USE OF A MELANOMA DIFFERENTIATION ASSOCIATED GENE (mda-7) FOR REVERSING A CANCEROUS PHENOTYPE

Description: TECHNICAL FIELD [Detailed description of the invention]

Use of melanoma differentiation-related gene (mda7) to reverse cancerous phenotype This application is filed on June 16, 1996, US Serial No. 08 / 696,57. This is a partial continuation application of 3, and the content of the original application is a part of the disclosure content of the present application by reference. To do. The present invention disclosed herein is the Department Health and Hu. Government assistance under NCI / NIH grant No. CA35675 from man Services) Made by. Therefore, the US Government has some rights to the invention. Throughout this application, various references are cited in parentheses. These publications All disclosures are by reference for a more complete description of the state of the art to which the invention belongs. It is a part of the disclosure content of this application. A complete bibliographic citation of these references is a series of It can be seen at the end of the experiment. Background of the invention Cancer has multiple remnants that function as positive and negative regulators of tumorigenesis Complex, multifactorial and multistage involving interrelated expression and suppression of genes It is a process (1-5). Based on the introduction of a dominant phenotype of transformation or tumorigenesis Direct cloning strategies identify oncogenes that act positively (6-9). In contrast, detection of genes that suppress the cancer phenotype and crony Ng was found to be more difficult and difficult to catch (10-15). Proliferation and differentiation Active approaches to the isolation of genes directly involved in regulation It irreversibly loses its ability to proliferate with a cDNA library constructed from proliferating cancer cells. And a cDNA libra constructed from cancer cells induced to undergo final differentiation Subtraction hybridization with Lee is required (13, 14) ). This experimental strategy has been applied to human melanoma cells and is recombinant Human interferon β (IFN-β) and mezerein (MEZ) ) Induced final differentiation and previously described in the DNA database No novel melanoma differentiation-related (mda) genes have been cloned (13, 14) .. Increase The direct role of certain mda genes in mediating the regulation of reproduction and cell cycle is mda. It was revealed by the identification and cloning of -6 (13-16), which is a cycle. It is identical to a universal inhibitor of cyclin-dependent kinase p21 (17). In growth control The importance of p21 has been fully described, and this gene has been developed using various approaches. Individually as WAF-1, CIP-1, and SDI-1 by some laboratories Isolated on (18-20). Based on these studies, specific genes involved in growth control Is induced and is involved in the process of growth arrest and final differentiation in human cancer cells It is suggested that it can be done. Humans whose mda-7 gene has been treated with a differentiation inducer (IFN-β plus MEZ) Cloned from the melanoma (H0-1) deduction library (13, 14) ). The full-length mda-7 cDNA is 1718 nucleotides and its major open The reading frame is a new tampa of 206 amino acids with Mr of 23.8 kDa. Codes quality (21). According to previous studies, mda-7 has human melanoma. It has been suggested that it is induced in vesicles as a role of growth arrest and induction of final differentiation. (14, 21). In addition, the expression of mda-7 is inversely related to the progression of melanoma. That is, normal human melanocytes that proliferate actively are metastatic human melanoma cells. It expresses more mda-7 than vesicles (21). Besides, mda-7 is a transient tran In the sulfation assay and dexamethasone (DEX) inducible mda- Suppresses the proliferation of human melanoma cells in stable transformed cells containing 7 genes Control (21). In these studies, mda-7 was found in human melanocytes and melanoma. It has been shown that it can contribute to the physiology of human melanoma. It has been shown to have growth inhibitory properties when overexpressed in cells. The mda-7 gene is also available in the International Patent Cooperation Treaty Application No. PCT / US94 / 121. Recorded in 60 (International Filing Date October 24, 1994, International Publication No. WO95 / 11986) It is included and this content is hereby incorporated by reference. In the present invention, mda-7 has various origins (chest, central nervous system, cervix, colon, prostate). And reports that it is an effective growth suppressor gene in cancer cells (such as connective tissue) I will tell you. Suppression of colonization is p53 and / or retinoblastoma (RB) remains Cancer cells with defective genes, or deficient expression of p53 and RB It occurs in cells. In contrast, normal human breast epithelial cells, human skin fibroblasts And the expression of mda-7 in the fibroblasts of rat embryos is quantitatively as high as that of cancer cells. Does not induce growth inhibition. mda-7 is a human cervical carcinoma (HeLa) and prostatic Stable expression in adenocarcinoma (DU-145) cells causes proliferation and transformation It has a negative effect on matation-related characteristics. What is the effect of mda-7 on HeLa cells? , Feeling of a genetically modified Ad5 vector expressing the antisense mda-7 gene It can be reversed after the MDA-7 protein is absent by dyeing. This From these observations, mda-7 is widespread in human cancers showing various genetic defects. It is suggested that it is a novel growth inhibitory gene having a range of inhibitory action. Outline of the invention The present invention is a method for reversing the cancerous phenotype of cancer cells and melano. Nucleic acid containing a differentiation-related gene (mda-7) under conditions in which the gene can be expressed. A method of reversing the cancerous phenotype of the cancer cells by introducing them into the cancer cells. I will provide a. The present invention also aims to introduce the above nucleic acid into cancerous cells of a subject. To provide a method for reversing the cancerous phenotype of cancer cells in a subject. To. The present invention is also a method for reversing the cancerous phenotype of cancer cells. Introducing the gene product of the melanoma differentiation-related gene (mda-7) into cancer cells Further, a method for reversing the cancerous phenotype of the cancer cells is provided. The present invention In addition, by introducing the above gene product into the cancerous cells of the subject, the subject can be treated. Provided is a method for reversing the cancerous phenotype of cancer cells. The present invention is also effective for reversing the cancerous phenotype of cancer cells. A certain amount of nucleic acid containing a differentiation-related gene (mda-7) and a pharmaceutically acceptable carry Provided is a pharmaceutical composition containing a. The present invention also relates to a cancerous phenotype of cancer cells. A certain amount of the gene product of the above gene effective for inversion and pharmaceutically acceptable Provided is a pharmaceutical composition comprising a competent carrier. A brief description of the drawing Figure 1. mda-for the formation of hygromycin-resistant colonies in HeLa cells 7 Effect of expression. 10 μg pREP4 vector (RSV vector), pREP4 vector in HeLa cells Mda-7 (RSV-MDA-7-a) cloned into the ter with antisense orientation Mda cloned into a pREP4 vector with a sense orientation) Transfected with -7 (RSV-MDA-7-sense) and 100 μg high Selected in medium containing glomycin. Figure 2. expressing the pREP4 vector HeLa cl 1 cell and mda-7 (S) Effect of antisense mda-7 on monolayer proliferation of HeLa cl 2 cells. HeLa cl 1 (HeLa clone transformed with pREP4 vector) cells and He Lacl 2 (HeLa clone expressing mda-7) cells express antisense mda-7 Recombinant type 5 adenovirus (Ad5) [Ad.mda-7 (AS)] 1 0 Proliferate after or without infection with plaque-forming units / cells Ta. The result is the average cell number of 3 samples with a &lt;10% difference. Figure 3. High molecular weight in HeLa, HeLa cl 1 and HeLa cl 2 cells-MDA- Against 7 complex (HMC) protein, MDA-7 protein, actin protein The effect of antisense mda-7. HeLa cells and HeLa cl 1 cells (HeLa black transformed with pREP4 vector) ), [³⁵S] 10 plaque-forming units / cells labeled with methionine Infected with Ad.mda-7 (AS) for 96 hours (+) or uninfected (-), HMC, MDA-7 and actin protein levels measured by immunoprecipitation analysis I decided. For HeLa cl 2 (HeLa clone expressing mda-7), 10 pullers For protein levels of infection with Ad.mda-7 (AS) in the formation unit / mL After +24, +48, +72 and +96 hours [³⁵S] Methionine label Cell lysates were measured by immunoprecipitation analysis. HeLa cl 2 cell control The effect of infection with the roll mutant Ad5 (H5dl 434), 10 plaque formation After 96 hours of unit / cell infection, [³⁵S] Methionine-labeled cell lysate Was measured by immunoprecipitation analysis. Figure 4 In a DU-145 clone containing the A &amp; 4B.DEX-inducible mda-7 gene synthesis of mda-7RNA and protein. Figure 4 A.10^-6Proliferate cells for 96 hours in the presence or absence of M DEX , Isolate total RNA, perform Northern blotting, mda-7, neomycin resistant Sex (Neo^R) Gene and GAPDH were detected using a probe. Figure 4 B.10^-6Proliferate cells for 96 hours in the presence or absence of M DEX , Cellular protein [³⁵S] Labeled with methionine, MDA-7 and Acti Immunoprecipitation was performed using an antibody that recognizes the protein. Figure 5. Transthymation of established human cervical cancer (HeLa) in athymic nude mice Suppression of plant growth. Figure 6. Effect of Ad.mda-7 S on HeLa tumor volume ratio. The results showed that Ad.mda-7 S suppressed tumor progression in vivo in nude mice. It shows that it can be done. Detailed description of the invention It appears somewhat frequently to make it easier to understand the detailed explanation of the experiment described later. The method and / or terminology is explained in Sambrook et al. (45). The present invention is a method for reversing the cancerous phenotype of cancer cells and melano. Nucleic acid containing a differentiation-related gene (mda-7) under conditions in which the gene can be expressed. Inverting the cancerous phenotype of the cancer cells by introducing them into the cancer cells Provide a method provided with. The present invention is also for reversing the cancerous phenotype of cancer cells in a subject. A nucleic acid molecule containing a melanoma differentiation-related gene (mda-7), which is a method, is used by a subject. Introduced into cancerous cells of the subject under the condition that the gene can be expressed in the cells of A method comprising reversing the cancerous phenotype of the cancer cells by I will provide a. Methods of introducing nucleic acid molecules into cells are well known in the art. Naked nucleus Acid molecules can be introduced into cells by direct transformation. Alternatively, a nucleic acid molecule Can be embedded in liposomes. Therefore, the present invention is a naked DNA technique, Adenovirus Vector, Adeno-Related Viral Vector, Epstein-Barr Luvirus vector, herpes virus vector, attenuated HIV vector, retro Coated with viral vector, vaccinia viral vector, liposomes, antibody Nucleic acid molecules are introduced into cells by liposomes or mechanical or electrical means The above method is provided. The methods listed above are viable to introduce nucleic acid molecules into cells. It is just an example of such means. Other known methods are used in the present invention. You can also. In one aspect of the above method, the melanoma differentiation-related gene (mda-7) is responsible for its development. The present is linked to the regulatory sequence so that it is under the control of the regulatory sequence. Yet another aspect In, the regulatory sequence is inductive or constitutive. Inducible promoter Inducible regulatory sequences such as are known in the art. Directing constitutive expression Regulatory sequences, such as promoters, are also known in the art. In another embodiment, the regulatory sequence is a tissue-specific regulatory sequence. At this time, md Expression of the a-7 gene will be tissue-specific. In another aspect of the above method, the cancer cells are defective tumor suppressors within the cancer cells. It is characterized by the presence of a control gene. Defect tumor suppressor genes include p53, retinoblastoma (RB) or p16^ink4aContains genes, which Not limited. In one embodiment of the method, the cancer cells are dominantly acting (domi) within the cancer cells. nant acting) Characterized by the presence of oncogenes. Especially dominant work The oncogenes used are Ha-ras, mutant p53 or human papillomavirus remains. Can be a messenger. Ha-ras is the Harvey virus's ras oncogene. In one aspect of the method, the nucleic acid comprises a vector. The vector is Ade Novirus Vector, Adeno-Related Viral Vector, Epstein-Baru Ils vector, herpes virus vector, attenuated HIV vector, retroui Includes, but is not limited to, Ruth vector and Vaccinia virus vector. Good In a better embodiment, the adenovirus vector is replication defective expressing mda-7. It is an adenovirus vector and is called Ad.mda-7 S. Smell in another aspect The adenovirus vector is a replication competent adenovirus vector. To. The present invention is also a method for reversing the cancerous phenotype of cancer cells. Introducing the gene product of the melanoma differentiation-related gene (mda-7) into cancerous cells To provide a method comprising reversing the cancerous phenotype of the cancer cells. To. The present invention further reverses the cancerous phenotype of cancer cells in a subject. The gene product of the melanoma differentiation-related gene (mda-7) of the subject By introducing into cancerous cells, the cancerous phenotype of the cancerous cells can be reversed. And provide a method. In one embodiment of the method, the cancer cells are the chest, cervix, colon, prostate. Includes, but is not limited to, cells of the nasopharynx, lungs, connective tissue or central nervous system .. The cancer cells also include cells derived from glioblastoma polymorphism, lymphoma and leukemia. included. The present invention is also effective for reversing the cancerous phenotype of cancer cells. A certain amount of nucleic acid containing a differentiation-related gene (mda-7) and a pharmaceutically acceptable carry Provided is a pharmaceutical composition containing a. The term "pharmaceutically acceptable carrier" as used herein is any standard. Also includes a variety of pharmaceutical carriers. This pharmaceutical composition is for the dosage method of choice. Can be configured in any suitable form. Compositions suitable for oral dosing are in solid form (Pills, capsules, granules, tablets, and powders, etc.) and liquid forms (solvents, Includes syrups, elixirs, and suspensions). Effective form for parenteral medication The states are sterile solvents, emulsions, suspensions and the like. In some embodiments, the nucleic acid comprises a vector. Vector is adenovirus Vector, Adeno-Related Viral Vector, Epstein-Barr Virus Bek Tar, herpes virus vector, attenuated HIV virus, retrovirus vector -And, but not limited to, vaccinia virus vectors. preferable In an embodiment, the adenovirus vector is a replication defective a that expresses mda-7. It is a denovirus vector and is called Ad.mda-7 S. In another aspect , Adenovirus is a replication competent adenovirus vector. The present invention also provides a constant amount effective for reversing the cancerous phenotype of cancer cells. Gene product of melanoma differentiation-related child (mda-7) and pharmaceutically acceptable carrier To provide a pharmaceutical composition comprising and. In one embodiment of the method, the cancer cells are the chest, cervix, colon, prostate. Includes, but is not limited to, cells of the nasopharynx, lungs, connective tissue and central nervous system .. The cancer cells also include cells derived from glioblastoma polymorphism, lymphoma and leukemia. included. The present invention will be further understood by the detailed description of the experiments below. However For those skilled in the art, the specific method and the results being discussed will be covered by the claims described below. It will be readily appreciated that it is merely an example of the invention as fully described. Detailed description of the experiment Cancer is a disease characterized by deficiencies in growth control, and tumor cells often It often shows an abnormal pattern of cell differentiation. Recombinant human fibroblast interferon And the combination of the anti-leukemia drug mezerein is a human melanoma cultivator. Corrects these abnormalities in nourishing cells, resulting in irreversible growth arrest and final differentiation To reach. Humans that have stopped growing and are finally differentiated by subtractive hybridization -Melanoma differentiation-related gene (mda-7) with increased expression in melanoma cells ) Is identified. Human tumors in which mda-7 has multiple genetic defects of different origin When transfected into vesicles, colonization is reduced. In contrast, normal Cells (human breast epithelial cells, human skin fibroblasts and rat embryo fibroblasts) Proliferation and colony in a transient transfection assay using The effect of mda-7 on formation is quantitatively less than the effect found using cancer cells I. Tumor cells with increased expression of mda-7 are monolayered and scaffold-independent Shows suppression. Recombinant type 5 adenowi expressing antisense mda-7 Proliferated and transformed phenotype in vitro by infection with Ruth The suppression by mda-7 is removed. Retinoblastoma (RB) and p53 gene Increased in cancer cells that do not express both or have defects in both The ability of mda-7 to suppress growth is this in mediating mda-7-induced growth suppression. It shows that important tumor suppressors are not involved. previously described by mda-7 Absence of growth-suppressing genes and protein homology, and normal cells / Due to the differentiating effect of this gene on cells, mda-7 has a new class of cancer growth It is suggested that it corresponds to a growth-suppressing gene. Materials and methods Cell lineage and culture conditions Human carcinoma cell lineage, eg MCF-7 and T47D (chest), LS174 T and SW480 (colorectal), HeLa (cervix), DU-145 (prostate) , And HONE-1 (nasopharynx) (9, 22-25), 10% fetal bovine serum (D) In Dulbecco's improved Eagle medium supplemented with MEM-10) And 37 ° C, 5% CO₂/ 95% air-grown in a humidified incubator. Another Human cell types such as HBL-100 (normal breast epithelium), H0-1 and C 8161 (melanoma), GBM-18 and T98G (glioblastoma polymorphism), and Saos-2 (human osteosarcoma) was maintained under similar conditions. Normal human breast in early passage Epithelial cells (HMEC; passage 10-12), Clonetics Corporation (San Diego) , CA). HMEC cells described by Clonetics Corporation It was maintained in serum-free medium as described above. CREF-Trans 6 (Clone Fi Fischer rat embryo fibroblasts (9, 26) and CREF Ha-ra s (CREF cells transformed with the Ha-ras (T24) oncogene) (27) , DMEM-5 culture. HeLa cl 1 is hygromycin resistant (Hyg)^R)of Transformed with Rous sarcoma virus RSV vector (pREP4) (Invitrogen) It is a HeLa clone. HeLa cl 2 is Hyg^RHeLa claw expressing mda-7 Is. HeLa cl 1 and HeLa cl 2 cells were constructed as described in the literature (12, 21), maintained in DMEM-10 containing 100 μg / mL hygromycin Ta. DU-145 cl 6 and DU-145 cl 7 cells are (pMAMneo vectors) Includes (21) DEX-inducible mda-7 gene (Clontech) cloned into ), Maintained in DMEM-10 containing 200 μg / mL G418. Subtracted hybridization, plasmids, expression vector constructs, and no Zan hybridization Identification and cloning of mda-7 by subtraction hybridization is a statement. Achieved as described in the offering (13). Recombination I of full-length mda-7 cDNA Screen the H0-1 cDNA library treated with FN-β plus MEZ By using the cDNA terminal rapid amplification method as described in the literature (13). (15) Isolated. CDNA fragment of mda-7 containing open reading frame (Nucleotide position 176-960) amplified by PCR and pC by TA cloning RII^TMCloned to (Invitrogen). Limits the orientation of inserts in the vector Determined by mapping. Constructs of human cell expression PC with KpnI-XhoI fragment R^TMSense from vector to pREP4 vector (Invitrogen) [mda-7 (S)] Or with antisense [mda-7 (AS)] orientation, downstream of the RSV promoter Created by rolling. Separately, sense the fragment of the mda-7 gene. And antisense orientation cloned into pMAMneo (Clontech) vector .. RNA isolation and Northern blotting were performed as described (9,12,13, twenty one). Monolayer growth, scaffold-independent and DNA-transfection assays Monolayer and scaffold-independent growth assays, as previously described in the literature. Went to (8,12,26). To investigate the effect of mda-7 on monolayer colonization , Insertless vector, or mda-7 (S) or mda-7 (AS) expression construction The vector [pREP4 (RSV)] containing the substance is used by the lipofection method (GIBC). Transfected to various cell types by O / BRL) Hygromycin-resistant colonization or cell proliferation in thin was measured (12,2) 1). Construction of antisense-mda-7 adenovirus vector A recombinant replication defect Ad.mda-7 (AS) was created in two steps. First, m Crony the coding sequence of the da-7 gene into the improved Ad expression vector pAd.CMV (28). This is, in turn, 355 bp from the left end of the Ad genome, the site Pre-Early Promoter of Megalovirus (CMV), Splice Donating Site and Acceptance Part DNA encoding, cloning part for the desired gene (mda-7 in this case) Position, DNA encoding the poly A signal sequence derived from the β-globin gene, and E Contains an adenovirus sequence of approximately 3 kbp extending from within the IB coding region. This array is High levels by the CMV pre-early gene promoter of the cloned sequence Allows expression and RNA processing (28). Recombinant virus 293 cells by homologous recombination between mda-7-containing vector and plasmid JM17 Created in vivo in vivo (29), which was cloned into the modified pBR322 It contains the entire Ad genome (30). JM17 gives rise to Ad genome in vivo However, the Ad genome is too large to integrate. From this constraint, the choice By recombination with a vector to create an integrateable genome containing a gene Will be released (30). Recombinant virus is compounded in human cells other than 293 cells There is a manufacturing defect, which expresses adenoviruses E1A and E1B. Two pu After transfection with rasmid, the infectious virus is recovered and the genome is analyzed. The recombinant structure was confirmed, and then the virus was plaque-purified. All to standard method Got more (31). Peptide antibody production and immunoprecipitation analysis Peptide antibody prepared for PSQEN EMFSIRD as described in the literature (21). HeLa, HeLa Cl 1 (Hyg) that proliferates logarithmically^RPREP4 Vector Co By trolling HeLa clone) and HeLa cl 2 [pREP4-mda-7 (S) Transfected Hyg^RMda-7-expressing HeLa clone] cells untreated Or control adenovirus of 10 plaque-forming units (H5) Recombinant adenovirus expressing d1434) (32) or mda-7 (AS) [Ad.md a-7 (AS)] was infected. Mech the culture at various times after infection Methionine starvation for 1 hour at 37 ° C in onin-free medium to precipitate cells Concentrate by letting, 100 μCi (1Ci = 37GBq)³⁵S (NEN, Express ³⁵S ) Was labeled in 1 mL of the same medium for 4 hours at 37 ° C. 2 μg MDA-7 Petit Rabbit polyclonal antibody or actin monoclonal antibody (Oncoge) Immunoprecipitation analysis using ne Sciences) was performed as described in the literature (15,21). .. Experimental result Mda- in human cancer cells and Ha-ras-transformed rat embryo fibroblasts Increased growth inhibitory properties of 7 DNA transfection assay performed, mda-7 expression for cell proliferation The effect of the increase was evaluated. Mda-7 (S) construct in human cervical cancer (HeLa) cells When transfected, the mda-7 (S) construct becomes a pREP4 vector and And Hy g^RColonies are reduced 10 to 15 times (Fig. 1 and Table 1). Table 1. Human cancer cells, normal rat embryo fibroblasts (CREF) and Ha-ras Effect of mda-7 on monolayer colonization of transformed CREF cells<img file="JP2000516618A_D0001.tif" />a Logarithmically proliferating cells, 1 × 10 per 100-mm plate⁶Sow with, insert Vector, or 10 μg vector containing mda-7 (S) or mda-7 (AS) Transfected with ter. After 24 hours, 100 μg / mL hygro cells Approximately 2 x 10 per 100-mm plate in medium containing mycin⁵Seeded again with cells .. The medium is changed every 3 or 4 days, and the plate is replaced on the 14th or 21st day. It was fixed with Lumamide and stained with Giemsa. Count colonies containing more than 50 cells Ta. The values shown are the average Hyg formed on 4 or 5 plates.^RColony ± SD To. b Values in parentheses are transfected with RSV-mda-7 (AS) It shows the reduction multiple of colonization when compared with the cells. c MCF-7, T47D, HeLa, LS174T, DU-145 and HONE- 1 is a human carcinoma (Ca) cell lineage isolated from the indicated structural site. T98 G is the cell lineage of human glioblastoma polymorphism. CREF-ras is Ha-ras (T24) It is a CREF clone transformed with a gene. In addition to less colonization, mda-7 (S) colonies are generally pRE Obtained after transfection with P4 vector or mda-7 (AS) construct Corresponding Hyg^RIt is smaller than the colony (Fig. 1). Separate mda-7 (S) structure When transfected into human cancer cell lineage, Hyg^RNo colonization 3 And reduce it by 10 times (Table 1). These include human breast carcinomas (MCF-7 and T47D), colon cancer (LS174T and SW480), nasopharyngeal cancer (H) ONE-1), prostate cancer (DU-145), melanoma (H0-1 and C8) 161), glioblastoma polymorphism (GBM-18 and T98G) and osteosarcoma (Saos-2) Is included. Transfected with mda-7 (S) construct, as observed in HeLa cells Hyg formed after action^RAverage colony size is empty pREP4 vector Or from those formed after transfection with the mda-7 (AS) construct small. These results show that mda-7 has a wide range of histologically different human cancer odors. When overexpressed, it demonstrates that it is an effective growth-suppressing gene. mda- Whether 7 suppresses the growth of normal cells, and this effect is observed in human cancer cells Transient DNA tigers to determine if they are quantitatively identical to the effects Expression assay, passage 10-12 normal human breast epithelium (HMEC) ) Cells, normal thoracic epithelial cell lineage HBL-100, normal human skin fibroblasts (passage) 21) and cloned normal rat embryo fibroblast lineage (CREF-Tra) This was done using ns6) (7,8). HMEC, HBL-100 and normal human skin Fibroblasts frequently form distinct colonies, even when using the feeder layer Since it does not, transfect with a different RSV construct and hygro The effect on the total cell number after growing in mycin for 2-3 weeks was measured. this Transfect with mda-7 (AS) or pREP4 vector using approach Approximately 1.1 to 1 in mda-7 (S) and HMEC for normal cells .6-fold reduction, 1.1-1.2-fold reduction in HBL-100, normal human skin fibroblasts 1.3 to 2.1-fold reduction in numbers (in 3 independent experiments with each cell type) It was observed. In contrast, T47D human breast cancer using a similar experimental protocol For tumor cells, vector and antisense transfected cells Approximately 3.2-5 after transfection with the mda-7 (S) construct compared to .2-fold growth was suppressed. For CREF-Trans6 cells, 6 independent transfections Hyg for Action Assay^RIn colonization, mda-7 (AS) and The difference between mda-7 (S) for Kutar-transfected cells is 0.5 It extends from to 2.8 times (Table 1). In contrast, the Ha-ras form of the mda-7 (S) structure Transfection into altered CREF cells reduces colonization 6-8 fold (Table 1). These results show human cancer cells and Ha-ras transformed la In normal human cells and normal rodent cells, mda-7 is as much as embryonic cells. Shows that it is not quantitatively effective in reducing proliferation and colonization .. Stable and inducible effect of mda-7 expression on cell proliferation, and mda-7 expression Anne The effect of Chisense inhibition on cell proliferation, as well as the transformed phenotype. Infrequent HeLa cell survival after transfection with the mda-7 (S) gene After transfection with the mda-7 (S) construct, 1 0 independent Hyg^RColonies were isolated. Mda-7 by Northern blotting Of the 10 clones analyzed for expression, 7 clones were detectable mda-7 m Not expressing RNA, 2 clones express low levels of mda-7 mRNA One clone (named HeLa cl 2) expresses high levels of mda-7 mRNA. Was there. In contrast, all of this clone is Hyg^RAnd glyceraldehyde 3- Showed comparable levels of expression of the phosphodehydrogenase (GAPDH) gene .. Compared to parental HeLa cells or pREP4 vector HeLa clones (named HeLa cl 1) By comparison, HeLa cl 2 (mda-7 expressing) cells proliferated at a low rate (Fig. 2). On agar When proliferated, uncloned HeLa and HeLa cl 1 cells are about 42 Proliferated with an efficiency of%, but HeLa cl 2 (mda-7 expressing) cells increased with an efficiency of about 25% The average size of the bred and colonies is the parental HeLa and pREP4 vector HeLa cl 1 It was smaller than observed for cells. From these results, the tiger on mda-7 HeLa survival after inhibition is predominantly deficient or low mda-7 expression It has been shown to occur due to levels of mda-7 expression. But stable mda-7 In monolayer culture and scaffold-independent in HeLa cells expressed at high levels Proliferation is reduced. Decreased in vitro growth observed in HeLa cl 2 (mda-7 expression) and To determine if transformation inhibition is a direct result of mda-7 expression, md An antisense strategy was used to directly suppress a-7 expression. Ann Recombinant Ad5 vector containing the mda-7 gene cloned in the chisense orientation [Ad.mda-7 (AS)] was constructed. HeLa cl 2 by Ad.mda-7 (AS) ( Infection with mda-7 expression) increases growth rate and agar cloning efficiency (from about 25% to about) Increased (to 44%), but up to HeLa cl 1 (pREP4 vector, mda-7 not expressed) Or the parent HeLa is not (Figure 2). In contrast, it does not contain the mda-7 gene. The mutant Ad5 vector (H5dl434) of the trolley is the parent HeLa, HeLa cl. Does not affect monolayer or agar proliferation of 1 or HeLa cl 2 cells (data) Not shown). Mda-7-specific pebutide antibody produced in rabbits and immunoprecipitation analysis were used However, in HeLa cl 2 (mda-7 expression) cells, MDA of about 24 kDa protein -7 and levels of ultra-high molecular weight complex (HMC) proteins of about 90-110 kDa Is increasing (Fig. 3). Infection with Ad.mda-7 (AS) is 24 kDa MD Causes a temporary drop in both A-7 and HMC proteins, This is not the case with the mda-7 non-expressing virus of the H5dl434 control (21) (Fig. 3). .. Decreased levels of both proteins were 48 hours after infection with Ad.mda-7 (AS). Seen in, and remains suppressed for 96 hours. In contrast, actin levels Remains unchanged after virus infection. These observations are for HeLa cl 2 (mda- Antisense inhibition of MDA-7 protein expression in 7 expression) leads to mda-7 It was shown that the suppression of growth and the suppression of scaffold-independent growth by scaffolding can be directly eliminated. There is. To confirm the inhibitory effect of mda-7 on cell proliferation, DU-145 human prostate cancer Cells were engineered to express the DEX-inducible mda-7 gene.

[DEX inducibility Contains the mda-7 (S) gene] DU-145 cl 6 or cl 7 cells, 10^-6M DEX When grown for 24-96 hours in the presence of mda-7 mRNA and (HMC tongue) Proteins (including proteins) are induced, but not in parental DU-145 cells (Fig. 4). In contrast, DEX is neo in DU-145 cl 6 and cl 7 cells. Mycin resistance (Neo^R) Does not alter gene expression or on all tested cells It does not change the expression of GAPDH (Fig. 4). DU-145 cl 6 and cl 7 In cells, 10^-6Induction of mda-7 expression by proliferation in M DEX is non-DEX Compared to proliferation in the presence, it reduces the number of cells after 96 hours by about 50%. In contrast, Parental DU-145 or pMAMneo vector transformed DU-145 cells, 10^-6MD Growth in medium containing EX for 96 hours does not cause significant growth inhibition (day) Not shown). These data show that ectopic expression of mda-7 is present in prostate cancer cells. It is shown that cell proliferation can be directly changed. Experimental consideration Human mice that have stopped growing and are finally differentiated by subtractive hybridization. Increased expression of the mda gene was identified in lanoma cells (13, 14, 21) .. Determining the function of these mda genes is responsible for human melanoma and other cells. It may be important in defining the molecular basis for growth control and final differentiation in type U. The mda-7 gene (14, 21) is also transient in a wide range of human cancer cell lines. Is shown today to be a universal growth-suppressing gene when expressed stably. This finding is a characteristic of suppressing the growth of MDA-7 protein in human melanoma cells. It extends the previous observations of sex (21). For its effect on cancer cells In contrast, full-fledged human breast epithelium, normal human skin fibroblasts and normal rat embryos. Transfection of mda-7 into fibroblasts quantitatively lowers growth inhibition Give rise. Similar to another mda gene, mda-6 (p21), the expression of mda-7 is also measured. Inversely related to the progression of lanoma, at both mda-6 (p21) and mda-7 levels The increase is present in normal human melanocytes compared to metastatic human melanoma cells (14-16, 21). Decreased rate for melanoma cells, but normal Both mda-6 (p21) and mda-7 because lanin cells still retain proliferative capacity Negative regulation of melanoma progression in melanocytes / melanoma lineal cells It can function as a lator (14-16, 21). Besides, the final differentiation In human melanoma cells that have irreversibly stopped growing, mda-6 (p21) Increased expression of both and mda-7 allows these genes to express their final differentiation It can be suggested that it is an important regulator of the type (13-16, 21). The mechanism by which mda-7 elicits its growth-suppressing effect on human cancer cells is currently known. Not known. The structure of mda-7 suggests a potential mode of action It doesn't provide insight into potential features because it doesn't exist. Cell proliferation The effect of mda-7 on is the extensively studied tumor suppressor gene p53 Can be distinguished (33, 34). Carcinoma of the human chest containing mutant p53 Transient expression of p53 in cell line T47D causes growth inhibition, but in the wild Wild-type p53 gene in human breast carcinoma cell lineage MCF-7 containing type p53 Transfection does not cause growth inhibition (34). In contrast, mda- 7 causes similar growth inhibition in both T47D and MCF-7 cells (Table 1). Growth inhibition by mda-7 is due to retinoblastoma gene (pRB), pRb Related p107 gene and putative tumor suppressor gene p16^ink4Observed about It can also be considered separately from things (25, 35). Excessive occurrence of pRb and p107 Currently, it suppresses cell growth in a cell cycle-dependent manner in a particular cell type. (35-37). Cell lineage of human glial blastoma containing an apparently normal RB gene T Transfection of pRb or p107 against 98G (25) Therefore, growth inhibition is not caused (35, 37), but transient expression of mda-7 (S) Reduces T98G colonization (Table 1). At present, suppression of proliferation of mda-7 The effect is RB family member p130 / pRb2 (this is on T98G cells) However, it is indistinguishable from growth inhibition induced by (inhibiting proliferation). twenty five). p16^ink4The gene arrests growth in cells containing the functional RB gene Causes (35, 37), but inhibition of mda-7 growth is normal, abnormal, or non-functional It occurs in cells containing the RB gene. Human progenitor containing mutated RB gene Expresses adenocarcinoma cell lineage DU-145 (38) and RB (or wild-type p53) Not by transfecting human osteosarcoma cells Saos-2 with mda-7 , Causes suppression of colonization (Table 1). Similarly, stable DEX-induction Induction of mda-7 expression in DU-145 clones transformed with sex mda-7 Causes growth inhibition. Based on these discoveries, regarding the suppression of proliferation by mda-7 , Shows no dependence on the functional RB gene. Comprehensive of these studies Considered, the suppressive effect of mda-7 is the two most widely studied tumor suppressor genes. P53 and pRB, as well as the putative tumor suppressor gene p16^ink4What is the mode of action of It is explained that it is caused by a different mechanism. Shows increased expression as a function of growth arrest or DNA damage in mammalian cells Several genes have been identified (39,40). Stop growth and damage DNA Three (gadd) genes to induce (gadd45, gadd153 and gadd34), closely related Early response to bone marrow differentiation (MyD118) gene (41), as well as repressive inheritance of wild-type p53 Child mdm-2 (42) treated with the DNA damage agent methylmethane sulfonate (MMS) Is upregulated intracellularly by (40). gadd45 and growth arrest specific The gene (gas1) (43,44) is serum starvation by keeping cells dense. Induced by cells or by proliferating cells with low serum (40,43,44) .. In contrast, mRNA expression of mda-7 is associated with methyl methane sulfonate (MMS). Smell in human melanoma cells after treatment with or after maintaining the cells in a congested state Is not induced (21). Moreover, after 96 hours of growth in serum-free medium, HO-1 hi To Only a slight increase in mRNA expression of mda-7 occurs in melanoma cells I (21). Differences in the regulation of mda-7 compared to gadd, MyD118 and gas-1 genes Shows that mda-7 may be representative of a novel class of growth arrest genes .. In summary, it contains both normal and mutant p53 and RB genes Negative growth regulator, or md, that induces growth suppression in human cancer cells a-7 is explained. The genostructural characterization of mda-7 is that this gene suppresses tumors For normal functioning as a control gene and for comparison between normal cells and tumor cells Leave will be important in determining whether a mutation in this gene is present .. Identification of the promoter region of mda-7 further indicates that this gene is associated with a particular cell type. Mechanics that are specifically expressed and induced by IFN-β plus MEZ It will enable the analysis of rhythm. mda-7 has more cancer cells and traits than normal cells The discovery that it is growth-inhibitory for converted cells is potentially important and research It guarantees the development of. In this regard, for the treatment of specific human malignancies In a manner similar to that of the wild-type p53 gene currently being tested. , Mda-7 as part of a gene-based therapeutic strategy for cancer treatment It will prove to be effective.<img file="JP2000516618A_D0002.tif" /><img file="JP2000516618A_D0003.tif" /><img file="JP2000516618A_D0004.tif" />Second series of experiments Melanoma differentiation-related gene-7 (mda-7) in recombinant adenovirus , Nude mouse strain suppresses the growth of human tumors. Hi of various origins, as shown by reduced colonization in monolayer cultures Previous studies have shown that ectopic expression of mda-7 in tumor cells suppresses proliferation. More documented (Jiang et al., PNAS, 93: 9190-9165, 1996). Contrast In addition, mda-7 does not significantly alter the proliferation of normal human epithelial cells or fibroblasts .. These observations support the hypothesis that mda-7 is a universal cancer growth inhibitory gene. I have it. The ability of mda-7 to selectively suppress the growth of cancer cells is due to this gene being a human cancer It suggests that it will bring therapeutic benefits in treatment. Learn more about this possibility To investigate, we created an adenovirus that expresses replication-defective mda-7. That Rotocol is an adenovirus that expresses antisense mda-7 (Ad.mda-7 A) Similar to the one used to build S) (Jiang et al., PNAS, 93 91 60-9165, 1996). Recombinant replication defect Ad.mda-7 S was prepared in two steps. First, the mda-7 gene is sense-oriented to the improved Ad expression vector pAd.CMV. I cloned it. The virus, in turn, originates from the left end of the Ad genome 355b p, Cytomegalovirus (CMV) pre-early promoter, β-globin gene DNA encoding the next poly A signal sequence, and extending from within the E1B coding region Contains an adenovirus sequence of about 3 kbp. This sequence is the pre-CMV early gene High levels of expression of cloning sequences by promoters, and suitable RNA Enables processing. This recombinant virus contains mda-7-containing vector and J Created in vivo in 293 cells by homologous recombination with M17, which Contains the entire Ad genome cloned into the modified pBR322. JM 17 gives rise to the Ad genome in vivo, but is too large to pack .. From this constraint, to create an integrateable genome containing selected genes Released by recombination with the vector. Recombinant virus is human except 293 A replication defect in cells that expresses adenoviruses E1A and E1B To do. After transfection of the two plasmids, all by standard methods Dyeing Collect the sex virus, analyze the genome to confirm the recombinant structure, and then the virus Was purified into plaque. Various human cancer cell lines, as observed with transfection with mda-7 Infection with Ad.mda-7 S in the line suppresses proliferation, but suppresses it in normal cell lines Absent. These results show that this virus is observed in the mda-7 plasmid construct. Prove that you have sex. Many cancer cells, such as breast carcinoma (MC) F-7 and T47D), glioblastoma (GBM-18 and T98G) Infection with Ad.mda-7 S, such as in melanoma (HO-1 and C8161) Causes the induction of programmed cell death (apoptosis). This effect is In normal cells, even after numerous infections with Ad.mda-7 S (100 pfu / cell) It was not pulled out. In other cancer cell types (coloni in monolayer culture) Proliferative inhibition (as indicated by inhibition of formation) is a morphological change in the nucleus, nucleoso As indicated by the formation of a ladder or a positive TUNEL reaction, It is clear even if there are no signs of apoptosis. These results are Ad.mda-7 Show that S virus can selectively suppress the growth of human cancer cells in vitro There is. Moreover, in certain cancer cell types, growth inhibition induces apoptosis. It is related to guidance. These observations show that mda-7-induced suppression of cancer growth It is suggested that it can occur by multiple routes. Ad.mda-7 S in vivo using a nude mouse human tumor xenograft model Was determined to be able to suppress the growth of human cancer cells. Obtained from Taconic Labs 100 in PBS mixed with matrigel in thymus nude mice Thousands of human cervical cancer (HeLa) cells were injected subcutaneously (final volume 0.4 mL; Trigel to PBS ratio 1: 1). Tumor average volume 100-200 mm³(10 after inoculation It was grown until it reached (or 21st day). Then randomly select 2 groups of mice Divided into: Group 1: Replication defect Ad deficient in the mda-7 gene; null Virus (null); and Group 2: Ad.mda-7 S. Processing is for 4 weeks Intratumoral injection of null or Ad.mda-7 S, 3 times a week (100 μL / injection at 4 sites) ) Consists of. Tumors were measured with calipers 2-3 times a week. Tumor volume is a general formula Calculated using: pi / 6 x large diameter x (small diameter)².. 4 weeks after treatment, animals Also It was observed for another week and sacrificed. The final tumor volume divided by the first tumor volume is the cancer progression Corresponds to the tumor volume ratio specified as an index of. Strained HeLa xenotransplantation treated with Ad.mda-7 S suppresses proliferation throughout the study Tumors that were controlled but treated with null virus continued to progress and grow (Figure 5 and 6). The inhibitory effect of mda-7 was significant when the p value &lt;0.05. This study is repeated No, the same result was obtained. From this data, ectopic expression of mda-7 cures human cancer It is suggested that treatment can bring therapeutic benefits. The experiment was a strained human chest carcinoma It is ongoing today in nude mice with ulcers, MCF-7 and T47D.