The present invention further relates to a method for identifying genetic instability and screening for genes involved in genetic alterations by using genes that are capable of inducing genetic instability. More specifically, the present invention provides a method for generating genetically unstable cell lines by using the human metastasin 1.

BACKGROUND OF THE INVENTION Neoplastic cells typically possess numerous lesions, which may include sequence alterations such as point mutations, small deletions, insertions and/or gross structural abnormalities in one or more chromosomes such as large-scale deletions, rearrangements, or gene amplifications (Hart and Saini, 1992 Lancet, 333: 1453-61; Seshadri et al., 1989 Int. J. Cancer, 43: 270-72. Based upon this general observation, it has been suggested that cancer cells are genetically unstable and that acquisition of genomic instability may represent an early step in the process of carcinogenesis and a general feature of many human tumors (Liotta et al., 1991 Cell, 64: 327-36. The ensuing genetic instability drives tumor progression by generating mutations in oncogenes and tumor-suppressor genes, which leads to the clonal outgrowth of a tumor (Ponta et al., 1994 BBA, 1198: 1-10; Berstein and Liotta, 1994 Current Opt. In Oncology ; 6: 106-13; Brattein et al., 1994 Current Opt. In Oncology ; 6: 477-81 ; Fidler and Ellis, 1994 Cell, 79: 315-28). These mutant genes provide cancer cells with a selective growth advantage by promoting resistance to immune-based destruction, allowing disobeyance of cell cycle checkpoints that would normally induce apoptosis,

facilitating growth factor/hormone-independent cell survival, supporting anchorage- independent survival metastasis, reducing dependence on oxygen and nutrients, and conferring resistance to cytotoxic anticancer drugs and radiation. Therefore, elucidation of the genes that cause genetic instability in cancer cells, as well as identification of the genes that are most susceptible to the alterations, represents a promising approach for development of new strategies for combating cancer and for its diagnosis at early stages of development.

Genomic Instability Genomic instability in its broadest sense is a feature of virtually all neoplastic cells. In addition to the mutations and/or gene amplification that appear to be a prerequisite for the acquisition of a neoplastic phenotype, human cancers exhibit other markers of genomic instability, and in particular, a high degree of aneuploidy. Many studies have shown that aneuploidy is an almost invariant feature of cancer cells, and it has been argued by some that the emergence of aneuploid cells is necessary step during tumorigenesis. The functional link between genomic instability and cancer is strengthened by the existence of several"genetic instability"disorders of humans that are associated with a moderate to severe increase in the incidence of cancers. These disorders include ataxia telangiectasia (Gonzalez-del Angel, A. et al.. 2000 Am. J.

Med. Genet., 90: 252-4), Bloom's syndrome (Karow, J. K. et al., 2000 Proc. Natl.

Acad. Sci. USA, 97: 6504-8), Faconi anemia (Leteutre, F. et al., 1999 Brit. J Hematol., 105: 883-93), xeroderma pigmentosum, and Nijmegen breakage syndrome (Mathur, R. et al., 2000 Indian Pediatr. 37: 615-25), of all which are very rare and are inherited in a recessive manner. Analysis of the cells from such cancer prone individuals is clearly a potentially fruitful approach for delineating the genetic basis for instability in the genome. It is assumed that by identifying the underlying cause of genetic instability in these disorders, one can derive valuable information not only about the basis of particular genetic diseases, but also about the underlying causes of genomic instability in sporadic cancers in the general population.

The leading role that genomic instability plays in tumor progression and formation of metastatic cancer involves activation of oncogenes, rearrangement of chromosomes, karyotipic, genetic and epigenetic instability and amplification of genes

(Hart and Saini, 1992 Lancet, 339: 1453-61; Seshadri et al., 1989 Int. J Cancer, 43: 270-72). The metastatic process depends not only on transformation, but also on a chain of interactions between tumor cells with the host's cells and tissues (Ponta et al., 1994 BBA, 1198: 1-10; Berstein and Liotta, 1994 Current Opt. In Oncolgy, 6: 106-13; Brattein et al., 1994 Current Opt. In Oncolgy, 6: 77-81; Fidler and Ellis, 1994 Cell, 79:315-28).

For cancer cell expansion at least two conditions should be met, deregulated cell proliferation and suppressed apoptosis. In their landmark review"The Hallmarks of Cancer,"Hanahan and Weinberg (2000) delineated the principle mechanisms restraining somatic cell proliferative autonomy that must be abrogated for cancers to arise. Normal somatic cell proliferation is shackled both by an absolute need for external mitogens and sensitivity to inhibition by multiple growth-suppressive signals.

Consequently, deregulation of cell proliferation requires acquisition of both autonomy from exogenous mitogens and refractoriness to normal growth inhibitory signals.

Tumor progression is characterized by complex processes such as cell motility and invasiveness, as well as cell proliferation. A number of genes have become correlated with the formation and metastasis of tumors. For example, several normal cellular genes become oncogenes by incorporation into a retroviral genome. Due to the juxtaposition of new promoter elements, such incorporation frequently allows a potential oncogene to be expressed in inappropriate tissues or at higher levels than it normally would be expressed. It appears from work with tumorigenic retroviruses as well as other systems that mis-expression of many cellular proteins, particularly those involved in the regulation of the cell cycle, cell mobility, or cell-cell interaction may lead to a cancerous phenotype.

Evidence is accumulating for an important role of members of the protein S 100 family in these processes. The S100 proteins represent a subfamily from a large family of calcium binding proteins involved in numerous functions ranging from control of cell cycle progression and cell differentiation to enzyme activation and regulation of muscle contraction. The human protein metastasin-1, a (hereinafter,"mts-1"protein), a member of the S 100 family also known as S 100A4, as well the rodent homolog of the human protein has attracted attention from cancer researchers.

The human mts-1 gene has been mapped on chromosome 1. It is clustered with 12 other genes, belonging to the S100 family, on the lq21 region that is altered in several cancer types (Engelkamp et al., 1993 Proc Natl Acad Sci USA, 90: 6547-51 ; Ridinger et al., 1998 Biochim Biophys Acta, 1448 : 254-63). In contrast to other gene clusters, however, the S 100 family genes retain a specific pattern of expression, and they are most likely characterized by independent regulation mechanisms.

The expression of mts-1 is observed in various aggressive cell strains (Baraclough et al., 1987 J Mol. Biol., 29: 293-98; De Vouge and Mukerjee, 1992 Oncogene, 7: 109-19). Moreover, it has been demonstrated that transfection of malignant rodent cell strains with the mts-1 gene may enhance metastasis.

Transfection experiments show that human mots-l and the rat homolog can induce a metastatic phenotype in previously nonmetastatic rat mammary cells (Davies et al., 1993 Oncogene, 8 : 999-1008 ; Lloyd et al., 1998 Oncogene, 17: 465-73). Similarly, transfection of the rodent homolog ti mts-l, scr gene into the B16 murine melanoma (Parker et al., 1994 DNA Cell Biol, 13: 1021-28) and into human breast cancer MCF-7 cells (Grigorian et al., 1996 Int J Cancer, 67: 831-41) increased the capability to metastasize to the lungs. (Grigorian et al., 1993 Gene, 135: 229-38 and Davies et al., 1993 Oncogene, 8: 999-1008. These findings are supported by the demonstration of a marked up-regulation of mts-1 at the mRNA and protein level in murine NIH3T3 fibroblasts or normal rat kidney cells on transformation with oncogenes, such as v-K- ras, v-Ha-ras, or v-src (De Vouge and Mukherjee, 1992 Oncogene, 7: 109-l9 ; Takenaga et al., 1994 Jpn J Cancer Res, 85: 831-39) A possible mechanism for the association between mts 1 and cell proliferation may involve binding of mts-1 protein to the tumor-suppressor protein p53. Mutations in p53/MDM2 pathway prevent apoptosis in cancer cells. Amplification and/or overexpressit of anti-apoptotic molecules is a common feature of many cancer cells. The known anti- apoptotic molecules include several Bcl-2 family members, Bcl-2 itself, Bcl-x (l), Mcl-1, Bc] w, and Al, and IAP (inhibitor of apoptosis protein) family, consisting of IAP1, IAP2, and XIAP proteins which act as endogenous caspase inhibitors. Another way to block apoptosis is overexpression of TNF decoy receptors or production of soluble TNF receptors which are

capable to block binding of TNF ligands with corresponding receptors and thus prevent the initial apoptosis signalling apoptosis.

Grigorian et al demonstrated that transfection of mts-1-negative cells with mtsl constructs led to clonal death, and this death could be prevented by co- transfection with the anti-apoptotic gene bcl-2. The binding of mts-1 to the extreme end of the C-terminal regulatory domain of wild-type p53 was also demonstrated by.

In addition, it was shown that, via interaction with p53, mts-1 differentially modulates the transcription of p53-regulated genes, such as p21/WAF and bax. It was concluded that mts lcooperates with wild-type p53 to stimulate apoptosis, and that this process, at an early stage of tumor development may accelerate the loss of wild-type p53 functions, and consequently lead to the selection of more aggressive cell clones.

(Grigorian et al., 2001 J Biol Chem, 276: 22699-08). Mts 1 may also modulate the functions of at least some p53 mutants and therefore play important roles in advanced cancer stages (Chen et al., 2001 Biochem Biophys Res Commun, 286 : 1212-17).

Angiogenesis is critical for tumor growth and cancer metastasis. Experiments with mts-linducible cell lines grown at high density suggest that mts-1 strongly down- regulates the thrombospondin 1 (THBS1) gene (Roberts, 1996 FASEB J, 10: 1183-91) another p53 target, which is known to repress tumor progression by inhibition of angiogenesis (Grigorian et al., 2001 J Biol Chem, 276: 22699-08) Thus, it is conceivable that mts-1 also promotes angiogenesis in vivo by preventing the anti- angiogenic effect of THBS1. Further, preliminary experiments suggest that mts protein may act directly as an angiogenic factor (Ambartsumian et al., 2001 Oncogene, 20: 4685-95).

The association between mts-1 protein expression and tumor progression raises the question whether this protein represents a useful prognostic marker in clinical practice to patients at increased risk of metastasis who should be considered for more aggressive therapy. Several studies indicate mts-1 over expression in aggressive tumors may identify an at risk subgroup of patients (see e. g. Albertazzi et al., 1998 DNA Cell Biol, 17: 335-42) Davies et al., 1993 Oncogene, 8: 999-08 Platt-Higgins et al., 2000 Int J Cancer, 89 : 198-208 ; Rudland et al., 2000 Cancer Res, 60: 1595-60 ; Pedrocchi et al., 1994 Int J Cancer 1994,57: 684-90). On the other hand, some

members of the S 100 protein family, such as S 1 OOA2, are down-regulated in neoplastic breast cells compared to normal cells and the expression of mts-1 protein decreases quantitatively from low-grade human astrocytomas to high-grade anaplastic astrocytomas and to glioblastomas (Camby et al., 1999 Brain Pathol, 9: 1-19). These results highlight the complexity of the biological functions of the S 100 protein family members, which presumably have reciprocal regulation mechanisms and may influence cell behavior in opposite directions. It is unlikely that mts-1 expression will, in the future, act as an unequivocal biomarker able to accurately discriminate between neoplastic and nonneoplastic cells. Studies on the distribution of mts-1 protein in normal tissues have been hampered by technical problems related to the cross- reactivity of the available antibodies. Thus the need exists for more specific and reliable markers in the diagnosis and prognosis of various types cancers.

SUMMARY OF INVENTION The present invention describes a method of treatment of the diseases associated with genetic instability by suppressing Mts-1 gene or its analogs or the genes that are up-regulated by Mts-1 via novel methods. The present invention further involves a method for detecting genes associated in genetic alterations by using specific genes capable of inducing genetic instability such as mts-1 and their protein products.

The present inventors discovered that cells that were transfected with mots-l and later expressed mts-l, resulted in acquired genomic instability. Based on this finding, the present invention provides a method for using the human mts-1 gene or its protein product, or their analogs, as well as other genes, their protein products and their analogs that have a similar function to that of mts-I gene, to induce genetic instability in tissues and cell lines. Further, the the present invention provides a method for identifying unstable genes susceptible to mts-1 induced instability by comparing the phenotype and gene expression pattern of subclones that express mts-1.

The present invention also provides a method for diagnosing and identifying genetic elements that are involved in a pathological process, as well as a tool for facilitating genome instability research. In addition, the present invention further

provides a method for using these genetically induced unstable cell lines in kits for analysis of genome instability by genome dose variability array (GDVA).

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for identifying genes that are involved in genetic instability comprising the steps of transfecting cells with a vector expressing a gene capable of inducing genetic instability; selecting the cells that express the incorporated gene capable of inducing genetic instability; subcloning the cells; comparing the phenotypes of the subclones that express the incorporated gene capable of inducing genetic instability; and comparing the pattern of genetic expressions of the subclones. The present invention demonstrates that the mots-l gene and its analogs and their protein products can be used to induce genome instability in stable cells and tissues.

While the mst-1 gene is well established as tumor marker for variety of diseases, the ability of this gene to induce genetic instability that is known as major driving force of tumor progression was previously unknown. Therefore, the present invention provides a new method of treatment of cancer and other diseases associated with genetic instability by suppressing the development of genetic instability via modulation of a gene capable of inducing genetic instability in susceptible genes by modulating that gene or modulating genes susceptible to mts-1 induction of instability.

In particular, the present invention teaches the modulation of the mts-1 gene or its protein product or analogs, or genes and their protein products that are up-regulated by the Mts-1 gene to suppress genetic instability in cancer and other diseases associated with genetic instability.

According to the present invention, Human breast carcinoma MCF7 cells were stably transfected with a vector expressing human mts-l. More importantly, the present invention demonstrates that genomic instability is induced by cells that express the mots-l gene. In particular, selected MCF7 cell clones expressing mots-l showed a remarkable variation in their phenotype in terms of their morphology, proliferation rate, sensitivity to estradiol and pattern of gene expression. Among the obtained cell clones, 40% demonstrated multilayer cell growth, indicating immortalization. After re-cloning, these cell strains showed a continued diversification of cell clones with respect to their phenotypes.

In a preferred embodiment, cells are transfected with a vector containing the human metastasin (mst-1) gene and its analogs and their protein products. Cells are selected for expression of mst-1 and subcloned to several generations and analyzed for diversity in phenotype and genetic expression patterns.

It was noted that in the subclones expressing mts-1, the proviral genome IAP (HERV-67) was eliminated whereas the pS2-gene was amplified. In addition, the pS2 and p53 genes, respectively showed a loss of correlation in a similar manner when compared to the dose of certain genes (i e., uPA, TIMP-1, IAP and Bcl-x), whereas subclones of parental MCF7 cells and MOCK-transfected MCF7 control cells demonstrated a good internal correlation at comparisons between all genes. The loss of correlation between dose of different genes in mts-I-expressing cells indicates that mts-1 causes an unstable genome, which might explain the observed progressive diversification of phenotypes.

In another embodiment, patterns of genetic expression are analyzed using a known marker gene such as but not limited to the proviral genome IAP (HERV-67), uPA, TIMP-1, IAP, Bcl-x, p53 and pS2. Genomic DNA samples are isolated from clones, subclones, subpopulations of cells, solid tumors or from subpopulation of non- solid tumors in which genomic instability is induced by mts-1 gene or by its functional analogs. These samples may be labeled with dye or radioisotope, or any other tag known to one skilled in the art. The labeled samples are hybridized with different sets of cDNA, mRNA, oligonucleotide probes or non-coding DNA and RNA probes that are immobilized to an array. Commercial arrays can be used without limitation, such as Human Broad Coverage cDNA Microarray; Human Apoptosis; Human Cancer; Human Cell Cycle; Human Cell Interaction; Human Cytokine/Receptor; Human Hematology; Human Neurobiology ; Human Oncogene/Tumor Suppressor; Human Stress; Human Cardiovascular; Human Trial Kit; Human Cancer 1.2; (3-Kinase ; ß- Signal Transduction; P Metabolism; (3 Diagnostic expressed sequence tags; Human 1.2 I ; Human 1.2 II ; Human Toxicology II; Human 1.2 III ; Human Functional cDNA Microarray; Atlas Glass Human 1.0 Array; Mouse cDNA Broad Coverage MicroArray; Mouse Stress; Mouse 1.2; Mouse 1.2 II ; Mouse 1.2 Cancer; Rat Stress; Rat 1.2; Rat 1.2 II ; Rat Toxicology II; E. coli Gene Arrays; H. pylori Gene Arrays; B.

subtilis Gene Arrays; H. Pylori ORFmers sets; B. subtilis ORFmer sets; E. coli ORFmer sets.

Further, the probe sets could be assembled using various sources such as, without limitation, cancer related genes, retroviral DNA or RNA, DNA fragments, cDNA and RNA extracted from various viruses, microorganisms, plants, insects and animals, synthetic oligonucleotides and their analogs or mimics. Analysis of instability is performed by calculating correlation coefficients (r) for each couple of probes, or by other statistical methods as described below.

In another embodiment, the genomic DNA samples are immobilized to an array and hybridized with sets of cDNA, mRNA, oligonucleotide probes, as well as non-coding DNA and RNA probes that are labeled with a dye or isotope, or any other tag. The probe sets are assembled using various sources such as, without limitation, cancer related genes, retroviral DNA or RNA, DNA fragments, cDNA and RNA extracted from various viruses, microorganisms, plants, insects and other animals, synthetic oligonucleotides and their analogs and mimics. Analysis of instability is done by calculating correlation coefficients (r) for each couple or by other statistical methods as described below.

Analysis of instability can be performed by using statistical methods, for example, by calculating correlation coefficient (r) for each couple of genomic DNA samples. The following correlation analysis methods can be used without limitation: multiple regression; cluster analysis; factor analysis; classification trees; canonical analysis; multidimensional scaling; correspondence analysis; linear regression analysis.

In still another embodiment, analysis of genetic instability in the samples is performed by using comparative genomic hybridization; microsatellite analysis; differential display analysis; or any other similar methods that allow identification of genetic alterations.

In yet another embodiment, analysis of genetic instability of tumor tissues or other pathological tissues is performed by comparing patterns of gene expression using two or more known marker genes. These marker genes may be identified by employing the method of this invention. Therefore, another embodiment of the present invention is a method for identifying unstable genes in cells that are involved

in genomic instability comprising transfecting cells with a vector expressing human mts-1 or its functional analogs; selecting for the cells that express mts-1 ; subcloning the cells that express mts-l ; comparing the phenotype of the subclones that express mts-1, comparing the pattern of gene expression of subclones with two or more marker genes; and identifying the cells as possessing unstable genes if the phenotype of the subclones that express mst-1 continues to demonstrate diversity and the pattern of gene expression for the subclones that express mst-1 show a loss of correlation with the marker gene when compared to other subclones that express mst-1. Genomic DNA, mRNA or cDNA isolated from samples obtained from the analyzed tissue samples are immobilized on arrays and hybridized with the marker genes that are labeled with radioactive or fluorescent probes or any other suitable tag. The correlation analysis between the doses of the marker genes in the samples is performed as described above. Such an analysis can be used without limitation for genetic diagnostics of the disease, evaluation of its development stage and its progression prognosis.

The results of the analysis generated from the present method can be used for evaluation of genetic instability of new genetically altered organisms and cell lines. In another embodiment a single gene or groups of genes that are identified by the above- described strategies, or their expression products are used as targets for therapeutic agents or for preparation of therapeutic and preventive vaccines against the disease.

In still another embodiment, genetic instability is stimulated ex vivo by using unstable genes of the present invention in tissue samples obtained from an individual patient to promote genetic alterations that are characteristics for disease progression in this particular individual. The single antigens or groups of antigens, or genetic material such as mRNA or cDNA, are further used to prepare therapeutic and preventive vaccines against the disease.

In another embodiment, the present invention can be used for diagnostics and identification of genetic elements involved in a pathological process related to genome instability.

In a particular embodiment, different cancer or normal cells lines (LoVo, H23, CaCo-2, MCF7, ME-SA, etc.) are transfected with genetic construction expressing wild type or mutant mtsl gene or its functional analogs and stably transformed

immortalized sub-lines are selected. These sub-lines are used for evaluation of new anti-cancer drugs, drugs for cardiovascular, CNS, and other diseases, as sources of genetical material for further investigations, etc. and for screening of resistance to known drugs.

In another embodiment, total DNA purified from parental and mtsl transfected cell sub-lines is used for the identification of amplified or lost chromosome regions, analysis of gene copy number changes, endogenous virus elimination, chromosome rearrangements (translocations, inversions, deletions) by comparative genomic hybridization, FISH analysis, Southern hybridization, hybridization with commercial or custom made cDNA or oligonucleotide microarrays, or microarrays comprised of BAC/PAC genomic clones, or microarrays comprised of chromosome specific genomic clones.

Total or mRNA is purified from parental and mtsl transfected cell sub-lines and gene expression patterns are analyzed by hybridization with cDNA or oligonucleotide microarrays, Northern hybridization, quantitative RT-PCR, and mRNA protection assay. Genes specifically up-or down-regulated by mtsl transfection are also identified by differential display, subtractive hybridization, SAGE analysis, or sequencing of cDNA libraries prepared from parental and mtsl transfected cell lines, in silico analysis of available expression databases. In addition, total protein extracted from parental and mtsl transfected cell sub-lines is used for the analysis of protein expression by Western blot, 2D electrophoresis, and proteomics arrays. Changes in the expression or intracellular localization of individual proteins in mtsl-transfected cells are analyzed by immunohystochemistry/immunocytochemistry, immuno electron microscopy.

Thus, according to the present invention genes and their protein products specifically amplified/lost or up-/down regulated in mtsl transfected cells are identified. This set of unstable genes and chromosome regions comprises several genes and chromosome regions that are known to be implemented in different cancers and pinpoints many new genes whose role in cancer development and progression was not previously known. Pro-inflammatory and anti-apoptotic genes constitute a substantial portion of all amplified and/or up-regulated genes. Mtsl and these amplified/up-regulated pro-inflammatory and anti-apoptotic genes and their protein

products are used as drug targets for screening pharmaceutical compositions suspected to have therapeutic utility for the treatment of metastatic cancer and other diseases accompanied by genetic instability, inflammation-related diseases, cardiovascular, CNS, and other diseases.

Mtsl gene and its protein product and unstable genes and their protein products identified by the present invention are used as markers of cancer progression or progression of other diseases (inflammation, cardiovascular, CNS, etc.). Antibodies to these proteins are used as diagnostic tools for clinical investigations. cDNAs and proteins are used for the production of diagnostic microarrays (array of unstable genes), diagnostic PCR or RT-PCR, ELISA analysis, etc.

Libraries of biological agents (small molecules, peptides, drugs) are screened for the compounds that interact with mts 1 and/or its protein product or functional analogs thereof using Elisa assay, phage display and other binding assays. These libraries are also searched for compounds capable of disrupting the homo/heterodimer formation between mtsl and its functional analogs using energy transfer, fluorescence quenching, etc. Mtsl is used as a bite in the two-yeast binding screening system, in phage display screening, and in screening of cDNA expressing libraries to identify its new binding partners.

Compounds that are generated from gene products or selected from libraries according to the invention are screened for the ability to inhibit mtsl-induced up- regulation of anti-apoptotic and pro-inflammatory genes identified by the present inventtion The selected compounds are used for treatment of mtsl-transfected cancer sub-lines (breast, lung, colon, etc.) and the expression of anti-apoptotic and pro- inflammatory genes is monitored.

These compounds may also be screened for the ability to inhibit mtsl-induced immortalization of cells.

The compounds that are selected are screened for the ability to inhibit inflammation reaction induced by mts-1 and other inflammation inducers.

Apoptosis induced by taxol or other drugs or other chemical or physical stimulus is inhibited in mtsl transfectants. Using FACS analysis, TUNNEL assay, cytotoxicity tests, the compounds that are identified and selected according to the

present invention are screened for the ability to inhibit mtsl-mediated suppression of apoptosis in relevant models. Selected compounds interact with mtsl protein product or functional analogs of thereof and are potent inhibitors of mtsl-mediated suppression of apoptosis. These compounds are screened for their ability to induce apoptosis directly in different cancer cell lines or assist in the induction of apoptosis by known pro-apoptotic drugs.

The compounds that are generated or selected from libraries are also screened as anti-inflammatory drug candidates using appropriate models.

The biological agents that show pro-apoptotic or anti-inflammation activities are administered into human or animal patients using an appropriate pharmaceutical composition.

The present invention contemplates treating cancers and other diseases associated with genetic instability by inactivating, destroying or nullifying the function of mts-1 or an unstable gene identified by the present invention. For example, the antibodies, prepared as described above, may be utilized to inactivate mts-1 protein expressing cells that contain unstable genes: either unconjugated antibodies or anti- mts-1 or unstable gene antibodies conjugated to a toxin may be employed in the therapy of cancer.

The present invention contemplates pharmaceutical compositions containing, for example, an antibody reactive with a mammalian mts-1 or mts-1 up-regulated polypeptide or protein, or an antisense nucleic acid. The active ingredients of a pharmaceutical composition containing the protein products of mts-1-transfected cells possessing unstable genes, antibodies to these protein products and antisense nucleic acids are contemplated to exhibit effective therapeutic activity, for example, in eliciting an immune response, or inhibiting mts-1 functions respectively. Thus the active ingredients of the therapeutic compositions containing such active agents are administered in therapeutic amounts which depend on the particular disease. For example, from about 0. 511g to about 2000 mg per kilogram of body weight per day may be administered. The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or

the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

Depending on the route of administration, the active ingredients which comprise gene protein products or reagents that interact with them may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, the low lipophilicity of mts-1 protein, and some anti-cancer reagents, may allow them to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide or nucleotide bonds and in the stomach by acid hydrolysis. In order to administer the active agents by other than parenteral administration, they should be coated by, or administered with, a material to prevent inactivation. For example, they may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes.

The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the mts-1 protein or anti-cancer reagents are suitably protected as described above, the active compound may be orally administered, for example, with

an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.5 t. g and 2000 llg of active compound.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from about. 5 ig to about 2000 Rg. Expressed in proportions, the active compound is generally present in from about 10 u. g to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

As used herein"pharmaceutically acceptable carrier"includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and adsorption delaying agents, and the like. The use of such media agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

Moreover, the present invention provides a method of suppressing the development of genetic instability in disorders associated with genetic instability, such as cancer, autoimmune disorders, inflammatory disorders, and neurodegenerative disorders in a susceptible cell by providing to the susceptible cell a nucleic acid encoding an antisense nucleotide sequence. For example, such an antisense nucleic acid can comprise SEQ ID NO : 1, SEQ ID NO : 2. SEQ ID NO : 3. or SEQ ID NO : 4. Preferably, the antisense mts-1 nucleic acids of the present invention have at least 15 or 17 nucleotides.

According to the present invention, antisense mts-1 nucleic acids can inhibit the development of genetic instability in susceptible cells by binding to sense mts-1 mRNA. Such binding can either prevent translation of mts-1 protein or destroy mts-1 sense mRNA, e. g., through the action of RNaseH. Accordingly, less mts-1 protein is available to susceptible cells and the development of instability in these cells is prevented.

EXAMPLES Example I.

Construction of mots-l expression vectors In order to facilitate subcloning, a mts-1 fragment was PCR-amplified using primers containing restriction sites. The pCMVmts-1 plasmid was obtained by inserting in the sense orientation, a 320 bp BamHl/Xbal fragment (nucleotides-10 to +310) into the pCMV vector in which expression is under the control of the cytomegalovirus (CMV) promoter (Baker et al., 1990).

A Tet-Off plasmid was obtained by inserting in the sense orientation, a 320 bp BamHl fragment of mts-1 gene (nucleotides-10 to +310) into the pUHDlO-3 which expression is under the control of the CMV minimal promoter which is under the tetracycline operator control.

A linear construct containing pCMV promoter, mts-1 gene, rabbit B-globin intron II and polyadenylation signal was obtained from pCMV mts-1 plasmid.

Genomic DNA fragment of mts-1 or cDNA of mts-1 can be used for insertion into the genome for stimulation of instability.

Example II.

Preparation of genetically instable cell subclones Malignant MCF7 cells (ATCC HTB 22) were cultured in plastic flasks in Eagle medium in modification by Dulbecco (DMEM), containing 10% of calf serum.

The pCMVmts-1 plasmid was obtained by inserting in the sense orientation, a 320 bp BamHI/Xbal mtsl cDNA into the pCMV vector in which expression is under the control of the CMV promoter (Baker et al., 1990). MCF7 cells were electroporated

with a linearized pCMVmts-1 construct (l0ug) or linearized pCMV vector alone (10ug), using a BioRad apparatus at 400 volts and 125 mF. Transfectants were then selected with the neomycin analog G418 (400pg/ml, Gibco BRL) by means of the neomycin-resistance gene of the pCMV vector.

Cloning of MCF7 cells transfected by pCMV and pCMVmts-1 construction was performed in petri dishes (Falcon 100 mm) at the estimated density of 30 cells per dish. The visual control was performed under an inverted microscope (Nicon).

Total RNA was prepared from cultured cells using a single step procedure (Chomczynski and Sacchi, 1987). RNA was fractionated by electrophoresis on a 1% agarose gel in the presence of formaldehyde, and transferred to nylon membranes (Hybond N, Amersham). Filters were acidified for 10 min in 5% CH3COOH and stained for 10 min (0.004 % methylene blue, 0. 5M CH3COONa, pH 5.0) prior to hybridization. Northern blots were hybridized under stringent conditions (50% formamide, 42°C) with mts-1 fragment and 36B4 cDNA probes 32P-labeled by random priming. Washing was performed in 2 X SSC, 0.1% SDS at 22°C, followed by 0.1 X SSC, 0.1% SDS at 55°C.

The transgene expression was assayed using Western blot analysis. Confluent cells were trypsinized and centrifuged at 500g for 5 min; at 20°C. Cell pellets were washed in PBS buffer and analyzed by SDS-polyacrylamide gel electrophoresis (15%) under reducing conditions. Protein concentration was determined using the BioRad kit (BioRad Laboratories). For immunoblot analysis, proteins were transferred after electrophoresis to nitrocellulose filters that were incubated with a mouse monoclonal antibody specific for human mts-1 (a generous gift from Dr. Lukanidin). Bound antibodies were visualized using a peroxidase-labeled goat antibody raised against mouse IgM, followed by Enhanced Chemiluminescence detection (ECL kit, Dupont NEN).

Among forty-eight primary cell clones with G418 resistance, eighteen clones were found to express mts-1 RNA and protein, whereas mtsl RNA and the protein were not detectable in the parental MCF7 cells, in the pCMV3 and pCMV4 clones transfected with the pCMV vector alone or in the pCMVmts-1 transfected clone 28A6.

Example III.

Characterization of the primary mts-1 positive cell clones The original MCF7 cells and the obtained cell clones were grown in 6-wells falcon plates with or without estradiol in DMEM medium supplemented with 10% FCS without the"phenol red". To remove estradiol and other steroids, the culture medium was treated with activated charcoal. The proliferation rates of the cells were analyzed by counting trypsinized cells every 24 hours using a Coulter cell counter (Coultronics, France). Cell proliferation was estimated as the ratio of the number of cells growing for 24 hr to the number of cells at the beginning of the experiment. The cell motility was analyzed using the"wound"test. To that end, a 1x10 mm parafilm bands were placed on the bottom of each well of 6-wells falcon plate, pressed down, and the analyzed cell suspensions were added. After the cells reach confluence the parafilm bands were removed, whereby allowing a cell-free space in the wells. The starting time point was the moment the parafilm bands were remove. The cell motility rates were measured using the following formula: V = a (ll-k)/2N, where V is the monolayer border motility ( m/24 hr); 11-the distance between borders of the monolayer at the beginning of the experiment; 12-the distance between the right and the left monolayer borders in N days; a-proportionality coefficient measured experimentally using an internal standard scale bar. The results are presented in Table 1.

Table 1. Phenotypic characterization of the primary cell clones Clone (Mts-1 Proliferation Index Cell Motility, pm/day expression) Estradiol + Estradiol- 15A5 (+) 1. 69 1.38 113~12 15B2 (+) 1.45 0. 96 250+31 28A1 (+) T26LOO1824 28B6 (+) 1. 11 0. 80 98+9 28A6 (-) 1. 55 0. 99 64+6 MCF7 (-) 1. 61 1. 00 112+13

In addition to the above experiments, a visual observation on the cell growth and phenotype was performed by means of daily photographing of the same group of cells.

A multilayer cell growth was observed in 40% of cases of mts-1 transfectants, but not in control clones,. The initial MCF7 cell line did not show multi-layer growth during the culturing.

Example IV.

Subcloning of the primary mts-1 positive clones The primary cell clones of the Example I were subcloned further. The subclones that were obtained after the third subcloning demonstrated a high morphological diversity indicating a progressive change in phenotype within each obtained cell subclone expressing mts-1. The morphological variations in the subclones were represented by different colony sizes ranging from several cells to several hundred cells in one colony after two weeks of growth; different colony shapes and different cell sizes. Some colonies demonstrated internal heterogeneity such as several different cell shapes within a single colony. Universal feature observed for all subclones expressing mts-1 was an overgrowth of fillapodies, which was not observed in the parental MCF7 cells.

To confirm that the cells present in the same subclone are generated from the single parental primary clone, the location of the mts-1 insertion in the genome was analyzed. The cells from each subclone were grown in 75 mm2 flasks to a confluent monolayer and washed twice with PBS. After the addition of 2 ml of buffer for genomic DNA extraction containing 10 mM Tris-HCl, pH 8.0; 0. 1M Na2-EDTA, pH 8.0; 0.5% SDS; 2011gel ribonuclease A; 100, ug/ml proteinase K, the flasks were incubated at 56°C for 12 hours. The genomic DNA was precipitated with 1 volume of isopropanol. After washing with 70% ethanol, DNA was dried in the air and dissolved in TE buffer at 4°C. For determination of the insertion localization, 101lg genomic DNA from each sample were treated with endonuclease XhoI. For the control of the restriction completeness, lg -phage DNA was added to the reaction mixture. DNA fractionation was performed in 0.8% agarose with subsequent transfer onto Hybond

N followed by hybridization with mtsl cDNA. The resulted Southern blot analysis confirmed that the insertion sites within the analyzed subclones were the same.

Therefore, the subclones represent pure cell strains and are not a result of contamination by other transfectants.

Example V.

Analysis of the gene doses of the selected gene panel in the cell subclones The following genes were analyzed in the subclones of the Example III: Cadherin, Bcl-x, PUMP-1,36B4, Col72, uPA, pS2, mtsl, p53, pS2, HERV-67. The same gene panel was analyzed in the cell subclones derived from the non-transfected MCF-7 cells and MCF-7 cells transfected with the MOCK plasmid without Mts-1 insert as control.

Cells were grown in 75 mm2 flasks to a confluent monolayer and washed twice with PBSxl. After addition of 2 ml of buffer for extraction (10 mM Tris-HCl, pH 8.0; 0. 1M Na2-EDTA, pH 8.0; 0.5% SDS; 20 pg/ml ribonuclease A; 100tg/ml proteinase K), the flasks were incubated at 42°C for 12 hours. The genomic DNA was precipitated with 1 volume of isopropanol. After washing in 70% ethanol, DNA was dried in the air and dissolved in TE buffer at 4°C. For dot blot analysis, 1 0pg genomic DNA was denatured in 0.4 M NaOH at 65°C for 1 hour and deposited on 10 identical Hibond Ns blots, 1 llg on each. The quantitative estimation of hybridization was carried out using a phosphoimager.

Southern dot blot analysis of the obtained subclones and of control subclones of MOCK-transformed MCF7 cell subclones was performed. We prepared 9 identical 96 wells-dot blots with immobilized DNA from subclones expressing and not expressing mts-1. As a control, genome DNA from BALB/c mouse and plasmid DNA containing Cadherin, Bcl-x, PUMP-1,36B4, Col72, uPA, pS2, mtsl, p53, pS2, HERV-67 genes were used. The dot blots were hybridized to the following 32p_ labeled cDNA probes: TIMP-1, IAP (HERV-67), mtsl, pS2; E-Cadherin, 36B4, Bcl- x, p53, uPA. A quantitative estimation of hybridization signals was carried out using a phosphoroimager. The results were grouped as shown in Table 2. Table 2. Results of the hybridization analysis (relative units) A. Mtsl transfected MCF7 subclones Clone/gene uPA ITMPI PS2 Mtsl HERV CADH 36B4 Bcl-x P53 C1 16 46 1472 18 29 107 90 12 17 C2 14 44 2285 22 25 115 111 13 21 C3 30 63 2362 21 30 179 150 14 23 C4 13 60 2407 23 22 143 113 9 21 C5 15 59 2497 33 27 157 136 10 22 C6 17 61 2053 24 27 149 125 11 21 C7 17 42 2166 21 19 114 105 13 18 C8 16 47 2303 21 17 105 102 10 23 C9 16 45 2038 25 19 123 126 11 20 C10 14 45 1391 20 21 98 97 11 21 C11 23 119 3122 52 65 197 183 15 25 C12 14 59 683 15 37 70 75 10 15 C13 15 58 556 12 50 67 78 11 14 C14 13 41 931 15 33 83 87 11 16 C15 14 48 1258 19 31 100 92 8 19 C16 13 86 1030 17 47 98 89 9 18 C17 13 94 1273 22 62 126 115 11 19 C18 24 77 2250 40 38 211 138 15 21 C19 20 71 1882 33 41 165 120 15 23 C20 17 54 1128 27 26 135 83 11 17 C21 23 87 2731 40 42 255 170 15 22 C22 17 54 737 31 30 111 118 11 17 C23 18 72 1488 35 42 98 93 14 19 C24 16 59 565 25 43 118 126 12 16 C25 16 55 1091 23 39 86 85 12 17 C26 20 56 1188 32 42 189 163 11 22 C27 18 56 1186 26 39 139 111 10 19 C28 16 50 724 23 30 119 100 11 21 C29 14 63 287 12 49 64 58 11 14 C30 17 58 865 29 35 133 83 10 19 C31 20 57 1463 31 41 122 115 9 17 C32 19 60 867 27 51 108 107 14 15 C33 18 57 869 26 44 102 138 12 16 C34 24 55 117 9 39 76 40 9 9 C35 15 54 467 20 45 78 101 14 14 C36 15 56 1247 28 39 126 120 11 18 C37 14 45 678 26 41 99 95 11 15 C38 15 40 799 19 28 89 78 10 16 C39 17 54 615 24 38 86 82 9 16 C40 16 46 1816 24 25 120 88 9 19 B. MCF7 subclones Clone/gene uPA IMP1 PS2 Mts1 HERV CADH 36B4 Bcl-x P53 C1 14 61 279 9 39 97 92 8 12 C212341487214542910 C3 16 64 920 8 16 124 103 10 22 C4 10 27 91 7 10 34 22 8 10 C5 15 61 975 9 32 160 128 12 20 C6 16 62 1389 8 30 131 126 12 22 C7 16 54 1888 8 37 150 151 11 22 C8 17 67 687 10 43 132 123 13 23 C. MOK transfected MCF7 subclones Clone/gene UPA TIMP1 PS2 Mtsl HERV CADH 36B4 Bcl-x P53 C1 18 56 684 9 37 156 137 11 21 C2 17 60 292 9 45 133 123 12 21 C3 18 54 766 9 38 133 115 9 21 C4 18 54 687 9 35 127 126 11 22 C5165360494115691921 C6 14 57 464 9 42 133 75 8 21 C7 14 69 460 9 34 122 82 9 17 C8 19 60 678 8 36 137 113 11 19 C9 20 62 1694 8 32 115 155 13 16 C10 20 70 1745 9 37 161 132 11 19

Example VI.

Linear regression analysis of results of Example V The hybridization data of the Example IV were subjected to a linear regression statistical analysis. The resulted correlation coefficients (r) reflecting the relative doses of each couple of analyzed genes in the subclones are shown in tables 3-5. These results show that lack of correlation between the gene doses was observed only in the mts-1 transfected subclones (Table 3), while in the mts-1 negative subclones (MOCK transfected subclones, Table 5), as well as in the subclones derived from the parental MCF-7 cells (Table 4) all the analyzed gene doses were in correlation.

Table 3. Mtsl expressing MCF7 subclones*. TIMP pS2 mtsl IAP Cadh 36B4 Bclx p53 0. 36 0. 19 0.30 0.13 0.60 0.46 0. 49 0.12 uPA 0.22 0.50 0.75 0.52 0.49 0. 41 0. 23 TIMP 0.52-0.34 0.67 0.62 0.22 0. 82 pS2 0.23 0.71 0. 76 0.38 0. 82 mtsl 0.09 0.09 0. 27-0. 28 IAP 0.83 0. 47 0. 77 Cadh 0. 52 0. 70 36B4 0.14 Bclx Table 4. MCF7 cell subclones TIMP pS2 Mtsl IAP Cadh 36B4 Bclx P53 0.91 0. 83 0.61 0.58 0.92 0.94 0.74 0. 91 UPA 0. 58 0. 50 0. 61 0. 86 0. 84 0. 58 0. 76 TIMP 0.30 049 084 090 081090 PS2 0. 74 0. 73 0. 67 0. 35 0. 42 Mtsl 0.63 0. 72 0. 37 0. 32 IAP 0.97 0.82 0.90 Cadh 0. 79 0. 88 36B4 zu Bclx Table 5. MCF7 MOCK transfected subclones. TIMP pS2 mtsl IAP Cadh 36B Bclx p53 4 0. 64 0. 87 0.64 0.60 0. 70 0.85 0.65 0.67 UPA 0.76 0.45 0.63 0. 74 0. 68 0.47 0. 73 TIIP 0.55 0.50 0.89 0.84 0.80 0.79 pS2 0.49 0.59 0.26 0.59 0.36 mts1 0. 59 0. 66 0.54 0.38 IAP 0.78 0.46 0.80 Cadh 0.78 0. 76 36B4 0.69 Bclx

*Correlation coefficients between different genes when the gene dose within subclones was compared. Table 3: mts-1-expressing subclones (n=41) ; Table 4: parental MCF7 derived subclones (n=7); Table 6: MOCK-transfected subclones (n=18). The names of the cDNAs used are given above each column and to the right of each row. Correlation coefficients correspond to each pair of genes located in a cross between a column and a row. Correlation coefficients <0.3, which are considered to show, as a loss of correlation, has been marked in bold.

Example VII.

Cluster analysis of the results Example V The hybridization data of the Example V were subjected to cluster statistical analysis- (Squared Euclidian distances method). The following gene groups were established: A: CADH; 36B4 B: TIMP ; IAP

C: UPA; Mts-1 ; Bcl-x; p53 D: PS2 Example VIII.

Cluster analysis of the results of Example V The hybridization data of the Example 5 were subjected to clustering tree statistical analysis. The following gene groups were established: A: CADH; 36B4 B: TIMP ; IAP C: UPA ; Mts-1 ; Bcl-x; p53 D: PS2 Example IX.

Preparation of p53 plasmid construct for expression in animal tissues A p53 fragment was PCR-amplified using primers containing restriction sites in order to allow its subcloning. The pCMVmts-1 plasmid was obtained by inserting in the sense orientation, a 1.4 kb HindIII/Bgl II fragment into the pCMV vector in which expression is under the control of the cytomegalovirus (CMV) promoter.

Example X.

Preparation of p53 linear construct for expression in animal tissues A p53 fragment was PCR-amplified. Linear construction which expression is under the control of the cytomegalovirus (CMV) minimal promoter, intron II and polyadenylation signal of Rabbit p-globin was used for intramuscular immunization of mice.

Example XI.

Preparation of p53 adenoviral construct for expression in animal tissues A p53 fragment was PCR-amplified using primers containing restriction sites in order to allow its subcloning. The Adenovirus-mts-l construction was obtained by

inserting in the sense orientation, a 1.4 kb fragment into Adenovirus by using Adeno- XTM expression system (Clonetech).

Example XII.

Protective immunization of CT-26 tumor bearing Balb C mice with the plasmid encoding p53 A plasmid (pCMV-p53) encoding p53 gene driven by the cytomegalovirus immediate early region promoter and enhancer was used for intramuscular immunization of mice implanted with CT-26 cells.

The plasmid was expanded in E. coli DH5 strain and prepared using a Qiagen Endotoxin-free plasmid Giga-prep kit according to the supplier's protocol.

The purified plasmid DNA was resuspended in sterile saline (Gibco-BRL) and kept frozen in aliquots at a concentration of 5 mg/ml.

Before each intramuscular injection, the Balb C mice were anesthetized with a mixed solution of ketamine and xylazine. The animals were injected on days 0 and 14 with 5 and 50 ug of pCMV-p53. At day 20, each animal was challenged with 1 x 106 of CT-26 cells implanted subcutaneously and survival rates were monitored. The median survival times (MST) determined during this study were as follows: control group (non-immunized animals)-22.4+9 days ; experimental group (immunized animals)-535. 7 days.

Example XIII Microarrav comparative genomic hybridization To estimate the alterations in gene copy numbers in mtsl transfected subclones genomic DNA from parental cell lines LoVo and H23 and from several mtsl transfected subclones was randomly labeled with 32P-dCTP or digoxigenin-dUTP using Easy-Strip or DECAprime II kits (Ambion). Labeled DNA from parental and mtsl transfected clones was subsequently hybridized to commercial human cancer arrays 1.2 (Clontech) and custom-made arrays containing apoptosis and growth factor related cDNAs. For hybridization and pre-hybridization, ULTRArray Hyb buffer (Ambion) was used. Membranes were pre-hybridized at 42°C for 2 h in a hybridization

oven (Techne), hybridizations were performed at 42°C overnight. The concentration of labelled probes in the hybridization mixture was 20 ng/ml, before hybridization the probes were denaturated at 98°C for 10 min. After hybridization, membranes were rinsed twice with lxSSC, 0.1% SDS for 15 min at room temperature, and then with prewarmed 0. lxSSC, 0.1% SDS for 15 min at 68°C. After equilibration for 5 min in washing buffer (0.3% Tween 20 in maleic buffer (0.1 M maleic acid, 0.15 M NaCI, pH 7.5)), membranes were blocked for 2 h in 1% blocking solution (Roche) under slight agitation, and then treated for 30 min in 10 ml of alkaline phosphatase conjugated anti-digoxigenin antibody (Roche, Laval, QC) diluted 1: 2500. Following antibody incubation, membranes were rinsed three times for 15 min in washing buffer, equilibrated for 2 min in detection buffer (0.1 M Tris-HCl, 0.15 M NaCl, pH 9.5), and stained overnight with 175 llg/ml 5-Bromo- 4-chloro-3-indolyl-phosphate, toluidine salt (BCIP), and 330 ug/ml Nitro blue tetrazolium chloride (NBT) in detection buffer. Arrays were scanned using Phosphorlmager (Molecular Dynamics) for 32p labelling or flat bed scanner for digoxigenin labelling. For comparison of hybridization patterns in parental cell lines and in mtsl transfectants different software can be used, for example AtlasImage 1.5 (Clontech) or ScanAlyze (http://rana. lbl. gov/EisenSoftware. html). Table## lists amplified genes which were identified in mtsl transfected subclones. The remarkable feature of several genes amplified in mtsl transfectants is their participation in inflammatory reactions, inhibition of apoptosis, or signal transduction. Table 7. Genes amplified in mtsl-transfected subclones, their localization in genome, and function. Gene amplified in Genome Function mtsl transfectants region Iapl, baculoviral iap 1 lq22 apoptotic suppressor. the bir motifs region repeat-containing interacts with tnf receptor associated factors 1 and protein 2, birk2,2 (trafl and traf2) to form an heteromeric complex, (inhibitor of which is then recruited to the tumor necrosis factor apoptosis protein 1) receptor 2 (tnfr2). (hiapl) (c-iap2) Iap2, baculoviral iap 11 q22 apoptotic suppressor. the bir motifs region repeat-containing interacts with tnf receptor associated factors 1 and protein 3, birk3,2 (trafl and traf2) to form an heteromeric complex, (inhibitor of which is then recruited to the tumor necrosis factor apoptosis protein 2) receptor 2 (tnfr2). (hiap2) (c-iapl) Bcl2, B-cell 18q21 suppresses apoptosis in a variety of cell systems, CLL/lymphoma 2 regulates cell death by controlling the mitochondrial membrane permeability. TNFRSF6B, tumor 20ql3. 3 binding Fas ligand (TNFRSF6) and inhibiting necrosis factor FasL induced apoptosis, amplified in primary lung receptor and colon tumors superfamily, member 6B Traf4, tumor 17ql 1 Member of a family of proteins that interact with necrosis factor TNF receptors; expressed in breast carcinoma (TNF)-receptor-epithelial cells associated factor 4 Traf6, tumor llql3. 3 Involved in the activation of NFKB by TNFRSFs ; necrosis factor interacts with the serine/threonine kinase IRAK (TNF)-receptor- (IRAK1) in response to interleukin-1 (IL1) associated factor 6 stimulation; TNFRSF8, tumor lp36 receptor for a cytokine ligand known as cd301. necrosis factor may play a role in the regulation of cellular growth receptor and transformation of activated lymphoblasts. superfamily, member regulates gene expression through activation of 8, (CD30) nfkb SHC1, transduction lq21 may couple activated growth factor receptors to a adaptor protein SHC signaling pathway that regulates the proliferation of mammalian cells. shc might participate in the transforming activity of oncogenic tyrosine kinases IL1RL2, interleukin 2ql2 receptor for interleukin-1 alpha (il-la), beta (il-lb), 1 receptor related and interleukin-1 receptor antagonist protein (il- protein 2 lra). binding to the agonist leads to the activation of nf-kappa b. IL6ST, interleukin 5ql 1 signal-transducing molecule. the receptor systems 6, signal transducer for il-6, lif, osm, cntf, il-11 and ct-1 can utilize (GP130) gpl30 for initiating signal transmission. binds to il-6/il-6-r (alpha chain) complex, resulting in the formation of high-affinity il-6 binding sites, and transduces the signal. does not bind il-6. may have a role in embryonic development (by similarity). TNFRSF14, tumor lp36. 3 receptor for tnfsfl4. involved in lymphocyte necrosis factor activation. plays an important role in hsv receptor pathogenesis because it enhanced the entry of superfamily, several wildtype hsv strains of both serotypes into member 14 cho cells, and mediated hsv entry into activated (herpesvirus entry human t cells. mediator) NfkappaB2, nuclear 10q24 plO0 is the precursor of the p52 subunit of the factor of kappa light nuclear factor nf-kappa-b, which binds to the polypeptide gene kappa-b consensus sequence 5'-ggrnnyycc-3', enhancer in B-cells located in the enhancer region of genes involved in 2 (p49/plO0) immune response and acute phase reactions. the precursor protein itself does not bind to dna ephrin-B 1 Xql2 transmembrane ligand of Eph-related receptor tyrosine kinases, has a role in cell adhesion Ephrin-Al lq21 ligand of Eph-related receptor tyrosine kinases, induced by by tnf-alpha and interleukin-1 beta IL8RA, interleukin 2q35 receptor to interleukin-8, which is a powerful 8 receptor, alpha neutrophils chemotactic factor, involved in angiogenesis of endothelial cells IL18R1, interleukin 2ql2 member of the interleukin-1 receptor family; binds 18 receptor 1 interleukin-18 (IL 18)

Example XIV Comparison of gene expression in parental and mtsl-transfected cell lines Gene expression was analyzed in the original LoVo and H23 cells and the obtained mtsl-transfected cell clones. Total RNA was isolated using TRIzol Reagent according to manufacturer's instructions (Gibko). The RNA concentrations were determined by its absorbance at 260 nm. The quality of the RNA was verified by the integrity of 28S and 18S rRNA after ethidium bromide staining of RNA samples that were subjected to 1 % agarose gel electrophoresis. Before microarray hybridizations, total RNA was additionally purified and DNase treated on RNeasy columns (Qiagen, Mississauga, ON) according to manufacturer's recommendations. Digoxigenin (DIG)-labeled probes were produced in reverse transcription reaction using EndoFree RT kit (Ambion) with oligo (dT) anchored primer and 2 llg of total RNA from cell lines.

Gene expression was analyzed using Clontech membranes (human cancer arrays 1.2) or custom-made microarrays. Conditions for hybridization were the same as for microarray comparative genomic hybridization in example 1. Comparison of genes identified as up-regulated and amplified genes from Example 1 shows their substantial

overlap. For example, up-regulation was detected for such amplified genes as Traf4, Traf6, CD30, NikappaB2, gpl30, SHC1, IAP1, IAP2, and Bcl2. Analysis of gene expression revealed also up-regulated genes that were not amplified, i e. TNFAIP1, API4 (surviving), CD40. At the same time there are genes which were amplified but do not show overexpression, i. e. ZEB, TCF4, IL9, TNFRSF5, TNFRSF11A, Hrk.

Example XV Quantitative RT-PCR assay Altered expression of genes in mts 1 transfected cells was analyzed by quantitative RT- PCR.

First-strand cDNA was synthesized using 1 llg of total RNA with MMLV SuperScript II reverse transcriptase (Gibco BRL, Burlington, ON) according to manufacturer's directions. Each 15 ulPCRreactionwasperformedin 10mMTris-HCl, pH8. 6,50 mM KC1, 0.1% Triton X-100,1.5 mM MgCk, with 0.5 mM of each dNTP, 20 pM of gene- specific primer, 2.5 U of Taq DNA polymerase (Amersham Pharmacia Biotech, Baie d'Urfe, QC), and 1 u. l of reverse transcription reaction. Reactions were thermal cycled in a PTC200 PCR system (MJ Research) as follows: first denaturation at 94°C for 3 min, 30 cycles at 94°C for 45 sec, at 60°C for 1 min and at 72°C for 45 sec, the last extension at 72°C for 7 min. For quantitative PCR, each cDNA was 5 times serially 4-fold diluted and aliquots of these dilutions were used as templates for simultaneously performed PCR reactions with gene-specific primers and GAPDH-or 18S rRNA-specific primers as internal control (Ambion). Ethidium bromide stained agarose gels were quantitated using Scionhnage software (http://www. scioncorp. com). Linear range of PCR was identified for each gene and intensities in linear range were normalized according to intensities of GAPDH or 18S RNA. Comparison of gene expression in parental cell line LoVo and in mtsl-transfected subclones demonstrates variable upregulation of antiapoptotic (IAP1), proinflammatory (TNFRSF14, CD30, RelB, gpl30, NfkappaB2), and signalling (Ephrin B 1) genes in mtsl transfectants. The results of this analysis are represented in Table 2.

Table 8. Relative expression of selected genes in mtsl transfected subclones in comparison with parental cells LoVo. Expression in parental cell line is equal to 1.

Subclone/gene BCL2 TNFRSF6BTNFRSF14CD30EphrinBlRelB gp130NFkB2 IAP1 1505.10 1 1 4 4 4 1 16 4 64 1505.12 1 1 1 1 4 4 4 16 64 1505.16 1 1 1 1 1 16 4 4 16 1505.21 1 1 1 4 4 16 4 16 16 Example XVI, Cell Transfection by antisence oligonucleotide against Mts-1.

Cell Lines and Cell Culture. MCF7 bres carcinoma cells and MCF7 transfected by mtsl cells grown in Dulbecco's modified Eagle medium in low-glucose (DMEM). All cell culture media were supplemented with 10% FCS (fetal calf serum, D. Dutscher, Brumath, France), 2 mM L-glutamine (Gibco BRL, Cergy-Pontoise, France), 100 units/mL penicillin (Gibco BRL), and 100 pg/mL streptomycin (Gibco BRL). Cells were maintained at 37 °C in a 5% C02 humidified atmosphere. When 80% confluent, they were detached with saline trypsine/EDTA (Gibco, BRL) and grown in new flasks at a 1/10 dilution.

Cell Transfection. Adherent cells were seeded in 24-well plates (Costar, D. Dutscher) the day before transfection so they could reach 60-70% confluence during transfection. All experiments were done in triplicate. Prior to transfection, cells were rinsed and 1 mL of culture medium complemented with 10% FCS was added. Two micrograms of the desired oligonucleotide was diluted into 50 pL of 0. 15 M NaCl.

The desired amount of 25 kDa PEI [from 10 mM stock solutions of PEI in water at pH 7.5; 1 NIP equivalent corresponds to the amount of polymer necessary to have one amino group (43 Da mean Mw for PEI per phosphate of nucleic acid (330 Da mean Mw) (Boussif et al., 1996)] was diluted into 50 pL of 0. 15 M NaCl, vortexed gently, and spun down. Fifteen minutes later, the cationic vector was added at once to the plasmid solution [and not the reverse order; see Boussif et al. (1996)], mixed, vortexed, and spun down. The amounts and volumes given above refer to those in a

single well and were actually 3-fold larger and distributed in three wells. After ca. 10 min, the resulting mixture was added to the cells and the cell supernatant was uniformly distributed with a gentle horizontal hand rotation. Immediately after, the cell culture dish was centrifuged for 5 min at 1500 rpm (ca. 280g). The cells were cultured for 24 h and then tested for gene expression by using Northern blot analysis.

The invention relates to oligonucleotides of the formula ATCACACGTACTATAGCAACA (SEQ. ID. NO. 1) TCTCCAGAGGGCACGCCATGACA (SEQ.ID.NO.2) TCATTTCTTCCTGGGCTGCTTATCT (SEQ.ID.NO.3) CCAACCACATCAGAGGAGT (SEQ. ID. NO. 4) Table 9.

Antisense Oligonucleotide reduction of mtsl mRNA level in mtsl transfected MCF7 cells.

RNA was extracted as described previously. Mtsl expression analyzed by Northern blot and normalized to 36B4 RNA content as described previously.

Example XVII Development of resistance to paclitaxel upon Mts-1 transfection The cells (parental MCF-7 and stable Mts-1 transfectants 15B4; 15A5; and 28B3) were cultured to achieve a sub-confluence condition. After that, the cells were treated with trypsin and were transferred to 96-well plates at the density of 10,000 cells per well. The cells were cultured in the presence of various concentrations of paclitaxel at 5% COa and at 37°C for 48h and then medium was changed to DMEM, 10% FCS without Taxol and incubated additional 48h. After that the medium was changed and standard XTT test was performed. The absorbance of the samples was analyzed by ELISA reader at a wavelength of 450 nm. The results were expressed as percentage of cell growth inhibitions and IC-50 values, resistance indexes were calculated. The results are shown in Table 10.

Table 10. Comparison of sensitivity of parental MCF-7 cells and stable Mts-1 transfectants to cytotoxic effect of paclitaxel Cell line MCF7 15B4 15A5 28B3 IC-50,pM 0. 3 3 6 12 Resistance 1 10 20 40 Index Example XVIII Decreased paclitaxel-induced apoptosis in mtsl-transfected cells.

Cells were treated with different concentrations of paclitaxel (Taxol) essentially as described before. After tripsinization, cells were fixed by 4% formaldehyde in PBS.

DNA labeling by terminal transferase (TdT) and flow cytometry analysis were performed according to protocol provided by Roche Diagnostics for cell death labeling TUNEL kit. Data of the analysis are presented in Table 11. Apoptosis induced by

paclitaxel treatment was substantially lower in both mts-1 transfected clones of MCF7 cells.

Table11A. MCF7 cells treated with paclitaxel Living cells % Akpoptotic cells % Dead cells % Control 78. 38 +1. 05 21. 62+1.06 0.00 Taxol 0. 1, uM 43. 18+4.06 29. 55+1.10 27.27+5.16 Taxol 0.05µM 28. 57+2.13 57.14+0.94 14.29+3.07 Taxol 0.025µM 43. 10+2.33 48.28+0. 25 8.62+2.08 TablellB. 15A5 subclone (MCF7-mtsl cells) treated with paclitaxel Living cells % Apoptotic cells % Dead cells % Control 94. 44+0.33 5.56+0.41 0.00 Taxol 0.1µM 45. 10+4.75 21.57+1.06 33. 33+5. 70 Taxol 0. 05tM 68. 18+2.43 25.00+0.62 6. 82+2.71 Taxol 0. 025µM 90.91+0.51 9. 09+1.050. 00 Table11C. 28B3 subclone (MCF7-mtsl cells) treated with paclitaxel Living cells % Apoptotic cells % Dead cells % Control 90. 53+1.53 9.47+0.91 0.00 Taxol 0.1µM 31. 04+2.92 44.60+1.6 24.37+2.62 Taxol 0. 05gM 70. 18+1. 28 29. 82+2.24 0.00 Taxol 0.025µM 92. 34+0.44 7.66+. 0.50 0.00

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