Technology, Gaithersburg, MD).
- Five cDNA fragments representing the complete anx7 protein coding sequence are amplified using pfu DNA polymerase. Since pfu DNA polymerase has proof-reading activity, it is less error prone as compared to Taq DNA polymerase.
- the PCR fragments are subjected to "Cold SSCP" with temperature optimized for each fragment. Aberrant bands from SSCP gels are reamplified and sequenced.
- the scraped tissue are digested with proteinase K, extracted with phenol/chloroform, followed by ethanol precipitation.
- the integrity and concentration of genomic DNA from frozen tissue is determined on agarose gels.
- the matching normal DNA is extracted from histologically normal tissue sections that did not contain tumor cells.
- Southern blot analysis is carried out using high molecular weight DNA digested with restriction enzymes. The bands are fractionated by electrophoresis on 0.6% agarose gel and transferred onto nylon membranes. The nylon membranes are hybridized with nick translated cDNA anx7 probe. The polymorphic changes in the disease samples are then analyzed.
- PCR and polymorphism analysis Primers flanking the dinucleotide repeat sequences have been identified in the anx7 gene (Shirvan et al., 1994). The PCR is performed on the genomic DNA samples using the following conditions: 5 nanograms (ng) of DNA template, 50ng of each primer, 0.5 unit of AmpliTaq Gold (Perkin Elmer Emeryville, CA), Ix PCR buffer, 200 ⁇ l dNTP mix in a 50 ⁇ l final volume. PCR conditions are identical for all primers used. PCR cycles includes 1 cycle of 95°C for 10 min. followed by 25 cycles of 95°C for 30 sec, 55°C for 45 sec. and 72°C for 1 min. One of the primers is end labeled using 32 P-ATP and T4 polynucleotide kinase kit (Life Technology, Gaithersburg, MD).
- PCR products are subjected to electrophoresis on a 7% acrylamide/urea/formamide gel. The gel is dried and processed for autoradiography.
- PCR products representing anx7 cDNA are denatured with methyl mercury hydroxide and electrophoresed through pre-made 20% polyacrylamide minigels (Novex) at a high voltage and a constant temperature. Temperature of the buffer and gel is accurately maintained by constant temperature water circulation through a specially designed cooling system. It is also important to point out that SSCP is more sensitive than direct DNA sequencing in detecting mutant anx7 alleles in the presence of wt anx7 sequences. These wt anx7 sequences are unavoidably present due to the presence of normal cells in tumor tissue architecture, or due to tumor cell heterogeneity where all the cells in tumor tissue may not contain anx7 mutations.
- Silver staining or SYBR green staining of the gels are responsible for the increased sensitivity of detection of SSCP bands on gel.
- the aberrant SSCP bands identified by "Cold SSCF" procedure are cut from the gel and reamplified using the same primers.
- PCR products are purified and sequenced using the (Rhodamine-terminator cycle sequencing kit (PE Applied Biosystem) following the supplier's recommended methods. DNA sequences are analyzed on an automated DNA sequencer (310
- LOH "Loss of Heterozysosity" of tumor samples will be initially determined visually by comparing the intensity of bands representing the alleles between normal DNA and tumor DNA. A decrease of >50% signal intensity in tumor DNA, as compared to normal, on more than one of the alleles in tumor DNA is scored as LOH. Quantitation is undertaken by exposing the dried gels to phosphor storage screens for 4-5 hours and images are collected on a Molecular Dynamics Phosphorimager and analyzed with Image Quant software (Sunnyvale, CA). Quantitation is done by subtracting the background and by the volume integration method within equal-sized rectangular regions that are placed manually over bands.
- ANX7 protein expression was determined in a prostate tissue microarray containing 301 specimens taken from all stages of human prostate tumor progression. As shown in Figure 16A, significant reductions in ANX7 expression were found to occur in a stage-specific manner. ANX7 expression was completely lost in a high proportion of metastases (57%) and in local recurrences of hormonal refractory prostate cancer (63%). By contrast, ANX7 occurs at close to normal levels in benign prostate glands, high grade prostatic intraepithelial neoplasms (PIN), and stage T2 and T3/4 primary tumors (all in the range of 89-96%).
- PIN prostatic intraepithelial neoplasms
- Ki67 immunostaining as an index of tumor cell proliferation, we found a positive correlation between a high Ki67 labeling index and a lack of ANX7 expression, as well as a correlation with advanced stage prostate cancer and high Gleason score.
- ANX7 positive human prostate cancer cells have significantly fewer cells with high levels of immunostaining by Ki67 antibody (red bar), as compared to the percentage of cells with a low level of immunostaining (purple bar).
- ANX7 negative human prostate cancer cells have a higher percentage of cells with high levels of immunostaining by Ki67 antibody (red bar).
- FIG 17B shows example histological images taken from the samples used in Figure 17A.
- the samples in the left column were stained by Ki67, which is indicative of the proliferative state.
- the samples in the right column are consecutive sections stained for ANX7 protein.
- Samples exhibiting benign prostatic hypertrophy (BPH) and primary intraepithelial neoplasms (PIN) were low in Ki67, but high in ANX7 protein.
- samples exhibiting metastatic prostate cancer (MET) had high levels of Ki67, but virtually no ANX7 staining.
- BPH benign prostatic hypertrophy
- MET metastatic prostate cancer
- ANX7 gene expression As seen above, in the case of prostate cancer, the normal prostate has high levels of ANX7, while metastatic and hormone insensitive local recurrences have very low levels of ANX7. By contrast, normal breast tissue has quite low levels (see Figure 26), while cancerous forms have increasingly more ANX7 levels (see figure 19). We also show in figures 19-25 that increasing levels of ANX7 protein expression in breast cancer strongly correlates with lower likelihood of survival.
- Example XIV Example XIV
- ANX7 protein expression was determined in a variety of other tumor types in which the normal tissue was found to have a low level of ANX7, as well as tumor types that tend to have higher levels. Data are given as percent of tumor cells positive for ANX7 protein. These tumor types include sarcoma, lung cancer, and testes, and the results are depicted in Figure 26. Normal adult lung was found to be virtually deficient in ANX7, while fetal lung was 25% positive. Carcinoid, small and large cell lung cancers are profoundly distinct from normal tissue ANX7 levels.
- ANX7 normal tissue gave high levels of ANX7, but some of the tumors tend to have low levels.
- the tissues represented in Figure 27 include skin, lymphoid tissue, prostate (see earlier parts of this description for detailed studies on the prostate), and nerve tissue.
- the tumor types had normal levels of ANX7 protein of ca. 50%. These included salivary gland tumors (adenocarcinoma is completely positive), renal, gynecological, and thyroid.
Abstract
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JP2001514749A JP2003506056A (en) | 1999-08-05 | 2000-08-07 | Knockout mouse of tumor suppressor gene anx7 |
EP00953846A EP1211931A4 (en) | 1999-08-05 | 2000-08-07 | A knockout mouse for the tumor suppressor gene anx7 |
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---|---|---|---|---|
EP1499180A2 (en) * | 2002-04-16 | 2005-01-26 | Dana-Farber Cancer Institute, Inc. | Cancer models |
EP3045470A1 (en) | 2015-01-15 | 2016-07-20 | Deutsches Krebsforschungszentrum Stiftung des Öffentlichen Rechts | Modulators of the function of the core domain of annexins, and uses thereof in autoimmune and/or cancer therapy |
US10947283B2 (en) | 2016-06-10 | 2021-03-16 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Core domain of annexins and uses thereof in antigen delivery and vaccination |
-
2000
- 2000-08-07 EP EP00953846A patent/EP1211931A4/en not_active Withdrawn
- 2000-08-07 CA CA002378292A patent/CA2378292A1/en not_active Abandoned
- 2000-08-07 JP JP2001514749A patent/JP2003506056A/en not_active Withdrawn
- 2000-08-07 WO PCT/US2000/021409 patent/WO2001010199A1/en active IP Right Grant
- 2000-08-07 AU AU66224/00A patent/AU779905B2/en not_active Ceased
Non-Patent Citations (7)
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1499180A2 (en) * | 2002-04-16 | 2005-01-26 | Dana-Farber Cancer Institute, Inc. | Cancer models |
EP1499180A4 (en) * | 2002-04-16 | 2006-09-13 | Dana Farber Cancer Inst Inc | Cancer models |
EP3045470A1 (en) | 2015-01-15 | 2016-07-20 | Deutsches Krebsforschungszentrum Stiftung des Öffentlichen Rechts | Modulators of the function of the core domain of annexins, and uses thereof in autoimmune and/or cancer therapy |
US10947283B2 (en) | 2016-06-10 | 2021-03-16 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Core domain of annexins and uses thereof in antigen delivery and vaccination |
Also Published As
Publication number | Publication date |
---|---|
EP1211931A1 (en) | 2002-06-12 |
AU779905B2 (en) | 2005-02-17 |
EP1211931A4 (en) | 2003-04-16 |
AU6622400A (en) | 2001-03-05 |
CA2378292A1 (en) | 2001-02-15 |
JP2003506056A (en) | 2003-02-18 |
WO2001010199A9 (en) | 2002-03-21 |
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