SENSITIZATION OF HER-2/NEU OVEREXPRESSING CANCER CELLS TO CHEMOTHERAPY

Abstract: The present invention relates to methods for the inhibition, of the gene product of the neu oncogene, p185neu tyrosine kinase. Overexpression of the neu oncogene leads to chemoresistance. The methods disclosed involve the novel use of E1A and/or LT in combination with chemotherapeutic drugs to treat carcinoma. Furthermore, E1A surprisingly potentiates the antineoplastic effects of the chemotherapeutic agents. The inventors propose that E1A sensitizes cancer cells such that they become amenable to treatment by chemotherapeutic drugs.

Description: DESCRIPTION SF.NSITIZATION OF HER-2/NEU OVEREXPRESSING CANCER CELLS TO CHEMOTHERAPY BACKGROUND OF THE INVENTION A. FIELD OF THE INVENTION The present invention relates to methodology and associated genetic constructs for the suppression of oncogene-mediated, transformation, tumorigenesis and metastasis. In particular, this invention relates to the suppression of oncogenesis that is mediated by the HER-2/c-erb B-2/neu oncogene, an oncogene which has been correlated with a poor prognosis of breast and ovarian carcinoma in humans. B. BACKGROUND OF THE RELATED ART During the last decade, a number of human malignancies have been discovered to be correlated with the presence and expression of "oncogenes" in the human genome. More than twenty different oncogenes have now been implicated in tumorigenesis, and are thought to play a direct role in human cancer (Weinberg, 1985). Many of these oncogenes apparently evolve through mutagenesis of a normal cellular counterpart, termed a "proto-oncogene", which leads to either an altered expression or activity ofthe expression product. There is considerable data linking proto-oncogenes to cell growth, including their expression in response to certain proliferation signals (see, e.g., Campisi et al., 1983) and expression during embryonic development (Muller et ai, 1982). Moreover, a number ofthe proto-oncogenes are related to either a growth factor or a growth factor receptor. The c-erbB gene encodes the epidermal growth factor receptor (EGFr) and is highly homologous to the transforming gene ofthe avian erythroblastosis virus (Downward et a!.. 1984). The c-erbB gene is a member ofthe tyrosine-specific protein kinase family to which many proto-oncogenes belong. The c-erbB gene has recently been found to be similar, but distinct from, an oncogene referred to variously as c-erbB-2, HER-2 or neu oncogene (referred to herein simply as the neu oncogene), now known to be intimately involved in the pathogenesis of cancers ofthe human female breast and genital tract. The neu oncogene, which encodes a pi 85 tumor antigen, was first identified in transfection studies in which NIH 3T3 cells were transfected with DNA from chemically induced rat wewroglioblastomas (Shih et al., 1981). The pi 85 protein has an extracellular, transmembrane, and intracellular domain, and therefore has a structure consistent with that of a growth factor receptor (Schechter et al., 1984) The human neu gene was first isolated due to its homology with v-erbB and EGF-r probes (Senba et al. , 1985). Molecular cloning ofthe transforming neu oncogene and its normal cellular counterpart, the neu proto-oncogene, indicated that activation ofthe neu oncogene was due to a single point mutation resulting from one amino acid change in the transmembrane domain ofthe neu encoded pi 85 protein (Bargmann et ai, 1986; Hung et al, 1989). The neu oncogene is of particular importance to medical science because its presence is correlated with the incidence of cancers ofthe human breast and female genital tract. Moreover, amplification/overexpression of this gene has been directly correlated with relapse and survival in human breast cancer (Slamon et al, 1 87). Therefore, it is an extremely important goal of medical science to evolve information regarding the neu oncogene, particularly information that could be applied to reversing or suppressing the oncogenic progression that seems to be elicited by the presence or activation of this gene. Unfortunately, little has been previously known about the manner in which one may proceed to suppress the oncogenic phenotype associated with the presence of oncogenes such as the neu oncogene. - J An extensive body of research exists to support the involvement of a multistep process in the conversion of normal cells to the tumorigenic phenotype (see, e.g., Land et al, 1983). Molecular models supporting this hypothesis were first provided by studies on two DNA tumor viruses, adenovirus and polyomavirus. In the case of adenovirus, it was found that transformation of primary cells required the expression of both the early region 1 A (EIA) and IB (ElB) genes (Houweling et al, 1980). It was later found that the El A gene products could cooperate with middle T antigen or with activated H-ras gene to transform primary cells (Ruley, 1985). These observations suggested that the involvement of multiple functions in the transformation process, and that various oncogenes may express similar functions on a cellular level. The adenovirus EIA gene codes for several related proteins to which a number of interesting properties have been attributed. In addition to its ability to complement a second oncogene in transformation, a closely related function allows El A to immortalize primary cells (Ruley, 1985). For example, introduction of El A gene products into primary cells has been shown to provide these cells with an unlimited proliferative capacity when cultured in the presence of serum. Another interesting action of EIA function is so-called "trans-activation", wherein EIA gene products stimulate transcription from a variety of viral and cellular promoters, including the adenovirus early and major late promoter. However, trans- activation is not universal for all promoters. In some instances, EIA causes a decrease in transcription from cellular promoters that are linked to enhancer elements (Haley et al, 1984). Recently, it has been shown that exogenously added El A gene can reduce the metastatic potential of rαs-transformed rat embryo fibroblast cells by activating the cellular NM23 gene that is associated with a lower metastatic potential (Pozzatti et al. , 1988; Wallich et fl/., 1985). The El A gene products are referred to as the 13S and 12S products, in reference to the sedimentation value of two mRNAs produced by the gene. These two mRNAs arise through differential splicing of a common precursor, and code for related proteins of 289 and 243 amino acids, respectively. The proteins differ internally by 46 amino acids that are unique to the 13S protein. A number of EIA protein species can be resolved by PAGE analysis, and presumably arise as a result of extensive post- translational modification ofthe primary translation products (Harlow et al, 1985) Another viral oncoprotein, the SV 40 large T antigen (LT) shares structural and functional homology to El A and c-myc (Figge et al, 1988) LT, EIA and c-myc have transforming domains which share amino acid sequence homology and similar secondary structure (Figge et al, 1988) All three proteins complex with the tumor suppressor, retinoblastoma gene product (Rb) (Whyte et al, 1988, DeCaprio et al, 1988, Rustgi et al, 1991), and the Rb binding domains of LT and EIA coincide with their transforming domains Based on this similarity, it has been thought that LT and El A transform cells by binding cellular Rb and abrogating its tumor suppressor function. LT, EIA and c-myc are also grouped as immortalization oncogenes as determined by the oncogene cooperation assay using rat embryo fibroblasts (Weinberg, 1985) In spite ofthe similarity between the Rb binding domains of LT and EIA, the two proteins differ substantially in other regards In fact, there is apparently only a short equivalent stretch of acidic amino acids (Figge et al, 1988) This stretch lies between amino acids 106-114 in LT and amino acids 121-1 9 in EIA. The large T antigen is encoded by the simian virus 40, a member ofthe polyoma virus family. In contrast, El A is encoded by adenovirus 5 virus, which is a member ofthe adenovirus family LT is 708 amino acids long, while El A is substantially shorter at 298 amino acids LT has been observed to bind directly to certain DNA sequences, however, EIA has not. LT binds with the tumor suppressors Rb and also with p53. EIA complexes with Rb but not with p53 EIA has been shown to induce apoptosis in cells, this has not been demonstrated for LT Further, LT is an apparent anomaly in the scheme of oncogenic classification Oncogenes are typically classified as being cytoplasmic or nuclear oncogenes However, LT, through the actions of a single protein, is able to introduce "nuclear" characteπstics such as immortalization and "cytoplasmic" characteristics such as anchorage independence in cells (Weinberg, 1985) LT antigen can be found in both the nucleus and at the plasma membrane, and mutations that inhibit the transport of LT into the nucleus appear to reduce its immortalizing ability while leaving intact its effect on anchorage independence and its ability to transform already immortalized cells Consequently, this oncogene is considered to be a member of both the nuclear and cytoplasmic oncogenic classes, since it sends its gene product to do work at two distinct cellular sites (Weinberg, 1985) In contrast, El A is known as a nuclear oncogene only Despite advances in identifying certain components which contribute to the development of malignancies, it is clear that the art still lacks effective means of suppressing carcinogenesis For example, there is as yet no particularly successful way of suppressing neu oncogene activation or the development of various cancers, such as those ofthe breast and genital tract, which are associated with this molecular event. SUMMARY OF THE INVENTION The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing methods for the suppression of wew-mediated oncogenesis Certain aspects ofthe present invention relate to the inventors' surprising discovery that, in contrast to previous characterizations ofthe EIA gene and the LT gene as being involved in promoting transformation, the El A and LT gene products can actually serve to suppress not only the expression ofthe neu oncogene, but suppress the oncogenic phenotype which accompanies neu oncogene activation Furthermore these gene products sensitize cancer cells to chemotherapeutic agents It is proposed that this exciting discovery opens the door to novel approaches to the treatment of neu oncogene-mediated cancers, as well as an improved understanding ofthe regulation of this oncogene in particular and the oncogenic phenotype in general The present invention thus arises out ofthe inventors' surprising discovery that products ofthe adenovirus EIA gene, a gene that is itself known to serve as an oncogene, can be effectively employed to suppress the transforming capability ofthe neu oncogene and sensitize chemoresistant cancer cells to chemotherapeutic regimens Accordingly, the invention can be characterized in a general sense as relating to a method of treating neu oncogene-mediated transformation of a cell, which method includes introducing an El A gene product into such a cell in a manner that is effective to suppress an oncogenic phenotype, as indicated by a reduction in transforming, tumorigenic or metastatic potential ofthe cell Further the introduction ofthe EIA gene product into the cell sensitizes it to conventional chemotherapeutic agents Hence gene therapy is used in conjunction with chemotherapy to effectively kill the cancer The invention also arises out ofthe inventors' surprising showing that introduction of LT antigen into cells leads to a significant decrease in the expression of neu encoded pi 85 and also sensitizes new-overexpressing cells to chemotherapeutic agents LT, like El A and c-myc, represses the upstream regulatory sequences of neu However, LT represses a different region ofthe neu regulatory sequences compared to EIA and c-myc, suggesting LT affects neu expression through a different pathway Thus the present invention, in a general and overall sense, concerns methods of inhibiting oncogene-mediated transformation of a cell and sensitizing the cell to chemotherapeutic agents using gene products These methods involve contacting the cell with a wew-suppressing gene product and a chemotherapeutic drug in amounts effective to inhibit the transformed phenotype The objects ofthe invention may be achieved by introduction of El A gene products or LT intracellularly in any convenient manner, including, for example, virus mediated gene transfer, DNA transfection via calcium phosphate or liposome methods, and even direct introduction of gene products by microinjection It is proposed that methods such as these will work adequately, e g , where one is seeking to study neu oncogene suppression However, where a treatment regimen is contemplated it will likely be necessary to introduce the selected EIA gene product or LT by intracellular introduction of a DNA segment which encodes the particular domain ofthe EIA protein or LT that is required for repression of neu In any event, since the El A gene products have been extensively characterized, and the gene itself has been cloned (see, e g , Berk et al, 1978), the starting materials, l e , the El A products and gene, are readily available to those of skill in the art who desire to practice the invention LT is also characterized and the gene has been cloned The entire SV40 nucleotide sequence is disclosed in the book Molecular Biology of Tumor Viruses, Part 2, 2d ed , Tooze, J , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1981), Appendix A, pgs 799-813 In addition to the genomic sequence, Molecular Biology of Tumor Viruses contains a map of S V40 landmarks including the location ofthe large T antigen within the SV40 genome [pg 813]. The references Fiers et al, 1978 and Reddy et al, 1978 also report the genetic sequences of SV40. The amino acid sequence of LT can be found in Molecular Biology of Tumor Viruses, pgs 854 and 857-861 Various mutant of native LT have been described For example, Kalderon et al. (1984) describe many LT mutations, which were the result of deletion and point mutations ofthe native LT gene The relevant amino acid sequences of each LT mutant reported in Kalderon et al are contained in Table 2 of that reference By combining the information in Kalderon et al. (1984) with the sequence information for native LT contained in Molecular Biology of Tumor Viruses, the sequence for any of these mutants can be determined. All ofthe genomic and amino acid sequences of native LT and LT mutants contained in the references cited in this paragraph are incorporated by reference in this specification Some embodiments ofthe invention involve methods of inhibiting oncogene- mediated transformation of a cell Generally, these methods comprise the step of contacting the cell with an oncogenic phenotype suppressing gene product and a chemotherapeutic drug in amounts effective to inhibit the transformed phenotype In a preferred embodiment, the oncogene-mediated transformation being inhibited will be neu oncogene-mediated transformation Also, preferably, the embodiments in which transformation is to be inhibited will comprises a tyrosine specific protein kinase encoded by neu Of course, the invention also applies to methods of inhibiting other oncogene-mediated transformation events, such as transformation by ras, src, yes, fps, fes, abl, ros,fgr, erbB,fms, os, ra etc Embodiments ofthe present invention involve chemotherapeutic agents These are compounds that exhibit some form of anti-cancer activity In some preferred embodiments, the chemotherapeutic drug is an alkylating agent, plant alkaloid, antibiotic, or antineoplastic agent In those embodiments ofthe invention where the chemotherapeutic is an alkylating agent, the alkylating agent may be, for example, mechlorethamine, cyclophosphamide, ifosfamide chlorambucil, melphalan, busulfan, thiotepa, carmustine, lomustine, and/or shreptozoin In those embodiments where the chemotherapeutic agent comprises a plant alkaloid, the plant alkaloid is, for example, vincristine, vinblastine or taxol In a preferred embodiment, the plant alkaloid is taxol In those embodiments ofthe invention where the chemotherapeutic agent is an antibiotic, the antibiotic may be, for example, dactinomycin, daunorubicin, idarubicin, bleomycin mitomycin or doxorubicin In most preferred embodiments the antibiotic is doxorubicin In other embodiments where the chemotherapeutic agent comprises an antineoplastic, the preferred antineoplastic is, for example, cisplatin, VPI 6 and TNF In certain embodiments ofthe invention, the El A or LT is administered to the cell prior to the administration ofthe chemotherapeutic agent In other aspects ofthe invention, the chemotherapeutic agent is administered to the cell prior to administration of the E 1 A or LT Alternatively the E 1 A or LT and the chemotherapeutic drug are administered simultaneously In some embodiments ofthe invention, the cell is located within an animal and effective amounts ofthe El A or LT and the chemotherapeutic drug are administered to the animal In certain embodiments ofthe invention, the chemotherapeutic drug and the EIA or LT are suitably dispersed in a pharmacologically acceptable formulation In certain preferred embodiments where the cell is an animal cell, the animal cell is a human cell In other preferred embodiments the cells is a lung, cancer cell , ovarian cancer cell, or a breast cancer cell In some embodiments ofthe present invention the cell is contacted with a single composition comprising the El A or LT in combination with a chemotherapeutic agent In such cases, the composition may be suitably dispersed in a pharmacologically acceptable formulation The invention contemplates embodiments comprising sensitizing a cancer cell to a chemotherapeutic drug These embodiments comprise exposing the cell with an effective amount ofthe El A or LT In some such embodiments inhibition of neu- mediated cancer is accomplished by administrating an effective combination ofthe EIA or LT and chemotherapeutic drug to an animal having or suspected of having cancer in an effective amount to inhibit the cancer In embodiments where the composition is administered to an animal, the animal is typically a mammal In such cases, the invention will be of particular use in the treatment and prevention of «ez/-mediated transformation in humans Certain embodiments ofthe present invention comprise injecting a therapeutically effective amount ofthe El A or LT into an animal and contacting the animal with a chemotherapeutic drug In certain embodiments ofthe invention the cancer site is contacted with a chemotherapeutic drug by administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising a chemotherapeutic drug wherein the chemotherapeutic drug is for example cisplatin, doxorubicin, VP 16, taxol or TNF The inventors have also enabled the production of pharmaceutical compositions comprising an El A or LT and a chemotherapeutic drug in a pharmacological carrier. Those of skill will understand the nature of such pharmacological carriers based on the teachings of this specification and the current knowledge in the art. The pharmaceutical compositions ofthe invention may contain any ofthe El A or LT and chemotherapeutic drugs mentioned above or elsewhere in this specification, or know to those of skill in the art. They may also contain emodin and/or an emodin like compound. In a preferred pharmaceutical composition the chemotherapeutic drug is cisplatin, doxorubicin, etoposide, taxol or TNF. In some preferred embodiments, the new-suppressing gene product is El A. In some preferred embodiments the new-suppressing gene product is LT. The invention also encompasses pharmaceutical combinations comprising an a new-suppressing gene product and a chemotherapeutic drug. In certain preferred combinations, the new-suppressing gene product is El A. In certain other preferred combinations, the new-suppressing gene product is LT. The chemotherapeutic drug may be any that is listed elsewhere in this specification or known to those of skill in the art at the present or in the future. Exemplary chemotherapeutic drugs for us in the pharmaceutical combinations ofthe present invention are cisplatin, doxorubicin, etoposide, emodin and or emodin like compounds, taxol and TNT. In certain embodiments ofthe invention the pharmaceutical combination may contain the El A or LT and the chemotherapeutic drug within the same pharmaceutical composition. In other embodiments, the pharmaceutical combinations will comprise separate pharmaceutical compositions for each ofthe El A or LT and the chemotherapeutic drug. These separate compositions may be combined internal to or external to a body to create the pharmaceutical combination. Other embodiments ofthe invention include therapeutic kits comprising in suitable container, a pharmaceutical formulation of an the EIA or LT preparation, a pharmaceutical formulation of a chemotherapeutic drug, and/or a pharmaceutical formulation comprising both the El A or LT and a chemotherapeutic drug. Emodin and/or emodin like compounds may also be present in the kit either in combination with the gene products, chemotherapeutic agent, gene products and chemotherapeutic agent or indeed in a separate formulation. The kit may also contain instructions on how to administer the pharmaceutical formulation or formulations ofthe kit to an animal either alone, or in combination with formulations that one may obtain separately from the kit. The kit may also comprise instructions that explain how to use the kit but are provided separately from the container ofthe kit. The kit may comprise the EIA or LT, emodin and/or emodin like compound, and chemotherapeutic drug to be present within a single container or alternatively the kit could comprise the El A or LT and/or emodin and the chemotherapeutic drug are present within distinct containers. Some embodiments ofthe present invention relate to a method of sensitization of a cell to an anticancer drug, comprising contacting the cell with the El A or LT. These gene products are well-described in this specification. In preferred embodiments the oncogene-mediated transformation is new oncogene-mediated transformation. Also, preferably, the embodiments in which transformation is to be inhibited will comprises a tyrosine specific protein kinase encoded by new. The invention also contemplates pharmaceutical compositions, and kits comprising the El A or LT to suppress new- mediated transformation. Of course, the invention also applies to methods of inhibiting or suppressing other oncogene-mediated transformation events, such as transformation by ras, src, yes,fps,fes, abl, ros,fgr, erbB,fms, mos, raf Emodin-like tyrosine kinase inhibitors ofthe invention are those compounds that exhibit similar characteristics to those of emodin with regard to tyrosine kinase inhibition and the inhibition of new-mediated transformation. Of course the invention is not limited to the use of these inhibitors and other inhibitors that possess the structural and/or functional properties of emodin may be used. In some preferred embodiments, the emodin-like tyrosine kinase inhibitor is an anthraquinone-like tyrosine kinase inhibitor. The emodin-like tyrosine kinase inhibitor may be, for example, emodin, emodin-8-O-D-glucoside, chrysophanic acid, gluco-chrysophanic acid, physcion, or physcion-8-O-D-glucoside. In the most preferred embodiment the new tyrosine kinase inhibitor is emodin. Other embodiments ofthe present invention relate to a method of inhibiting oncogene-mediated transformation of a cell, comprising contacting the cell with the El A or LT, further contacting the cell with emodin and/or an emodin-like compound and further still contacting the cell with the chemotherapeutic agent The cell may be contacted with the gene product, the emodin and/or emodin like compound and the chemotherapeutic agent successively in any order Alternatively the cell is contacted with a combination of gene product and emodin, gene product and chemotherapeutic drug, emodin and chemotherapeutic drug followed or preceded by treatment ofthe third agent In yet another embodiment it is possible to contact the cell with the gene product, emodin and/or emodin like compound and chemotherapeutic agent concurrently with each other In some embodiments of this invention an a cell is contacted with between about 0 5 mg/kg total weight and 500mg/kg total weight ofthe emodin-like tyrosine kinase inhibitor Other embodiments wherein the cell is contacted with between about 0 5 mg kg total weight and 500mg/kg total weight of emodin In still other embodiments the cell is contacted with between about 0 5mg/kg total weight and 500mg/kg total weight of an emodin-like tyrosine kinase inhibitor Total weight may be defined as the total weight ofthe cell or cells in culture, or the body weight of an animal, including a human The present invention also contemplates compositions comprising a liposomal complex This liposomal complex will comprise a lipid component and a DNA segment encoding a new-suppressing gene The new-suppressing gene employed in the liposomal complex can be, for example, an LT gene or an EIA gene Liposomal complexes comprising LT mutants may have certain advantages These advantages may be particularly distinct when the LT gene encodes non-transforming LT mutant, such as Kl An EIA gene encoding either the El A 12S or El A 13S gene product, or both, may be complexed with a lipid to form the liposomal complex The lipid employed to make the liposomal complex can be any ofthe above- discussed lipids In particular, DOTMA, DOPE, and/or DC-Chol may form all or part ofthe liposomal complex The inventors have had particular success with complexes comprising DC-Chol In a preferred embodiment, the lipid will comprise DC-Chol and DOPE While any ratio of DC-Chol to DOPE is anticipated to have utility, it is anticipated that those comprising a ratio of DC-Chol DOPE between 1 20 and 20 1 will be particularly advantageous The inventors have found that liposomes prepared from a ratio of DC-Chol DOPE of about 1 10 to about 1 5 have been useful in the studies they have performed In most studies, the inventors have used a ratio of 1 2 μmol DC-Chol 8 0 μmol DOPE The present invention also comprises kits for the introduction of a new- suppressing gene product into a cell comprising a new-suppressing DNA liposome complex BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the specification and are included to further demonstrate certain aspects ofthe present invention The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiment presented herein FIG 1 A and FIG IB show EIA gene product effects on the new promoter FIG 1 A transcriptional repression of new promoter by El A gene products Rat-1 cells were transfected with 5 μg ofthe pNew-EcoRl-CAT construct, which contains the CAT gene driven by new oncogene promoter containing 2 2-kb upstream DNA sequences Lane 1 , basal new promoter activity (its relative CAT activity is defined as 100%), lanes 2-4, CAT activity after cotransfection with 10 μg of carrier DNA pSP64 vector (102%, lane 2), El A-expressing plasmid pElA (34%, lane 3), pEl Apr, a plasmid containing only the El A promoter (98%, lane 4) The CAT activities of a reporter plasmid, RSV-CAT, containing the CAT gene under the control of RSV LTR (10%, lane 5) were not significantly changed by cotransfection of 10 μg of pE 1 A (98%, lane 6) or 20 μg of pE 1 A (96%, lane 7) FIG IB shows the effect of various adenovirus early genes on new promoter activity The pnez/EcoRI-CAT was cotransfected with pSP64 vector or plasmid expressing various adenovirus early genes, EIA, ElB, E2A, and E3, as indicated The relative CAT activities are as follows SP64, 100%, E 1 A, 35%, E 1 B, 97%, E2A, 99%, E3, 102% RSV-CAT was used as a positive control FIG. 2A, FIG 2B FIG. 2C FIG 2D and FIG 2E show transient expression from neu promoter with cotransfection with increasing amounts of pElA (FIG 2 A), pElA-13S (FIG 2B), pElA-12S (FIG 2C), and pEl Ad 1346 (FIG 2D) A constant amount (5 μg) ofthe pnewEcoRl-CAT construct was cotransfected into Rat-1 cells with 5, 10, 15, and 20 μg ofthe test constructs The total amount ofthe transfected DNA were kept constant by adding the appropriate amount of carrier DNA pSP64 The relative CAT activities without EIA (lanes 0 in FIG 2 A, FIG 2B, 2C and FIG 2D) are defined as 100% The relative CAT activities with 5, 10, 15 and 20 μg of test constructs are as follows EIA, 68%, 35%, 26%, 17%, El A- 13 S, 72%, 48%, 36%, 24%, E1A-12S, 66%, 46%, 28%, 21%, ElAdl346, 102%, 103%, 99%, 102%, (FIG 2E) Summary ofthe effects of different EIA mutants on transient expression from the neu promoter Schematic structures ofthe proteins encoded by different EIA mutants are shown on the bar diagram Hatched areas represent the conserved protein regions ofthe EIA products Bar diagrams are not drawn to scale FIG 3 A and FIG 3B show localization of El A-responsive DNA element in the upstream region of new promoter FIG 3 A depicts schematic maps ofthe new promoter 5' deletion constructs that were fused individually to the CAT gene to create the plasmids as indicated by the names ofthe restriction enzymes used for generating the constructs FIG 3B Illustrates the level of expression ofthe CAT gene directed by each of the promoter fragment constructs after transfection of 5 μg ofthe plasmids into Rat-1 cells with 10 μg of cotransfected pEl A (E) or carrier DNA pSP64 (C) The names of restriction enzymes above each triplet assay refer to the constructs indicated in the maps FIG 4 A and FIG 4B show derepression of new by cotransfection of competing amounts of Stu -Xho I neu promoter fragments FIG 4 A Rat-1 cells were transfected with 5 μg of the pNewEcoRl-CAT plasmids giving basal new promoter activity (lane 1), the repressed CAT activity after cotransfection with 5 μg ofthe pElA is shown in lane 2 Plasmids pSP64/Stu-Xho containing the Stw I-Xho I neu promoter fragment cloned in pSP64 were cotransfected with pnewEcoRl-CAT and pEl A Lanes 3-6 show the competitive effects of increasing amounts (5, 10, 15, and 20 μg, respectively) of pSP64/Stu-Xho Plasmids pSP64/Rl -Xba containing the EcoRl-Xbu I new promoter fragment were also cotransfected with pnewEcoRl-CAT and pEl A Lanes 7-9 show CAT activities from new promoter by cotransfecting 5, 10, and 20 μg of pSP64/RI-Xba, respectively The relative CAT activities of lanes 1-9 are as follows 100%, 32%, 27%, 31%, 58%, 79%, 38%, 31%, 24% FIG 4B shows immunoblot for pi 85 protein in the cell lysates of SK-BR-3 breast cancer cells transfected by pnewEcoRV-CAT Seventy-five micrograms of protein from each sample was electrophoresed on 7% SDS/PAGE gels prior to transfer on nitrocellulose Filters were blotted with the primary antibody mAb-3 Lane 1, lysates of SK-BR-3 cells transfected with 5 μg of pEl A, lane 2, cotransfected with 5 μg EIA and 20 μg of pSP64/RI-XbaI, lane 3, cotransfected with 5 μg of El A and 20 μg of pSP64/Stu-Xho, lane 4, lysates of SK-BR-3 cells after mock transfection The protein size marker is shown on the right The arrow indicates the position of pi 85 protein The pi 85 protein bands were scanned by Bio-Rad video densitometer model 620 to determined the relative pi 85 protein level The pi 85 protein level in the mock transfection sample is defined as 100% and the relative amounts of pi 85 proteins in lanes 1-3 are 57%, 54%, and 89%, respectively FIG 5 shows removal ofthe ElA-mediated repression of new by cotransfection of a 20-mer oligonucleotide containing the consensus sequence Rat-1 cells were transfected with 3 μg of pnewEcoRV-CAT plasmids, giving basal new promoter activity (lane 1), CAT activity after cotransfection with 10 μg of pElA is shown in lane 4 Two micrograms ofthe 20-mer double-stranded oligonucleotide containing the consensus sequence (lane 2, Cons) was cotransfected with pnewEcoRV-CAT and pElA (molar ratio of oligomer pNewEcoRV-CAT = 35 1), resulting in significant derepression, cotransfection of 2 μg of a 22-mer random nonhomologous oligonucleotide with pnewEcoRV-CAT and pElA had no significant derepression effect (lane 3, None) The values for relative CAT activity are the average of three studies The upper strand sequence ofthe synthetic 20-mer oligonucleotide is shown at the bottom, the proposed El A-responding sequence is underlined FIG 6 A, FIG 6B, FIG 6C and FIG 6D show EIA gene presence and protein production in cells FIG 6A shows southern blot analysis of NIH3T3, B104-1-1 and their transfectants using an EcoRI-SstI El A DNA probe 10 μg of genomic DNA from the indicated cell lines were digested to completion with EcoRI + SstI restriction endonucleases and subjected to electrophoresis on a 1% agarose gel The DNAs were transferred to Nitran™ filter paper and hybridized with the El A probe The DNA markers are shown on the left FIG 6B shows immunoblot analysis for El A proteins in the cell lysates ofthe indicated cell lines 50 μg of each sample were electrophoresed on 10% SDS-PAGE prior to transfer to nitrocellulose Filters were incubated with the primary antibody M73 against EIA The protein molecular weight marker and the position of El A proteins are shown on the right 25 μg of Cell lysate from 293 cells was used as a positive control FIG 6C Immunoblot analysis for the new encoded pi 85 protein in the cell lysates ofthe indicated cell lines The studies were performed as described in section (FIG 6B) above The primary antibody was mAB-3 against pi 85, purchased from Oncogene Science Ine FIG 6D Southern blot analysis ofthe indicated cell lines using rat neu DNA probe The studies were performed as described in FIG 6A above The DNAs were digested with Bam HI restriction endonuclease FIG 7A, FIG 7B, FIG 7C, FIG 7D, FIG 7E, FIG 7F show morphologic effects of E 1 A expression in new-transformed B 104- 1 - 1 cells (FIG 7 A) B 104- 1 - 1 , (FIG 7B) B-ElApr, (FIG 7C) N-E1A-1 , (FIG 7D) B-E1A-1 , (FIG 7E) B-ElA-2, (FIG 7F) B-ElA-3 (Magnification X130) FIG 8 A and FIG 8B show EIA effects on DNA synthesis FIG 8 A [Η] Thymidine incorporation ofthe indicated cell lines 9 X IO 3 cells were plated in 96 well multiwell plates and cultured in Dulbecco's modified Eagle medium supplemented with 10% calf serum for 16, 40 and 64 hrs Cell received a 2 hr pulse of 1 μCi [ 3 H]-thymidine per well to label those that were synthesizing DNA prior to harvest Radioactivities of individual samples were counted by scintillation counter Average cpm counts were calculated from replicated samples FIG 8B Anchorage independent growth of ElA-transfected B 104-1-1 and NIH3T3 cells lxlO J cells were plated in 0 35% soft agar over a 0 7% agar lower layer Colonies were counted after 4 weeks A typical plate and the mean of triplicate samples plus or minus the standard error ofthe mean are shown for each group FIG 9 A and FIG 9B show the effects of a tumorigenicity study FIG 9A Summary of tumorigenicity of B 104-1-1, NIH3T3 and their transfectant 1 x 10 5 viable cells were injected subcutaneously into right and left flanks of female homozygous nu/nu mice, respectively Tumor formation was scored at indicated days as presence or absence of a visible tumor mass 16 days after injection, tumor volumes were estimated as the product of tri-dimensional caliper measurements (longest surface length and width, and tumor thickness) N D not detectable at the time of evaluation FIG 9B A representative result of tumorigenicity study From right to left the animals were injected with B 104- 1-1, B-E1A-2 or NIH3T3 cells 18 days prior to the photographing data FIG 10A, FIG lOB and FIG 10C, FIG 10D, FIG 10E, FIG 10F show EIA inhibition of new-transformed cells FIG 10A EIA gene products inhibited the cell motility ofthe new-transformed 3T3 cells N-E1A NIH3T3 cells transfected with EIA, B-neo. B104-1-1 cells transfected with neomycin resistant gene, B-E1A-1 to 5 five independent cell lines generated by transfecting EIA gene into B 104- 1 - 1 cells The motility assays were carried out by using a transwell unit with 5 μm pore size polycarbonate filter in 24 well cluster plate (Costar) Lower compartment ofthe transwell contained 600 μl of one of the chemoattractants 20 μm fibronectin (FN) or 100 μm FN dissolved in DMEM/F12, or hepatic endothelial cell conditioned media (HSE), or DMEM/F12 medial only as negative control The cells (3 x 10 4 /0 1 ml in DMEM F12) were plated in the upper compartment and incubated for 6 hrs at 37°C in a humidified 5% CO 2 atmosphere After the incubation, the filters were fixed with 3% glutaraldehyde in PBS buffer and stained with Geimsa Each sample was assayed in triplicate and cell motility was measured by counting the number of cells that had migrated to the lower side ofthe filter At least four HPFs were counted per filter The number of cells migrated to DMEM/F12 has been deducted from each sample to eliminate the background and all the assays were done in triplicates. FIG 10B shows EIA gene products inhibited the invasiveness ofthe new- transformed 3T3 cells. The assay of in vitro invasiveness was done basically as described by Albini et al, 1987 and Repesh, 1989 The basement membrane preparation, matrigel, was purchased from Collaborative Research, Ine Filters in the transwell unit (same as used in motility assay) were coated with 0.1 ml of 1.20 dilution of matrigel in DMDM/F12 media Lower compartment contained 0 6 ml of HSE as chemoattractant or DMEM/F12 as negative control The cells (5 x 10 4 /0.1 ml in DMEM/F12) were plated in upper compartment and incubated for 72 hrs. at 37°C in a humidified 5% CO 2 atmosphere Cells were fixed, stained and counted as described in 1.a All the assays were done in triplicate and assays were repeated twice Gross appearance of lungs from the mice injected with B-neo cells (10C), N- E1A cells (10D), B-El A- 1 cells (FIG 10E), and B-El A-2 cells (FIG 10F), EIA gene products inhibited the lung colonization of new-transformed cells FIG 1 IA and FIG. 1 IB show that EIA suppresses new-induced tumor formation and metastasis in vivo in nude mice FIG 1 1 A Top, animal injected with B 104-1-1 cells, a new oncogene transformed NIH3T3 cell line; Bottom, animal injected with B-E1A2 cells, an EIA transfectant of B 104- 1-1 Photographs were taken 18 days after injection, and results are representative of other tumorigenicity studies FIG 1 IB Left, gross appearance of lungs from mice injected with B 104- 1-1 cells, Right, gross appearance of lungs from mice injected with the EIA transfected cells, B-E1A2 Mice were inoculated with 1 x 10 5 cells/0 1ml in PBS via the lateral tail vein at day 0, and were sacrificed 21 days after injection The numbers of lung tumor nodules were determined following infiltration with India ink, only those lung nodules greater than 1 mm in diameter were counted in the assay FIG 12 A- FIG 12B FIG 12C show the molecular characterization ofthe ipl, EIA and ipl .Efs transfectants described in Example V FIG 12A shows immunoblot analysis of El A proteins in the cell lysates of the indicated cell lines Seventy-five mg of proteins from each sample were subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel prior to transfer to nitrocellulose Filters were incubated with the primary antibody M73, which recognizes EIA proteins The position ofthe EIA proteins are indicated to the left of A FIG 12B shows immunoblot analysis ofthe c-erόB-2/new-encoded pl85 proteins in the cell lysates ofthe indicated cell lines. Seventy-five mg of proteins from each sample were subjected to electrophoresis on 10% sodium dodecyl sulfate- polyacrylamide gel prior to transfer to nitrocellulose Filters were incubated with the primary antibody c-/?ew-Ab-3 against pi 85 The position ofthe pi 85 proteins are indicated to the left of B FIG 12C shows Southern blot analysis of DNAs from the ipl EIA and ipl Efs transfectants Ten mg of genomic DNA from indicated cell lines were hybridized with the full-length c-erbB-2/neu cDNA probe DNA markers are shown to the right. 13A, FIG 13B and FIG 13C show the reduced growth rate ofthe ipl EIA transfectants versus control ipl Efs cells, the decreased [Η]thymιdine Incorporation by the ipl El A transfectants versus control ipl Efs cells, and significantly inhibited colony formation for the ipl El A transfectants versus control ipl Efs cells, respectively FIG 13 A shows reduced growth rate ofthe ipl EIA transfectants versus control ipl Efs cells The in vitro growth rates ofthe cell lines were assessed by measuring increases in cell number with the MTT assay (Alley et al, 1988) Cells (2 x 10 3 /well) were plated in 96-well culture plates in 0 2 ml of culture medium A total of 5 plates (9 wells/cell line/plate) were used One ofthe plates was analyzed at 24-h intervals after the addition of 40 μl MTT (Sigma Chemical Co , St Louis, MO) stock solution (1 25 mg MTT/ml of phosphate-buffered saline) to each well on the plate Cells were incubated at 37°C for 2 5 h, the medium was aspirated, and the cells were lysed in 100 μl of dimethyl sulfoxide Conversion of MTT to formazan by metabolically viable cells was monitored by a Dynatech MR 5000 fluorescence microplate reader at a wavelength of 450 nm Results were analyzed by regression analysis Each study was repeated for each cell line at least twice FIG 13B shows decreased [ 3 H]thymidine incorporation by the ipl EIA transfectants versus control ipl Efs cells For this assay, 10 replicated cell samples were plated into 96-well plates at a density of 8 X 10 3 cells/well in culture medium [ 3 H]Thymidine (1 μCi) was added to each well at 24, 48, and 72 h, respectively, with continuous incubation after each addition for 12 h at 37°C Cells were harvested, and cellular DNA was bound to fiberglass filters The radioactivity of each filter was counted with a scintillation counter Average cpm were calculated from ten replicate samples FIG 13C shows significantly inhibited colony formation for the ipl EIA transfectants versus control ipl Efs cells (P < 0 01) Soft agar assays were performed as previously described (Matin et al, 1990). Cells (1 X 10 3 cells/well) were plated in a 24-well plate in culture medium containing 0.35% agarose (BRL, Gaithersburg, MD) overlying a 0.7% agarose layer. The cells were then incubated at 37°C for 5 weeks, after which the plates were stained with /?-iodonitrotetrazolium violet ( 1 mg/ml) for 48 h at 37°C. Colonies greater than 100 μm were counted for each dish and cell line. The numbers of soft agar colony are shown in the FIG.. Studies were repeated four times for each cell line. FIG 14A and FIG. 14B show the EIA suppressed tumor formation by c-er?B2-/new-overexpressing ovarian cancer cells and the longer survival of mice given injections of ElA-expressing ipl . El A cells versus mice given injections of ipl . Efs human ovarian cancer cells. FIG. 14A shows EIA suppressed tumor formation by c-erbB2-lneu- overexpressing ovarian cancer cells. Four- to 6-week-old athymic female homozygous nu/nu mice were purchased from the Animal Production Area, National Canter Institute-Frederick Cancer Research Facility (Frederick, MD) or from Harlan Sprague Dawley, Inc., (Indianapolis, IN). The care and use ofthe animals was in accordance with institutional guidelines. For tumorigenicity assays, cells in log-phase growth were trypsinized, washed twice with phosphate-buffered saline, and centrifuged at 250 X g. The viable cells were counted; of those, 3 X 10 6 cells in 0.1 ml of phosphate-buffered saline were injected s.c. into both the right and left flanks of female mice under aseptic conditions. Tumor volumes were estimated as the product of three-dimensional caliper measurements (longest surface length and width; tumor thickness). The growth of tumors was monitored for a minimum of 80 days and a maximum of 160 days, as shown by the days indicated in the FIG.. FIG. 14B shows longer survival of mice given injections of ElA-expressing ipl El A cells versus mice given injections of ipl. Efs human ovarian cancer cells (P < 0.01) To assess the formation of malignant ascites after i.p. injection, suspensions of cells (harvested as above) at concentrations of 1 X 10 6 in 0 2 ml of Hank's balanced salt solution were injected i p into individual female nu/nu mice In two studies, totals of nine mice for the ipl Efs line, eight mice for the ipl El Al line, and nine mice fore the ipl E1A2 line were given injections. Mice were initially observed twice a twice a week for signs of tumor development and then daily when any or all ofthe following tumor symptoms appeared abdominal bloating, loss of subcutaneous fat, hunched posture, and decreased movement Mice were killed when they appeared moribund or, judging from the inventors previous experience, would not survive more than 24-48 h Symptom-free mice were killed 120 days after injection Autopsies were performed on all mice killed Similar results were obtained from the two studies, and results were combined for analysis FIG 15A- FIG 15B and FIG 15C show expression of new-encoded pi 85 and LT in B 104-1-1 cells stably transfected with plasmids encoding LT FIG 15A Immunoblotting for anti-pl85 of whole cell lysates from B 104-1-1 cells stably transfected with LT BTnlό (lane 1), BTnl4 (lane 2), BEn5 (lane 3) and NIH 3T3 (lane 4) cell lines Following transfer to nitrocellulose, the blots were probed with monoclonal anti-pl85 antibody (c-new, Ab-3, Oncogene Science) followed by goat anti-mouse conjugated to horse radish peroxidase The blot was subsequently developed using horse radish peroxidase substrate and hydrogen peroxide FIG 15B Immunoblot for LT of whole cell lysates ofthe stable transfectants Blots were probed with anti-LT (SV 40 T-Ag, Ab-2, Oncogene Science) and then with [125I]-protein A. Washed and dπed blots were exposed for autoradiography Lysates of BTnlό, lane 1, BTnl4, lane 2, BEn5, lane 3 and NIH 3T3 cell line, lane 4 FIG 15C Southern blotting for genomic new using 32P-labeled 0 4 and 0 8 kb Bam HI fragments (1 1) from neu cDNA probe to hybridize with Bam HI digested genomic DNA isolated from BTnl ό, lane 1, BTnl4, lane 2, BEn5, lane 3 and NIH 3T3 cells, lane 4 The rat new-specific bands are indicated by a triangle FIG 16 shows the effect of LT on the upstream regulatory sequences of new and epidermal growth factor receptor One mg of pNewEcoRlCAT (lanes 1 and 2) or pEGFrCAT (lanes 3 and 4) were cotransfected into ΝIH 3T3 cells with 10 mg of plasmid encoding LT, pVU-0 (lanes 2 and 4) or with control plasmid, pSV2E (lanes 1 and 3) which does not contain LT coding region Transfections and CAT assays were carried out as described previously (Yu et al. 1992) CAT assays were standardized to equal protein concentrations ofthe cell extracts The study was repeated 4 times and experimental error was within 13% One representative set of data is shown FIG 17 shows the effect of increasing concentrations of LT on the activity of the regulatory sequences of new Two and 10 mg of pVU-0 were cotransfected with 1 mg of pnewEcoRlCAT into ΝIH 3T3 cells The total amount of DΝA transfected was equal for all reactions, with the control plasmid, pSV2E, being used to make up a final DΝA concentration of 11 mg Lane 4, M, is control CAT assay of extracts from untransfected ΝIH 3T3 cells Representative data of 3 studies is shown, standard deviation was 11% FIG 18A and FIG 18B show data from serial deletions FIG 18A Series deletion-C AT constructs of the rat neu promoter FIG 18B Mapping of LT responding region in the new upstream regulatory sequence using the new deletion-C AT constructs One mg of each ofthe new deletion-CAT constructs were cotransfected into ΝIH 3T3 cells with 10 mg ofthe LT-producing plasmid, pVU-0 (indicated by +) or 10 mg of filler plasmid, pSV2E (indicated by -) Set 1 , pnewEcoRlCAT, set 2, pnewXbalCAT, set 3, ρnewEcoRV2CAT, set 4, pnewEcoRVCAT, set 5, pnewStulCAT, set 6, pnewXholCAT, M, control CAT assay of extracts from untransfected ΝIH 3T3 cells Each set (set 1, set 2, etc ) of CAT reactions with (+) and without LT (-) were standardized to equal protein concentrations FIG 19 shows gel shift assay demonstrating DNA-protein complex formed with the Xhol-Narl region ofthe new promoter The 32 P-labeled DNA is the 94 base pair Xhol-Narl fragment Lanes 1 and 2, nuclear extract from NIH 3T3 cells, lanes 3 and 4, nuclear extract from BTn 14 cell line Lanes 2 and 4 contain approximately 250-fold unlabelled Xhol-Narl fragment as specific competitor Lane 5, j2 P-labeled Xhol-Narl fragment only Incubation of probe (10 5 cpm) with nuclear extracts (3μg) were carried out as described (Dynlacht et al, 19 1) and samples were electrophoresed through a native 4 5% polyacrylamide gel (80 1 ; acrylamide bisacrylamide) containing 0 5 x TBE (45 mM boric acid, 1 mM EDTA, pH 8) for 2 5 h at 40°C F indicates free probe FIG 20A FIG 20B, and FIG 20C show the effect of mutant LT on new promoter activity FIG. 20 A Schematic diagram of LT showing Rb binding domain (shaded black) Kl encodes LT with single amino acid change (glu 107 to lys) in the Rb binding domain ofthe 708 amino acid LT protein FIG 20B Activity of pnewXhol CAT (with control plasmid pSV2E) and inhibition of activity in the presence of wild type LT (WT), and mutant LT (Kl) One mg pnewXhol CAT was cotransfected with 10 mg of filler plasmid, pSV2E, or wild type LT (pVU-0) or mutant LT (pKl) FIG 20C Effect of Kl on the transforming activity of activated new One mg of cNew-104 was cotransfected with 2 mg of Kl and 0 1 mg of pSV2neo into Rat-1 cells pSV2E was used as filler plasmid so that a final 5 mg DΝA was transfected into cells Cells were split 1 4 48 hours after transfection and duplicate plates were subsequently grown in regular medium (DMEM F12 plus 10% calf serum) or regular medium supplemented with 250 mg/mL G418 Foci and G418-resistant colonies were stained and counted after 3-4 weeks Results are expressed as ratio of foci to that of G418-resistant colonies from each transfection to correct for transfection efficiency The number of foci from transfecting cNew-104 alone was set at 100% FIG 21 shows liposome-deviated direct gene transfer techniques allow the delivery ofthe EIA gene to new-overexpressing SK-OV-3 human ovarian cancer cell The three mice were each injected with SK-OV-3 cells Five days later, the mice were injected with (1) El A DNA only, (2) complex of liposome and Efs DNA (an EIA frame shift mutant that does not cause active El A to be produced), and (3) complex of liposome and EIA DNA Booster injections ofthe same compositions were given each respective mouse on a weekly basis for the remainder ofthe mouse's life Mouse 1 developed extensive bloody ascites and died 65 days after SK-OV-3 injection Mouse 2 developed extensive blood ascites and a large tumor and died 76 days after the injection of SK-OV-3 cells Mouse 3 appeared healthy and was alive 160 days after SK-OV-3 injection FIG 22 shows the infection efficiency of adenovirus in ovarian cancer SK-OV- 3 (i.p.) SK-OV-3(i.p ) in 6 well plates (2 5 X 10 5 /well) were infected once by Ad RSVβgal at different virus/tumor cell ratios Two days later, cells were fixed and stained with X-gal Infection efficiency = No of positive cells/No of total cells X 100% FIG 23 shows a growth curve of SK-OV-3(i.p.) after treatment by Ad.ElA in vitro SK-OV-3(i p ) in 12 well plates (10 4 /well) were infected once by 2X10 5 adenovirus and cell growth was followed for 7 days FIG 24 shows colony formation in soft agarose. SK-OV-3(i p ) cells were infected once with adenovirus at a virus/tumor ratio of 20/1 Aliquots of 5 X IO 4 cells were mixed with 0.35% agarose in DMEM medium and plated over a base layer of 0 7% agarose Culture medium was allowed to harden in 6 well plates (n = 3) Colonies were stained and counted about 6 weeks later FIG 25 shows the Ad El A therapeutic effect on ovarian cancer SK-OV- 3(i p ) SK-OV-3(i p ) (10 6 /mouse) were injected i p in female nu/nu mice Five days later, mice were given i p injection of 0 1 ml of viral solution (titer 2 X IO 9 PFU/ml) once/day for three days, then once/week for 4 5 months The responses and survival rate were observed for more than one-half year (n = 5) FIG 26 A and FIG 26B show in vivo Ad RSVβgal-mediated transfer ofthe lacZ gene to intraperitoneal SK-OV-3(i p ), 26A, and to intratracheal H820, 26B FIG 26 A Mice were administered intraperitoneal SK-OV-3(i p ), two months later after tumor development, Ad RSVβgal was administered intrapeπtoneally Tumor and organs were evaluated for the presence of β-gal using X-gal The lacZ gene was localized in tumor cells and only slight β-gal activity was detected in normal organs FIG 26B Mice were administered intratracheally H820 Two months later, after tumor development, AD RSVβgal was administered intravenously Tumor and organs were evaluated for the presence of β-gal using X-gal The lacZ gene was localized in tumor cells and only slight β-gal activity was detected in some normal organs FIG 27 shows the survival of mice bearing ovarian cancer 2774 after treatment by Ad EIA Human ovarian cancer cell line 2774 which has low level expression of HER-2/new was injected i p into nu/nu mice (5 X lOVmouse) Five days later, mice were given i p injection of 0 1 ml of viral solution (titer 2 X 10 9 /ml) once/day for three days, then once/week for 4 5 months The responses and survival rate were observed AD El A(+) did not have significant therapeutic effect in 2774 Analysis of the results and the data of SK-OV-3(ι p ) which has high expression level of HER- 2/new indicate that AD E1A(+) can specifically inhibit the growth of tumor which has high expression level of HER-2/new FIG 28A, FIG 28B and FIG. 28C show a histoimmunochemical analysis of representative histological sections of treated and control mice FIG 28A Histological section from intraperitoneal SK-OV-3(i p ) stained with hematoxylin and eosin FIG 28B Expression level of HER- 21 neu PI 85 protein stained by polyclonal antibody against PI 85 with ABC alkaline phosphatase substrate kit Positive red color FIG 28C Expression of AD EIA protein stained by monoclonal antibody against AD EIA with ABC ACE substrate kit for horseradish peroxides Positive dark red color Ad EIA protein was detected in tumor tissue treated by Ad El A(+) in vivo The expression level of HER-2/new PI 85 was greatly inhibited in treated mouse tumor tissue FIG 29A FIG 29B and FIG 29C show representative mediastinal blocks of treated and control mice Arrow Tumor FIG 30A Effect of taxol (0 01-100 μM) on the cell growth of rat fibroblast with various HER-2/new and E 1 A expression Cell lysates were run through 8% SDS-PAGE and blotted with anti-pl 85 and anti-ElA antibody FIG 30B Effect of taxol (0 01-100 μM) on the cell growth of rat fibroblast with various HER-2/new and EIA expression B104-1-1, B neo, B.E1 Al Hy, and B El Al Dl were examined for the effect of taxol on cell growth by MTT assay, and the percentage of cell growth was calculated by defining the absorption of cells not treated with taxol as 100% Bars, SD The highest inhibition of cell growth was seen in B.E1 Al Hy with HER-2/new down-regulated by EIA, with taxol of 0.1 to 10 μM FIG 31 A Infection efficacy of Ad.RSV/αcZ Beta-galactosidase expression was examined after exposure ofthe MDA-MB-435, and 453, 231, and 361 with various V/T ratio Bars, SD. The infection efficiency was higher among HER-2/new-overexpressing breast cancer cell lines, MDA-MB-453 and MDA-MB-361 than basal level HER-2/new breast cancer cell line, MDA-MB-435 and MDA-MB-231 FIG 3 IB Infection efficacy of Ad. RSV/αcZ with high infection protocol, which depletes FBS during infection, infection efficiency higher than 95% can be obtained in MDA-MB-435, with a V/T ratio of 400.1 and MDA-MB-453, with a V/T ratio of 200 1 FIG. 32A Effect of adenovirus 5 EIA on the expression of HER-2/new among breast cancer cell lines MDA-MB-453 cell were treated with various V/T ratio, (0, 50 1, 100: 1, 150.1, 200: 1) of A .ElA(+) and Ad EIA (-). Cell lysates were run through 8% SDS-PAGE and blotted with anti-pl85 and anti-ElA antibody. Down- regulation of pi 85 expression is seen in a dose-dependent fashion in MDA-MB-453 FIG. 32B. Effect of adenovirus 5 EIA on the expression of HER-2/new among breast cancer cell lines. MDA-MB-435 cell were treated with V/T ratio, 400: 1 with Ad.ElA(+) and Ad EIA(-) using high infection protocol Cell lysates were run through 8% SDS-PAGE and blotted with anti-p 185 and anti-E 1 A antibody There was no difference in the basal level ofthe HER- 1 /new Comparable level of EIA expression was seen. FIG 33 Synergistic effect of El A and taxol against HER- 2/new- overexpressing breast cancel line MDA-MB-453. Adenoviral vector was delivered under high (FIG 4A and FIG 4B) or regular (FIG 33C and FIG 33D) infection protocol for 48 hours, then exposed to various concentration of taxol was applied The percentage of cell growth was calculated by defining the absorption of cells not treated with taxol or El A as 100% Bars, SD FIG 33 A Synergist effect of Ad E1A(+) V/T ratio of 200 1 and Taxol on MDA-MB-453 FIG 33B No synergistic effect of Ad El A(+) V/T ratio of 400 1 and taxol on MDA-MB-435 FIG 33C Synergist effect of Ad E1A(+) V/T ratio of 200 1 and Taxol on MDA-MB-453 FIG 33D No synergistic effect of Ad E1A(+) V/T ratio of 400 1 and taxol on the viable cell fraction of MDA-MB-435 FIG 34 Synergistic effect of EIA and taxol 10 μM against HER-2new- overexpressing breast cancel cell line MDA-MB-453 based on time scale Adenoviral vector was delivered under high (FIG 5 A and FIG 5B) or regular (FIG 5C and FIG 5D ) infection protocol for 48 hours, then exposed to taxol 10 μM the viability of cells were followed by MTT assay, Day 0 is when the taxol was applied The percentage of cell growth was calculated by defining the absorption of cells not treated with taxol or E 1 A as 100% Bars, SD FIG 34A Synergist effect of Ad E1A(+) alone shows cell growth suppression FIG 34B No synergistic effect of AD El A(+) V/T ratio of 400 1 and taxol on MDA-MB-435 Ad El A(+) does not suppress cell growth Low HER-2/new is more sensitive to taxol FIG 34C Synergist effect of AD E1A(+) V/T ratio of 200 1 and Taxol on MDA-MB-453 FIG 34D No synergistic effect of Ad El A(+) V/T ratio of 400 1 and taxol on the viable cell fraction of MDA-MBA-435 Regular infection protocol results in more higher degree of cell kill due to continuous existing of FBS, which results in activation of cell cycles FIG 35 Synergist effect of EIA and taxol 10 μM against HER-2/new- overexpressing breast cancer line MDA-MB-453 on clonogenic assay MDA-MB-453 shows synergistic suppression effect on the colony formation the soft agar with anchorage independent fashion by Ad E1A(+) V/T ratio of 200 1 and Taxol 20 μM, adenoviral vector was delivered under regular infection protocol and the percentage of colonies was calculated by defining the colony number not treated with taxol and EIA as 100% Bars, SD No synergistic effect of adenovirus 5 EIA V/T ratio of 400 1 and Taxol (1 μM) on the viable cell fraction of MDA-MA-435, adenoviral vector was delivered under high infection protocol and the percentage of colonies was calculated by defining the colony no not treated with taxol and El A as 100% Bars, SD FIG 36 Synergistic effect of EIA and taxol 10 μM against HER-2/new- overexpressing breast cancer cell line MDA-MB-453 on direct cell counting FIG 36A Growth curve of MDA-Mb-453 There was significant suppression of cell proliferation by Ad.El A(+) V/T ratio of 200 1 and taxol 10 μM Adenoviral vector was delivered without serum FIG 36B No synergistic effect of Ad.El A(+) V/T ratio of 400 1 and taxol (10 μM) on the viable cell fraction of MDA-MB-435, adenoviral vector was delivered under high infection protocol The percentage of cell growth was calculated by defining the absorption of cells not treated with taxol or EIA as 100%. Bars, SD. Viable cells were checked by trypan blue 0.5%. DETAILED DESCRIPTION OF THE INVENTION The present invention seeks to overcome drawbacks inherent in the prior art by providing new treatment methods, compositions and kits for increasing the efficacy of antineoplastic agents against new-mediated cancer. The invention provides methods for treating new-mediated cancers using a new- suppressing gene product and a chemotherapeutic drug in order to inhibit new-tyrosine kinase activity. The methods ofthe invention generally rest in using genes, for example, the El A or the LT gene in combination with an anti-cancer agent effective to treat the cancer cells associated with neu over-expression. Definitions and Techniques Affecting Gene Products and Genes. EIA Gene Products and Genes In this patent the terms "El A gene product" and "EIA" refers to proteins having amino acid sequences which are substantially identical to the native EIA amino acid sequence and which are biologically active in that they are capable of binding to Rb, suppressing new oncogene-mediated transformation, immortalizing cells, or cross- reacting with anti-El A antibody raised against EIA. Such sequences are disclosed, for example, in Berk et al, 1978 The term "EIA gene product" also includes analogs of EIA molecules which exhibit at least some biological activity in common with native EIA. Furthermore, those skilled in the art of mutagenesis will appreciate that other analogs, as yet undisclosed or undiscovered, may be used to construct EIA analogs. Such analogs may be generated in the manners described for the generation of LT mutants in Kalderon et al. (1984). There is no need for an "EIA gene product" or "EIA" to comprise all, or substantially all ofthe amino acid sequence ofthe native EIA gene Shorter or longer sequences are anticipated to be of use in the invention For example, the invention contemplates the use of a "mini-El A gene product" comprising less than the entire EIA amino acid sequence. For example, such a mini- El A gene product may comprise the N-terminal domain and the CRI domain of an EIA gene product. Such a mini-El A gene product may further comprise a spacer domain and/or a C-terminal domain ofthe El A-gene product. Other preferred mini- El A gene products are El A gene products from which the CR2 region has been ablated. The term "E 1 A gene" refers to any DNA sequence that is substantially identical to a DNA sequence encoding an El A gene product as defined above. The term also refers to RNA, or antisense sequences compatible with such DNA sequences. An "El A gene" may also comprise any combination of associated control sequences. The term "substantially identical", when used to define either an El A amino acid sequence or El A gene nucleic acid sequence, means that a particular subject sequence, for example, a mutant sequence, varies from the sequence of natural El A by one or more substitutions, deletions, or additions, the net effect of which is to retain at least some biological activity ofthe EIA protein Alternatively, DNA analog sequences are "substantially identical" to specific DNA sequences disclosed herein if (a) the DNA analog sequence is derived from coding regions ofthe natural EIA gene; or (b) the DNA analog sequence is capable of hybridization of DNA sequences of (a) under moderately stringent conditions and which encode biologically active EIA; or (c) DNA sequences which are degenerative as a result ofthe genetic code to the DNA analog sequences defined in (a) or (b). Substantially identical analog proteins will be greater than about 80% similar to the corresponding sequence ofthe native protein Sequences having lesser degrees of similarity but comparable biological activity are considered to be equivalents. In determining nucleic acid sequences, all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference nucleic acid sequence, regardless of differences in codon sequence. LT Gene Products and Genes In this patent the terms "LT gene product" and "LT" refers to proteins having amino acid sequences which are substantially identical to the native LT amino acid sequence and which are biologically active in that they are capable of binding to Rb, suppressing new oncogene-mediated transformation, immortalizing cells, inducing anchorage independency, or cross-reacting with anti-LT antibody raised against LT Such sequences are disclosed, for example, in Tooze - Molecular Biology ofthe Tumor Viruses, Fiers et al, 1978, and Reddy et al. 1978 The term "LT gene product" also includes analogs of LT molecules which exhibit at least some biological activity in common with native LT. Examples of such LT analogs are Kl and K7, which are defective for transformation of cells (Kalderon et al, 1984) Many other exemplary LT analogs are disclosed in Kalderon et al. 1984, particularly in Table 2 Furthermore, those skilled in the art of mutagenesis will appreciate that other analogs, as yet undisclosed or undiscovered, may be used to construct LT analogs There is no need for an "LT gene product" or "LT" to comprise all, or substantially all ofthe amino acid sequence ofthe native LT gene Shorter or longer sequences are anticipated to be of use in the invention The term "LT gene" refers to any DNA sequence that is substantially identical to a DNA sequence encoding an LT gene product as defined above The term also refers to RNA, or antisense sequences compatible with such DNA sequences An "LT gene" may also comprise any combination of associated control sequences. The term "substantially identical", when used to define either an LT amino acid sequence or an LT nucleic acid sequence, means that a particular subject sequence, for example, a mutant sequence, varies from the sequence of natural LT by one or more substitutions, deletions, or additions, the net effect of which is to retain at least some biological activity ofthe LT protein. Alternatively, DNA analog sequences are "substantially identical" to specific DNA sequences disclosed herein if: (a) the DNA analog sequence is derived from coding regions ofthe natural LT gene, or (b) the DNA analog sequence is capable of hybridization of DNA sequences of (a) under moderately stringent conditions and which encode biologically active LT; or (c) DNA sequences which are degenerative as a result ofthe genetic code to the DNA analog sequences defined in (a) or (b) Substantially identical analog proteins will be greater than about 8Ϊ)% similar to the corresponding sequence ofthe native protein Sequences having lesser degrees of similarity but comparable biological activity are considered to be equivalents In determining nucleic acid sequences, all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference nucleic acid sequence, regardless of differences in codon sequence Percent Similarity Percent similarity may be determined, for example, by comparing sequence information using the GAP computer program, available from the University of Wisconsin Geneticist Computer Group The GAP program utilizes the alignment method of Needleman et al, 1970, as revised by Smith et al, 1981 Briefly, the GAP program defines similarity as the number of aligned symbols (i e nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter ofthe two sequences. The preferred default parameters for the GAP program include (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non- identities) of nucleotides and the weighted comparison matrix of Gribskov et al, 1986, as described by Schwartz et al, 1979; (2) a penalty of 3.0 for each gap and an additional 0.01 penalty for each symbol and each gap, and (3) no penalty for end gaps Nucleic Acid Sequences In certain embodiments, the invention concerns the use of new-suppressing genes and gene products, such as the LT antigen gene product or the El A gene product, or both, that include within their respective sequences a sequence which is essentially that ofthe known LT antigen gene or El A gene, or the corresponding proteins. The term "a sequence essentially as that of LT antigen or EIA" means that the sequence substantially corresponds to a portion ofthe LT antigen or El A gene and has relatively few bases or amino acids (whether DNA or protein) which are not identical to those of LT or El A (or a biologically functional equivalent thereof, when referring to proteins). The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Accordingly, sequences which have between about 70% and about 80%; or more preferably, between about 81 % and about 90%; or even more preferably, between about 91% and about 99%; of amino acids which are identical or functionally equivalent to the amino acids of LT antigen or EIA will be sequences which are "essentially the same". LT antigen and EIA genes which have functionally equivalent codons are also covered by the invention The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids (Table 1). TABLE 1 FUNCTIONALLY EQUIVALENT CODONS. Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic Acid Asp D GAC GAU Glutamic Acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine He I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC ecu Glutamine Gin Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp w UGG Tyrosine Tyr Y UAC UAU It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one ofthe sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned The addition of terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either ofthe 5' or 3' portions ofthe coding region or may include various internal sequences, I e , introns, which are known to occur within genes The present invention also encompasses the use of DNA segments which are complementary, or essentially complementary, to the sequences set forth in the specification Nucleic acid sequences which are "complementary" are those which are capable of base-paiπng according to the standard Watson-Crick complementarity rules As used herein, the term "complementary sequences" means nucleic acid sequences which are substantially complementary, as may be assessed by the same nucleotide compaπson set forth above, or as defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein Biologically Functional Equivalents As mentioned above, modification and changes may be made in the structure of El A or LT and still obtain a molecule having like or otherwise desirable characteristics For example, certain amino acids may be substituted for other ammo acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, the new-gene Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain ammo acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like or even countervailing properties (e g , antagonistic v agonistic) It is thus contemplated by the inventors that various changes may be made in the sequence ofthe EIA or LT proteins or peptides (or underlying DNA) without appreciable loss of their biological utility or activity It is also well understood by the skilled artisan that, inherent in the definition of a biologically functional equivalent protein or peptide, is the concept that there is a limit to the number of changes that may be made within a defined portion ofthe molecule and still result in a molecule with an acceptable level of equivalent biological activity Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, ofthe amino acids may be substituted Of course, a plurality of distinct proteins/peptides with different substitutions may easily be made and used in accordance with the invention. It is also well understood that where certain residues are shown to be particularly important to the biological or structural properties of a protein or peptide, e g , residues in active sites, such residues may not generally be exchanged This is the case in the present invention, where it any changes in the new-binding region of either E 1 A or LT that render the peptide incapable of suppressing new-mediated transformation would result in a loss of utility ofthe resulting peptide for the present invention Amino acid substitutions, such as those which might be employed in modifying either El A or LT are generally based on the relative similarity ofthe amino acid side- chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like An analysis ofthe size, shape and type ofthe amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues, that alanine, glycine and serine are all a similar size, and that phenylalanine, tryptophan and tyrosine all have a generally similar shape Therefore, based upon these considerations, arginine, lysine and histidine, alanine, glycine and serine, and phenylalanine, tryptophan and tyrosine, are defined herein as biologically functional equivalents In making such changes, the hydropathic index of amino acids may be considered Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are isoleucine (+4 5), valine (+4 2), leucine (+3 8), phenylalanine (+2 8), cysteine/cystine (+2 5), methionine (+1 9), alanine (+1 8), glycine (-0 4), threonine (-0 7), serine (-0 8), tryptophan (-0 9), tyrosine (-1 3), proline (-1 6), histidine (-3.2), glutamate (-3 5), glutamine (-3 5), aspartate (-3 5), asparagine (-3 5), lysine (-3 9), and arginine (-4 5) The importance ofthe hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference) It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0 5 are even more particularly preferred It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity U S Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent ammo acids, correlates with its immunogenicity and antigenicity, i e with a biological property of the protein It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein As detailed in U S Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues arginine (+3 0), lysine (+3 0), aspartate (+3 0 ± 1), glutamate (+3 0 ± 1), serine (+0 3), asparagine (+0 2), glutamine (+0 2), glycine (0), threonine (-0 4), proline (-0.5 ± 1), alanine (-0 5), histidine (-0 5), cysteine (-1 0), methionme (-1 3), valine (-1 5), leucine (-1 8), isoleucine (-1 8), tyrosine (-2 3), phenylalanine (-2 5), tryptophan (-3 4) In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. While discussion has focused on functionally equivalent polypeptides arising from amino acid changes, it will be appreciated that these changes may be effected by alteration ofthe encoding DNA; taking into consideration also that the genetic code is degenerate and that two or more codons may code for the same amino acid. Sequence Modification Techniques Modifications to the El A and LT peptides may be carried out using techniques such as site directed mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis ofthe underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more ofthe foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence ofthe desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides ofthe deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. In general, the technique of site-specific mutagenesis is well known in the art as exemplified by publications (Adelman et al, 1983). As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage (Messing et al, 1981). These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage. In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart the two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the EIA gene or the LT gene. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example by the method of Crea et al. (1978) This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis ofthe mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement. Kalderon et al. (1984) report several mutagenic methods which have proved useful in mutating the native LT gene. Specifically, Kalderon et al. teach deletion mutations by displacement-loop mutagenesis and by the random insertion of EcoRI linkers into the LT gene. Further, point mutation by deletion-loop mutagenesis is taught. The reference also teaches screening procedures for determining the success of such mutations. The teachings of Kalderon et al. (1984) are incorporated by reference in this application. The preparation of sequence variants ofthe selected gene using site-directed mutagenesis is provided as a means of producing potentially useful EIA, LT, or other new-suppressing species and is not meant to be limiting as there are other ways in which sequence variants of these peptides may be obtained. For example, recombinant vectors encoding the desired genes may be treated with mutagenic agents to obtain sequence variants (see, e.g., a method described by Eichenlaub, 1979) for the mutagenesis of plasmid DNA using hydroxylamine. Other Structural Equivalents In addition to the E 1 A and LT peptidyl compounds described herein, the inventors also contemplate that other sterically similar compounds may be formulated to mimic the key portions ofthe peptide structure. Such compounds may be used in the same manner as the peptides ofthe invention and hence are also functional equivalents. The generation of a structural functional equivalent may be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope ofthe present invention. B. Chemotherapeutic Agents A wide variety of chemotherapeutic agents may be used in combination with the therapeutic genes ofthe present invention. These can be, for example, agents that directly cross-link DNA, agents that intercalate into DNA, and agents that lead to chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Agents that directly cross-link nucleic acids, specifically DNA, are envisaged and are shown herein, to eventuate DNA damage leading to a synergistic antineoplastic combination. Agents such as cisplatin, and other DNA alkylating agents may be used Agents that damage DNA also include compounds that interfere with DNA replication, mitosis, and chromosomal segregation. Examples of these compounds include adriamycin (also known as doxorubicin), VP-16 (also known as etoposide), verapamil, podophyllotoxin, and the like Widely used in clinical setting for the treatment of neoplasms these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg m 2 at 21 day intervals for adriamycin, to 35-100 mg/m 2 for etoposide intravenously or orally. (i) Antibiotics Doxorubicin Doxorubicin hydrochloride, 5,12-Naphthacenedione, (8s-c/s)-10-[(3-amino- 2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-7,8,9, 10-tetrahydro-6,8,l l-trihydroxy-8- (hydroxyacetyl)- 1 -methoxy-hydrochloride (hydroxydaunorubicin hydrochloride, Adriamycin) is used in a wide antineoplastic spectrum. It binds to DNA and inhibits nucleic acid synthesis, inhibits mitosis and promotes chromosomal aberrations. Administered alone, it is the drug of first choice for the treatment of thyroid adenoma and primary hepatocellular carcinoma. It is a component of 31 first-choice combinations for the treatment of ovarian, endometrial and breast tumors, bronchogenic oat-cell carcinoma, non-small cell lung carcinoma, gastric adenocarcinoma, retinoblastoma, newroblastoma, mycosis fungoides, pancreatic carcinoma, prostatic carcinoma, bladder carcinoma, myeloma, diffuse histiocytic lymphoma, Wilms' tumor, Hodgkin's disease, adrenal tumors, osteogenic sarcoma soft tissue sarcoma, Ewing's sarcoma, rhabdomyo sarcoma and acute lymphocytic leukemia. It is an alternative drug for the treatment of islet cell, cervical, testicular and adrenocortical cancers It is also an immunosuppressant. Doxorubicin is absorbed poorly and must be administered intravenously. The pharmacokinetics are multicompartmental. Distribution phases have half-lives of 12 minutes and 3.3 hr. The elimination half-life is about 30 hr. Forty to 50% is secreted into the bile Most ofthe remainder is metabolized in the liver, partly to an active metabolite (doxorubicinol), but a few percent is excreted into the urine. In the presence of liver impairment, the dose should be reduced. Appropriate doses are, intravenous, adult, 60 to 75 mg/m 2 at 21 -day intervals or 25 to 30 mg/m 2 on each of 2 or 3 successive days repeated at 3- or 4-wk intervals or 20 mg/m 2 once a week The lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs The dose should be reduced by 50% if the serum bilirubin lies between 1 2 and 3 mg/dL and by 75% if above 3 mg dL The lifetime total dose should not exceed 550 mg/m 2 in patients with normal heart function and 400 mg/m 2 in persons having received mediastinal irradiation Alternatively, 30 mg/m 2 on each of 3 consecutive days, repeated every 4 wk Exemplary doses may be 10 mg/m 2 , 20 mg/m 2 , 30 mg/m 2 , 50 mg/m 2 , 100 mg/m 2 , 150 mg/m 2 , 175 mg/m 2 , 200 mg/m 2 , 225 mg/m 2 , 250 mg/m 2 , 275 mg/m 2 , 300 mg/m 2 , 350 mg/m 2 , 400 mg/m 2 , 425 mg/m 2 , 450 mg/m 2 , 475 mg/m 2 , 500 mg/m 2 Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention In the present invention the inventors have employed EIA and LT as exemplary genes for therapy to synergistically enhance the antineoplastic effects ofthe doxorubicin in the treatment of cancers Those of skill in the art will be able to use the invention as exemplified potentiate the effects of doxorubicin in a range of different new-mediated cancers Daunorubicin Daunorubicin hydrochloride, 5, 12-Naphthacenedione, (8S-c/s)-8-acetyl-10-[(3- amino-2,3,6-trideoxy-a-L-lyxo-hexanopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8, l l- trihydroxy- 10-methoxy-, hydrochloride, also termed cerubidine and available from Wyeth Daunorubicin intercalates into DNA, blocks DAN-directed RNA polymerase and inhibits DNA synthesis It can prevent cell division in doses that do not interfere with nucleic acid synthesis In combination with other drugs it is included in the first-choice chemotherapy of acute myelocytic leukemia in adults (for induction of remission), acute lymphocytic leukemia and the acute phase of chronic myelocytic leukemia Oral absorption is poor, and it must be given intravenously The half-life of distribution is 45 minutes and of elimination, about 19 hr The half-life of its active metabolite, daunorubicinol, is about 27 hr Daunorubicin is metabolized mostly in the liver and also secreted into the bile (ca 40%>) Dosage must be reduced in liver or renal insufficiencies Suitable doses are (base equivalent), intravenous adult, younger than 60 yr. 45 mg/m 2 /day (30 mg/m 2 for patients older than 60 yr.) for 1, 2 or 3 days every 3 or 4 wk or 0 8 mg/kg/day for 3 to 6 days every 3 or 4 wk, no more than 550 mg/m 2 should be given in a lifetime, except only 450 mg/m 2 if there has been chest irradiation, children, 25 mg/m 2 once a week unless the age is less than 2 yr or the body surface less than 0 5 m, in which case the weight-based adult schedule is used It is available in injectable dosage forms (base equivalent) 20 mg (as the base equivalent to 21 4 mg ofthe hydrochloride) Exemplary doses may be 10 mg/m 2 , 20 mg/m 2 , 30 mg/m 2 , 50 mg m 2 , 100 mg/m 2 , 150 mg/m 2 , 175 mg/m 2 , 200 mg m 2 , 225 mg/m 2 , 250 mg/m 2 , 275 mg m 2 , 300 mg/m 2 , 350 mg/m 2 , 400 mg m 2 , 425 mg/m 2 , 450 mg/m 2 , 475 mg m 2 , 500 mg/m 2 Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention Mitomycin Mitomycin (also known as mutamycin and/or mitomycin-C) is an antibiotic isolated from the broth of Streptomyces caespitosus which has been shown to have antitumor activity The compound is heat stable, has a high melting point, and is freely soluble in organic solvents Mitomycin selectively inhibits the synthesis of deoxyribonucleic acid (DNA) The guanine and cytosine content correlates with the degree of mitomycin-induced cross-linking At high concentrations ofthe drug, cellular RNA and protein synthesis are also suppressed In humans, mitomycin is rapidly cleared from the serum after intravenous administration Time required to reduce the serum concentration by 50% after a 30 mg bolus injection is 17 minutes After injection of 30 mg , 20 mg , or 10 mg. I V , the maximal serum concentrations were 2 4 mg /mL, 1 7 mg /mL, and 0 52 mg /mL, respectively Clearance is effected primarily by metabolism in the liver, but metabolism occurs in other tissues as well The rate of clearance is inversely proportional to the maximal serum concentration because, it is thought, of saturation ofthe degradative pathways Approximately 10% of a dose of mitomycin is excreted unchanged in the urine Since metabolic pathways are saturated at relatively low doses, the percent of a dose excreted in urine increases with increasing dose In children, excretion of intravenously administered mitomycin is similar Actinomycin D Actinomycin D (Dactinomycin) [50-76-0], C6 2 H 86 Nι 2 Oι 6 (1255 43) is an antineoplastic drug that inhibits DNA-dependent RNA polymerase It is a component of first-choice combinations for treatment of choriocarcinoma, embryonal rhabdomyosarcoma, testicular tumor and Wilms' tumor Tumors which fail to respond to systemic treatment sometimes respond to local perfusion. Dactinomycin potentiates radiotherapy It is a secondary (efferent) immunosuppressive Actinomycin D is used in combination with primary surgery, radiotherapy, and other drugs, particularly vincristine and cyclophosphamide Antineoplastic activity has also been noted in Ewing's tumor, Kaposi's sarcoma, and soft-tissue sarcomas Dactinomycin can be effective in women with advanced cases of choriocarcinoma It also produces consistent responses in combination with chlorambucil and methotrexate in patients with metastatic testicular carcinomas A response may sometimes be observed in patients with Hodgkin's disease and non-Hodgkin's lymphomas Dactinomycin has also been used to inhibit immunological responses, particularly the rejection of renal transplants Half of the dose is excreted intact into the bile and 10% into the urine, the half- life is about 36 hr The drug does not pass the blood-brain barrier. Actinomycin D is supplied as a lyophilized powder (0/5 mg in each vial) The usual daily dose is 10 to 15 mg/kg, this is given intravenously for 5 days; if no manifestations of toxicity are encountered, additional courses may be given at intervals of 3 to 4 weeks Daily injections of 100 to 400 mg have been given to children for 10 to 14 days, in other regimens, 3 to 6 mg/kg, for a total of 125 mg/kg, and weekly maintenance doses of 7.5 mg/kg have been used Although it is safer to administer the drug into the tubing of an intravenous infusion, direct intravenous injections have been given, with the precaution of discarding the needle used to withdraw the drug from the vial in order to avoid subcutaneous reaction Exemplary doses may be 100 mg/m 2 , 150 mg/m 2 , 175 mg/m 2 , 200 mg/m 2 , 225 mg/m 2 , 250 mg/m 2 , 275 mg/m 2 , 300 mg/m 2 , 350 mg/m 2 , 400 mg/m 2 , 425 mg/m 2 , 450 mg/m 2 , 475 mg/m 2 , 500 mg/m 2 Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention Bleomycin Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus. It is freely soluble in water Although the exact mechanism of action of bleomycin is unknown, available evidence would seem to indicate that the main mode of action is the inhibition of DNA synthesis with some evidence of lesser inhibition of RNA and protein synthesis. In mice, high concentrations of bleomycin are found in the skin, lungs, kidneys, peritonewm, and lymphatics. Tumor cells ofthe skin and lungs have been found to have high concentrations of bleomycin in contrast to the low concentrations found in hematopoietic tissue. The low concentrations of bleomycin found in bone marrow may be related to high levels of bleomycin degradative enzymes found in that tissue. In patients with a creatinine clearance of >35 mL per minute, the serum or plasma terminal elimination half-life of bleomycin is approximately 115 minutes. In patients with a creatinine clearance of <35 mL per minute, the plasma or serum terminal elimination half-life increases exponentially as the creatinine clearance decreases In humans, 60%> to 70%> of an administered dose is recovered in the urine as active bleomycin Bleomycin should be considered a palliative treatment It has been shown to be useful in the management ofthe following neoplasms either as a single agent or in proven combinations with other approved chemotherapeutic agents in squamous cell carcinoma such as head and neck (including mouth, tongue, tonsil, nasopharynx, oropharynx, sinus, palate, lip, buccal mucosa, gingiva, epiglottis, larynx), skin, penis, cervix, and vulva It has also been used in the treatment of lymphomas and testicular carcinoma Because ofthe possibility of an anaphylactoid reaction, lymphoma patients should be treated with two units or less for the first two doses If no acute reaction occurs, then the regular dosage schedule may be followed Improvement of Hodgkin's Disease and testicular tumors is prompt and noted within 2 weeks If no improvement is seen by this time, improvement is unlikely Squamous cell cancers respond more slowly, sometimes requiring as long as 3 weeks before any improvement is noted Bleomycin may be given by the intramuscular, intravenous, or subcutaneous routes (ii) Miscellaneous Agents Cisplatin Cisplatin has been widely used to treat cancers such as metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications of 15-20 mg/m 2 for 5 days every three weeks for a total of three courses Exemplary doses may be 0.50 mg/m 2 , 1.0mg/m 2 , 1 50 mg/m 2 , 1.75 mg/m 2 , 2 0 mg/m 2 , 3.0 mg/m 2 , 4.0 mg/m 2 , 5 0 mg/m 2 , 10mg//m 2 Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally. In certain aspects of the current invention cisplatin may be used in combination with El A or LT in the treatment of breast carcinoma It is clear, however, that the combination of cisplatin and therapeutic genes could be used for the treatment of any other new-mediated cancer VP16 VPI 6 is also know as etoposide and is used primarily for treatment of testicular tumors, in combination with bleomycin and cisplatin, and in combination with cisplatin for small-cell carcinoma ofthe lung. It is also active against non-Hodgkin's lymphomas, acute nonlymphocytic leukemia, carcinoma ofthe breast, and Kaposi's sarcoma associated with acquired immunodeficiency syndrome (AIDS). VP 16 is available as a solution (20 mg/ml) for intravenous administration and as 50-mg, liquid-filled capsules for oral use. For small-cell carcinoma ofthe lung, the intravenous dose (in combination therapy) is can be as much as 100 mg/m 2 or as little as 2 mg/ m 2 , routinely 35 mg/m 2 , daily for 4 days, to 50 mg/m 2 , daily for 5 days have also been used When given orally, the dose should be doubled. Hence the doses for small cell lung carcinoma may be as high as 200-250mg/m 2 The intravenous dose for testicular cancer (in combination therapy) is 50 to 100 mg/m 2 daily for 5 days, or 100 mg/m 2 on alternate days, for three doses Cycles of therapy are usually repeated every 3 to 4 weeks The drug should be administered slowly during a 30- to 60-minute infusion in order to avoid hypotension and bronchospasm, which are probably due to the solvents used in the formulation. Tumor Necrosis Factor Tumor Necrosis Factor [TNF, Cachectin] is a glycoprotein that kills some kinds of cancer cells, activates cytokine production, activates macrophages and endothelial cells, promotes the production of collagen and collagenases, is an inflammatory mediator and also a mediator of septic shock, and promotes catabolism, fever and sleep Some infectious agents cause tumor regression through the stimulation of TNF production TNF can be quite toxic when used alone in effective doses, so that the optimal regimens probably will use it in lower doses in combination with other drugs Its immunosuppressive actions are potentiated by gamma-interferon, so that the combination potentially is dangerous A hybrid of TNF and interferon-α also has been found to possess anti-cancer activity (iii) Plant Alkaloids Taxol Taxol is an experimental antimitotic agent, isolated from the bark ofthe ash tree, Taxus brevifolia It binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules Taxol is currently being evaluated clinically; it has activity against malignant melanoma and carcinoma of the ovary Maximal doses are 30 mg/m 2 per day for 5 days or 210 to 250 mg m 2 given once every 3 weeks Of course, all of these dosages are exemplary, and any dosage in- between these points is also expected to be of use in the invention Vincristine Vincristine blocks mitosis and produces metaphase arrest It seems likely that most ofthe biological activities of this drug can be explained by its ability to bind specifically to tubulin and to block the ability of protein to polymerize into microtubules Through disruption ofthe microtubules ofthe mitotic apparatus, cell division is arrested in metaphase The inability to segregate chromosomes correctly during mitosis presumably leads to cell death The relatively low toxicity of vincristine for normal marrow cells and epithelial cells make this agent unusual among anti-neoplastic drugs, and it is often included in combination with other myelosuppressive agents. Unpredictable absorption has been reported after oral administration of vinblastine or vincristine. At the usual clinical doses the peak concentration of each drug in plasma is approximately 0.4 mM. Vinblastine and vincristine bind to plasma proteins. They are extensively concentrated in platelets and to a lesser extent in leukocytes and erythrocytes. Vincristine has a multiphasic pattern of clearance from the plasma; the terminal half-life is about 24 hours. The drug is metabolized in the liver, but no biologically active derivatives have been identified. Doses should be reduced in patients with hepatic dysfunction. At least a 50% reduction in dosage is indicated if the concentration of bilirubin in plasma is greater than 3 mg/dl (about 50 mM). Vincristine sulfate is available as a solution (1 mg/ml) for intravenous injection. Vincristine used together with corticosteroids is presently the treatment of choice to induce remissions in childhood leukemia; the optimal dosages for these drugs appear to be vincristine, intravenously, 2 mg/m 2 of body-surface area, weekly, and prednisone, orally, 40 mg/m 2 , daily. Adult patients with Hodgkin's disease or non-Hodgkin's lymphomas usually receive vincristine as a part of a complex protocol. When used in the MOPP regimen, the recommended dose of vincristine is 1.4 mg/m 2 . High doses of vincristine seem to be tolerated better by children with leukemia than by adults, who may experience sever newrological toxicity. Administration ofthe drug more frequently than every 7 days or at higher doses seems to increase the toxic manifestations without proportional improvement in the response rate. Precautions should also be used to avoid extravasation during intravenous administration of vincristine. Vincristine (and vinblastine) can be infused into the arterial blood supply of tumors in doses several times larger than those that can be administered intravenously with comparable toxicity Vincristine has been effective in Hodgkin's disease and other lymphomas Although it appears to be somewhat less beneficial than vinblastine when used alone in Hodgkin's disease, when used with mechlorethamine, prednisone, and procarbazine (the so-called MOPP regimen), it is the preferred treatment for the advanced stages (III and IV) of this disease In non-Hodgkin's lymphomas, vincristine is an important agent, particularly when used with cyclophosphamide, bleomycin, doxorubicin, and prednisone Vincristine is more useful than vinblastine in lymphocytic leukemia Beneficial response have been reported in patients with a variety of other neoplasms, particularly Wilms' tumor, newroblastoma, brain tumors, rhabdomyosarcoma, and carcinomas ofthe breast, bladder, and the male and female reproductive systems Doses of vincristine for use will be determined by the clinician according to the individual patients need 0 01 to 0 03mg/kg or 0 4 to 1 4mg/m 2 can be administered or 1 5 to 2mg/m 2 can also be administered Alternatively 0 02 mg/m 2 , 0 05 mg/m 2 , 0 06 mg/m 2 , 0 07 mg/m 2 , 0 08 mg/m 2 , 0 1 mg/m 2 , 0 12 mg/m 2 , 0 14 mg/m 2 , 0 15 mg m 2 , 0 2 mg/m 2 , 0 25mg/m 2 can be given as a constant intravenous infusion Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention Vinblastine When cells are incubated with vinblastine, dissolution ofthe microtubules occurs Unpredictable absorption has been reported after oral administration of vinblastine or vincristine At the usual clinical doses the peak concentration of each drug in plasma is approximately 0 4 mM Vinblastine and vincristine bind to plasma proteins They are extensively concentrated m platelets and to a lesser extent in leukocytes and erythrocytes After intravenous injection, vinblastine has a multiphasic pattern of clearance from the plasma, after distribution, drug disappears from plasma with half-lives of approximately 1 and 20 hours Vinblastine is metabolized in the liver to biologically activate derivative desacetylvinblastine Approximately 15% of an administered dose is detected intact in the urine, and about 10%> is recovered in the feces after biliary excretion. Doses should be reduced in patients with hepatic dysfunction At least a 50% reduction in dosage is indicated if the concentration of bilirubin in plasma is greater than 3 mg/dl (about 50 mM) Vinblastine sulfate is available in preparations for injection The drug is given intravenously, special precautions must be taken against subcutaneous extravasation, since this may cause painful irritation and ulceration The drug should not be injected into an extremity with impaired circulation After a single dose of 0.3 mg/kg of body weight, myelosuppression reaches its maximum in 7 to 10 days If a moderate level of leukopenia (approximately 3000 cells/mm 3 ) is not attained, the weekly dose may be increased gradually by increments of 0.05 mg/kg of body weight In regimens designed to cure testicular cancer, vinblastine is used in doses of 0 3 mg/kg every 3 weeks irrespective of blood cell counts or toxicity The most important clinical use of vinblastine is with bleomycin and cisplatin in the curative therapy of metastatic testicular tumors. Beneficial responses have been reported in various lymphomas, particularly Hodgkin's disease, where significant improvement may be noted in 50 to 90%> of cases The effectiveness of vinblastine in a high proportion of lymphomas is not diminished when the disease is refractory to alkylating agents It is also active in Kaposi's sarcoma, newroblastoma, and Letterer- Siwe disease (histiocytosis X), as well as in carcinoma ofthe breast and choriocarcinoma in women Doses of vinblastine for use will be determined by the clinician according to the individual patients need 0 1 to 0 3mg/kg can be administered or 1 5 to 2mg/m 2 can also be administered Alternatively, 0 1 mg/m 2 , 0 12 mg/m 2 , 0 14 mg/m 2 , 0 15 mg/m 2 , 0 2 mg/m 2 , 0.25 mg/m 2 , 0 5 mg/m 2 , 1 0 mg/m 2 , 1 2 mg m 2 , 1 4 mg/m 2 , 1 5 mg/m 2 , 2 0 mg/m 2 , 2 5 mg/m 2 , 5 0 mg/m 2 , 6 mg/m 2 , 8 mg/m 2 , 9 mg/m 2 , 10 mg/m 2 , 20 mg/m 2 , can be given Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention (iv) Alkylating Agents Carmustine Carmustine (sterile carmustine) is one ofthe nitrosoureas used in the treatment of certain neoplastic diseases It is 1,3 bis (2-chloroethyl)-l-nitrosourea It is lyophilized pale yellow flakes or congealed mass with a molecular weight of 214 06 It is highly soluble in alcohol and lipids, and poorly soluble in water Carmustine is administered by intravenous infusion after reconstitution as recommended Sterile carmustine is commonly available in 100 mg single dose vials of lyophilized material Although it is generally agreed that carmustine alkylates DNA and RNA, it is not cross resistant with other alkylators As with other nitrosoureas, it may also inhibit several key enzymatic processes by carbamoylation of amino acids in proteins Carmustine is indicated as palliative therapy as a single agent or in established combination therapy with other approved chemotherapeutic agents in brain tumors such as glioblastoma, brainstem glioma, medullobladyoma, astrocytoma, ependymoma, and metastatic brain tumors Also it has been used in combination with prednisone to treat multiple myeloma Carmustine has proved useful, in the treatment of Hodgkin's Disease and in non-Hodgkin's lymphomas, as secondary therapy in combination with other approved drugs in patients who relapse while being treated with primary therapy, or who fail to respond to primary therapy The recommended dose of carmustine as a single agent in previously untreated patients is 150 to 200 mg/m 2 intravenously every 6 weeks This may be given as a single dose or divided into daily injections such as 75 to 100 mg/m 2 on 2 successive days When carmustine is used in combination with other myelosuppressive drugs or in patients in whom bone marrow reserve is depleted, the doses should be adjusted accordingly Doses subsequent to the initial dose should be adjusted according to the hematologic response ofthe patient to the preceding dose. It is of course understood that other doses may be used in the present invention for example 10mg/m 2 , 20mg/m 2 , 30mg/m 2 40mg/m 2 50mg/m 2 60mg/m 2 70mg/m 2 80mg/m 2 90mg/m 2 100mg/m 2 The skilled artisan is directed to, "Remington's Pharmaceutical Sciences" 15th Edition, chapter 61 Some variation in dosage will necessarily occur depending on the condition ofthe subject being treated The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Melphalan Melphalan also known as alkeran, L-phenylalanine mustard, phenylalanine mustard, L-PAM, or L-sarcolysin, is a phenylalanine derivative of nitrogen mustard. Melphalan is a bifunctional alkylating agent which is active against selective human neoplastic diseases. It is known chemically as 4-[bis(2-chloroethyl)amino]-L- phenylalanine. Melphalan is the active L-isomer ofthe compound and was first synthesized in 1953 by Bergel and Stock; the D-isomer, known as medphalan, is less active against certain animal tumors, and the dose needed to produce effects on chromosomes is larger than that required with the L-isomer. The racemic (DL-) form is known as merphalan or sarcolysin Melphalan is insoluble in water and has a pKai of ~2.1 Melphalan is available in tablet form for oral administration and has been used to treat multiple myeloma Available evidence suggests that about one third to one half of the patients with multiple myeloma show a favorable response to oral administration ofthe drug Melphalan has been used in the treatment of epithelial ovarian carcinoma One commonly employed regimen for the treatment of ovarian carcinoma has been to administer melphalan at a dose of 0 2 mg/kg daily for five days as a single course. Courses are repeated every four to five weeks depending upon hematologic tolerance (Smith and Rutledge, 1975; Young et al, 1978) Alternatively the dose of melphalan used could be as low as 0 05mg/kg/day or as high as 3mg/kg/day or any dose in between these doses or above these doses Some variation in dosage will necessarily occur depending on the condition ofthe subject being treated The person responsible for administration will, in any event, determine the appropriate dose for the individual subject Cyclophosphamide Cyclophosphamide is 2H-l,3,2-Oxazaphosphorin-2-amine, N,N-bis(2- chloroethyl)tetrahydro-, 2-oxide, monohydrate, termed Cytoxan available from Mead Johnson; and Νeosar available from Adria Cyclophosphamide is prepared by condensing 3 -amino- 1 -propanol with N,N-bis(2-chlorethyl) phosphoramidic dichloride [(ClCH 2 CH 2 ) 2 Ν~POCl 2 ] in dioxane solution under the catalytic influence of triethylamine. The condensation is double, involving both the hydroxyl and the amino groups, thus effecting the cyclization Unlike other β-chloroethylamino alkylators, it does not cyclize readily to the active ethyleneimonium form until activated by hepatic enzymes Thus, the substance is stable in the gastrointestinal tract, tolerated well and effective by the oral and parental routes and does not cause local vesication, necrosis, phlebitis or even pain Suitable doses for adults include, orally, 1 to 5 mg/kg/day (usually in combination), depending upon gastrointestinal tolerance, or 1 to 2 mg/kg/day; intravenously, initially 40 to 50 mg/kg in divided doses over a period of 2 to 5 days or 10 to 15 mg/kg every 7 to 10 days or 3 to 5 mg/kg twice a week or 1 5 to 3 mg/kg/day A dose 250mg/kg/day may be administered as an antineoplastic Because of gastrointestinal adverse effects, the intravenous route is preferred for loading. During maintenance, a leukocyte count of 3000 to 4000/mm 3 usually is desired. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities. It is available in dosage forms for injection of 100, 200 and 500 mg, and tablets of 25 and 50 mg the skilled artisan is referred to "Remington's Pharmaceutical Sciences" 15th Edition, chapter 61, incorporate herein as a reference, for details on doses for administration. Chlorambucil Chlorambucil (also known as leukeran) was first synthesized by Everett et al. (1953). It is a bifunctional alkylating agent ofthe nitrogen mustard type that has been found active against selected human neoplastic diseases. Chlorambucil is known chemically as 4-[bis(2-chlorethyl)amino] benzenebutanoic acid. Chlorambucil is available in tablet form for oral administration. It is rapidly and completely absorbed from the gastrointestinal tract. After single oral doses of 0.6-1.2 mg/kg, peak plasma chlorambucil levels are reached within one hour and the terminal half-life ofthe parent drug is estimated at 1.5 hours. 0.1 to 0.2mg/kg/day or 3 to 6mg m 2 /day or alternatively 0.4mg/kg may be used for antineoplastic treatment. Treatment regimes are well know to those of skill in the art and can be found in the "Physicians Desk Reference" and in "Remingtons Pharmaceutical Sciences" referenced herein. Chlorambucil is indicated in the treatment of chronic lymphatic (lymphocytic) leukemia, malignant lymphomas including lymphosarcoma, giant follicular lymphoma and Hodgkin's disease. It is not curative in any of these disorders but may produce clinically useful palliation. Busulfan Busulfan (also known as myleran) is a bifunctional alkylating agent. Busulfan is known chemically as 1 ,4-butanediol dimethanesulfonate Busulfan is not a structural analog ofthe nitrogen mustards Busulfan is available in tablet form for oral administration Each scored tablet contains 2 mg busulfan and the inactive ingredients magnesium stearate and sodium chloride Busulfan is indicated for the palliative treatment of chronic myelogenous (myeloid, myelocytic, granulocytic) leukemia Although not curative, busulfan reduces the total granulocyte mass, relieves symptoms of the disease, and improves the clinical state ofthe patient Approximately 90% of adults with previously untreated chronic myelogenous leukemia will obtain hematologic remission with regression or stabilization of organomegaly following the use of busulfan It has been shown to be superior to splenic irradiation with respect to survival times and maintenance of hemoglobin levels, and to be equivalent to irradiation at controlling splenomegaly Lomustine Lomustine is one ofthe nitrosoureas used in the treatment of certain neoplastic diseases It is l-(2-chloro-ethyl)-3-cyclohexyl-l nitrosourea It is a yellow powder with the empirical formula of CoHi 6 ClN 3 O2 and a molecular weight of 233 71. Lomustine is soluble in 10%> ethanol (0 05 mg per mL) and in absolute alcohol (70 mg per mL) Lomustine is relatively insoluble in water (<0 05 mg per mL) It is relatively unionized at a physiological pH Inactive ingredients in lomustine capsules are magnesium stearate and mannitol. Although it is generally agreed that lomustine alkylates DNA and RNA, it is not cross resistant with other alkylators As with other nitrosoureas, it may also inhibit several key enzymatic processes by carbamoylation of amino acids in proteins Lomustine may be given orally Following oral administration of radioactive lomustine at doses ranging from 30 mg/m 2 to 100 mg/m 2 , about half of the radioactivity given was excreted in the form of degradation products within 24 hours The serum half-life ofthe metabolites ranges from 16 hours to 2 days Tissue levels are comparable to plasma levels at 15 minutes after intravenous administration Lomustine has been shown to be useful as a single agent in addition to other treatment modalities, or in established combination therapy with other approved chemotherapeutic agents in both primary and metastatic brain tumors, in patients who have already received appropriate surgical and/or radiotherapeutic procedures. It has also proved effective in secondary therapy against Hodgkin's Disease in combination with other approved drugs in patients who relapse while being treated with primary therapy, or who fail to respond to primary therapy The recommended dose of lomustine in adults and children as a single agent in previously untreated patients is 130 mg/m 2 as a single oral dose every 6 weeks. In individuals with compromised bone marrow function, the dose should be reduced to 100 mg/m 2 every 6 weeks When lomustine is used in combination with other myelosuppressive drugs, the doses should be adjusted accordingly It is understood that other doses may be used for example, 20mg/m 2 30mg/m 2 , 40 mg/m 2 , 50mg/m 2 , 60mg/m 2 , 70mg/m 2 , 80mg/m 2 , 90mg/m 2 , 100mg m 2 , 120mg/m 2 or any doses between these FIG s as determined by the clinician to be necessary for the individual being treated C. Pharmaceutical Compositions and Routes of Administration Compositions ofthe present invention will have an effective amount of a gene for therapeutic administration in combination with an effective amount of a compound (second agent) that is a chemotherapeutic agent as exemplified above Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or human, as appropriate As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-cancer agents, can also be incorporated into the compositions. In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used, including cremes, lotions, mouthwashes, inhalants and the like The expression vectors and delivery vehicles ofthe present invention may include classic pharmaceutical preparations Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra. The vectors of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection also may be prepared These preparations also may be emulsified. A typical compositions for such purposes comprises a 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as theyloleate Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases The pH and exact concentration ofthe various components in the pharmaceutical are adjusted according to well known parameters Additional formulations are suitable for oral administration Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders When the route is topical, the form may be a cream, ointment, salve or spray An effective amount ofthe therapeutic agent is determined based on the intended goal The term "unit dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity ofthe therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen The quantity to be administered, both according to number of treatments and unit dose, depends on the subject to be treated, the state ofthe subject and the protection desired Precise amounts of the therapeutic composition also depend on the judgment ofthe practitioner and are peculiar to each individual All the essential materials and reagents required for inhibiting tumor cell proliferation may be assembled together in a kit When the components ofthe kit are provided in one or more liquid solutions, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred For in vivo use, a chemotherapeutic agent may be formulated into a single or separate pharmaceutically acceptable syringeable composition. In this case, the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area ofthe body, such as the lungs, injected into an animal, or even applied to and mixed with the other components ofthe kit. The components of the kit may also be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means. The kits of the invention may also include an instruction sheet defining administration ofthe gene therapy and/or the chemotherapeutic drug. The kits ofthe present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. Irrespective ofthe number or type of containers, the kits ofthe invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement ofthe ultimate complex composition within the body of an animal. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle. Parenteral Administration The active compounds ofthe present invention will often be formulated for parenteral administration, e.g, formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. The preparation of an aqueous composition that contains a second agent(s) as active ingredients will be known to those of skill in the art in light ofthe present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified. Solutions ofthe active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. 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 or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The active compounds may be formulated into a composition in a newtral or salt form Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants The prevention ofthe action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various ofthe 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 freeze-drying techniques which yield a powder ofthe active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. In certain cases, the therapeutic formulations ofthe invention could also be prepared in forms suitable for topical administration, such as in cremes and lotions. These forms may be used for treating skin-associated diseases, such as various sarcomas. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, with even drug release capsules and the like being employable. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light ofthe present disclosure. For example, one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition ofthe subject being treated The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. D. In vivo Delivery and Treatment Protocols Where the gene itself is employed to introduce the gene products, a convenient method of introduction will be through the use of a recombinant vector which incorporates the desired gene, together with its associated control sequences The preparation of recombinant vectors is well known to those of skill in the art and described in many references, such as, for example, Sambrook et al. (1989), specifically incorporated herein by reference. In vectors, it is understood that the DNA coding sequences to be expressed, in this case those encoding the new-suppressing gene products, are positioned adjacent to and under the control of a promoter. It is understood in the art that to bring a coding sequence under the control of such a promoter, one generally positions the 5' end of the transcription initiation site ofthe transcriptional reading frame ofthe gene product to be expressed between about 1 and about 50 nucleotides "downstream" of (i.e., 3' of) the chosen promoter. One may also desire to incorporate into the transcriptional unit ofthe vector an appropriate polyadenylation site (e.g., 5'-AATAAA-3'), if one was not contained within the original inserted DNA. Typically, these poly A addition sites are placed about 30 to 2000 nucleotides "downstream" ofthe coding sequence at a position prior to transcription termination While use ofthe control sequences ofthe specific gene (i e , the EIA promoter for EIA and the LT promoter for LT) will be preferred, there is no reason why other control sequences could not be employed, so long as they are compatible with the genotype ofthe cell being treated Thus, one may mention other useful promoters by way of example, including, e g , an SV40 early promoter, a long terminal repeat promoter from retrovirus, an actin promoter, a heat shock promoter, a metallothionein promoter, and the like For introduction ofthe El A or LT gene, it is proposed that one will desire to preferably employ a vector construct that will deliver the desired gene to the affected cells This will, of course, generally require that the construct be delivered to the targeted tumor cells, for example, breast, genital, or lung tumor cells It is proposed that this may be achieved most preferably by introduction ofthe desired gene through the use of a viral vector to carry either the El A or LT sequences to efficiently infect the tumor, or pretumorous tissue These vectors will preferably be an adenoviral, a retroviral, a vaccinia viral vector or adeno-associated virus These vectors are preferred because they have been successfully used to deliver desired sequences to cells and tend to have a high infection efficiency Commonly used viral promoters for expression vectors are derived from polyoma, cytomegalovirus, Adenovirus 2, and Simian Virus 40 (SV40) The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the viral origin of replication Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems The origin of replication may be provided either by construction ofthe vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient A particularly desirable vector, at least as a starting point, is the El A containing retroviral vector, termed pSVXEl A-G, described by Robert et al, 1985. This vector comprises the El A gene which has been brought under the control ofthe SV-40 early promoter. For LT expression, the pZl 89 (driven by the SV-80 promoter) and the pVU-O vectors both contain LT LT mutants are contained in, for example, pKl and pK7 as well as other vectors described by Kalderon et al 1984. The inventors propose that these constructs could either be used directly in the practice of the invention, or could be used as a starting point for the introduction of other more desirable promoters such as those discussed above (i) Adenovirus One ofthe preferred methods for in vivo delivery involves the use of an adenovirus vector. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging ofthe construct and (b) to express an antisense polynucleotide that has been cloned therein In this context, expression does not require that the gene product be synthesized. Adenoviral transfer of El A is especially useful, because EIA is itself an adenoviral gene Therefore, there need be no non-viral genetic sequences inserted into an adenoviral vector to accomplish adenoviral delivery of El A Of course, LT-encoding DNA, or other new-suppressing gene product encoding sequences may be introduced via adenoviral vectors as well The expression vector comprises a genetically engineered form of adenovirus Knowledge ofthe genetic organization or adenovirus, a 36 kB, linear, double-strained DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992) In contrast to retrovirus, the infection of adenoviral DNA in host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans Adenovirus is a particularly suitable gene transfer vector because of its midsized genome, ease of manipulation, high titer, wide target-cell range and high infectivity Both ends ofthe viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging The early (E) and late (L) regions ofthe genome contain different transcription units that are divided by the onset of viral DNA replication The El region (EIA and ElB) encodes proteins responsible for the regulation of transcription ofthe viral genome and a few cellular genes The expression ofthe E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990) The products ofthe late genes, including the majority ofthe viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP) The MLP, located at 16 8 mμ is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5'-tripartite leader (TL) sequence which makes them preferred mRNA's for translation In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure. Use ofthe YAC system in is an alternative approach for the production of recombinant adenovirus. A preferred method of introducing the El A gene to an animal is to introduce a replication-deficient adenovirus containing the El A gene. An example of such an adenovirus is Ad.El A(+). Since adenovirus is a common virus infecting humans in nature and the El A gene is a gene that is present in native adenovirus, the use of a replication deficient El A virus to introduce the gene may efficiently deliver and express EIA into target cells. The replication-deficient EIA virus made by ElB and E3 deletion also avoids the viral reproduction inside the cell and transfer to other cells and infection of other people, which means the viral infection activity is shut down after it infects the target cell. The EIA gene still is expressed inside the cells. Also, unlike retrovirus, which can only infect proliferating cells, adenovirus is able to transfer the EIA gene into both proliferating and non-proliferating cells. Further, the extrachromosomal location of adenovirus in the infected cells decreases the chance of cellular oncogene activation within the treated animal. While the wild-type adenovirus may be used directly to transfer the El A gene into HER-2/new expressing cancer cells, wild-type virus will produce large amounts of adenovirus in the human body and therefore might cause potential side effects due to the replication competent nature ofthe wild type adenovirus. It is therefore an advantage to use the replication-deficient adenovirus such as ElB and E3 deletion mutant Ad.El A(+) to prevent such side effects. In fact, many modifications in the native adenovirus will result in a modified virus that will be useful for the purpose of the invention. Further modification of adenovirus such as E2A deletion may improve the EIA expression efficiency and reduce the side effects The only requirement of a native or modified adenovirus is that it should express an EIA gene in order to have the utility ofthe invention. Adenovirus can be used is by introducing an LT gene product into such a cell The LT gene product can be an LT mutant, especially a nontransforming mutant such as Kl. Such introduction can typically involve the introduction of an LT gene. In some preferred methods, the LT gene can be introduced by the use of an adenovirus that contains both the EIA gene and the LT gene In this case, adenovirus is a preferably a replication-deficient adenovirus such as the Ad.El A(+) adenovirus However, the introduction ofthe LT gene can be by any manner described in this specification or known to those of skill in the art such as viral, plasmid, retroviral vectors or liposomes. Introduction ofthe adenovirus containing the new-suppressing gene product gene into a suitable host is typically done by injecting the virus contained in a buffer The nature ofthe adenovirus vector is not believed to be crucial to the successful practice ofthe invention. Of course, as discussed above, it is adventageous if the adenovirus vector is replication defective, or at least conditionally defective, The adenovirus may be of any ofthe 42 different known serotypes or subgroups A-F Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector As stated above, a particularly useful vector according to the present invention is replication defective and will not have an adenovirus El region. Thus, it will be convenient to introduce the polynucleotide encoding the gene of interest at the position from which the E 1 -coding sequences have been removed However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention The polynucleotide encoding the gene of interest may also be inserted in lieu ofthe deleted E3 region in E3 replacement vectors as described by Karlsson et al. (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo This group of viruses can be obtained in high titers, e.g., 10 9 -10 u plaque-forming units per ml, and they are highly infective The life cycle of adenovirus does not require integration in to the host cell genome The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963, Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors Adendovirus have been used in eukaryotic gene expression (Levrero et al, 1991, Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992, Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991 , Stratford-Perricaudet et al. , 1990, Rich et al , 1993) Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al, 1991 , Rosenfeld et al, 1992), muscle injection (Ragot et al, 1993), peπpheral intravenous injections (Herz and Gerard, 1993) and stereotatic inoculation into the brain (Le Gal La Salle et al, 1993) (ii) Retroviruses The retroviruses are a group of single- stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA to infected cells by a process of reverse-transcription (Coffin, 1990) The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins The integration results in the retention ofthe viral gene sequences in the recipient cell and its descendants The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively A sequence found upstream from the gag gene, termed ψ components is constructed (Mann et al, 1983) When a recombinant plasmid containing a human cDNA, together with the retroviral LTR and ψ sequences is introduced into this cell line (by calcium phosphate precipitation for example), the ψ sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988, Temin, 1986, Mann et al, 1983) The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer Retroviral vectors are able to infect a broad variety of cell types However, integration and stable expression require the division of host cells (Paskind et al, 1975) A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used The antibodies were coupled via the biotin components by using streptavidin (Roux et al, 1989) Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus m vitro (Roux et al. , 1989) There are certain limitations to the use of retrovirus vectors in all aspects ofthe present invention For example, retrovirus vectors usually integrate into random sites in the cell genome This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al, 1981). Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact ψ sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al. , 1988; Hersdorffer et al, 1990). One limitation to the use of retrovirus vectors in vivo is the limited ability to produce retroviral vector titers greater than IO 6 infections U/mL. Titers 10- to 1, 000-fold higher are necessary for many in vivo applications Several properties ofthe retrovirus have limited its use in lung cancer treatment (Stratford-Perricaudet and Perricaudet, 1991 ; (i) Infection by retrovirus depends on host cell division. In human cancer, very few mitotic cells can be found in tumor lesions (Warner and Heston, 1991). (ii) The integration of retrovirus into the host genome may cause adverse effects on target cells, because malignant cells are high in genetic instability, (iii) Retrovirus infection is often limited by a certain host range. (iv) Retrovirus has been associated with many malignancies in both mammals and vertebrates, (v) The titer of retrovirus, in general, is 100- to 1, 000-fold lower than that of adenovirus. (iii) Other Viral Vectors as Expression Constructs Other viral vectors may be employed as expression constructs in the present invention Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and heψes viruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989, Ridgeway, 1988, Baichwal and Sugden, 1986, Coupar et al, 1988, Howrich et al, 1990) With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al, 1990) This suggested that large portions ofthe genome could be replaced with foreign genetic material The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer Chang et al. recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place ofthe polymerase, surface, and pre-surface coding sequences It was cotransfected with wild-type virus into an avian hepatoma cell line Cultures media containing high titers ofthe recombinant virus were used to infect primary duckling hepatocytes Stable CAT gene expression was detected for at least 24 days after transfection (Change et al, 1991) (iv) Liposomal Transfection In a further embodiment ofthe invention, the expression construct may be entrapped in a liposome Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium Multilamellar liposomes have multiple lipid layers separated by aqueous medium They form spontaneously when phospholipids are suspended in an excess of aqueous solution The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991) Also contemplated are lipofectamine-DNA complexes The present invention also provides particularly useful methods for introducing new-suppressing gene products into cells One method of in vivo gene transfer which can lead to expression of genes transfected into cells involves the use of liposomes Liposomes can be used for both in vitro and in vivo transfection. Liposome-mediated gene transfer seems to have great potential for certain in vivo applications in animals (Nicolau et al, 1987). Studies have shown that intravenously injected liposomes are taken up essentially in the liver and the spleen, by the macrophages ofthe reticuloendothelial system. The specific cellular sites of uptake of injected liposomes appears to be mainly spleen macrophages and liver Kupffer cells. Intravenous injection of liposomes/DNA complexes can lead to the uptake of DNA by these cellular sites, and result in the expression of a gene product encoded in the DNA (Nicolau, 1983). The inventors contemplate that new-suppressing gene products can be introduced into cells using liposome-mediated gene transfer. It is proposed that such constructs can be coupled with liposomes and directly introduced via a catheter, as described by Nabel et al. (1990). By employing these methods, the new-suppressing gene products can be expressed efficiently at a specific site in vivo, not just the liver and spleen cells which are accessible via intravenous injection. Therefore, this invention also encompasses compositions of DNA constructs encoding a new- suppressing gene product formulated as a DNA/liposome complex and methods of using such constructs. Liposomal transfection can be via liposomes composed of, for example, phosphatidylcholine (PC), phosphatidylserine (PS), cholesterol (Choi), N-[l-(2,3- dioleyloxy)propyl]-N,N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylethanolamine (DOPE), and/or 3β[N-(NN-dimethylaminoethane)- carbarmoyl cholesterol (DC-Chol), as well as other lipids known to those of skill in the art. Those of skill in the art will recognize that there are a variety of liposomal transfection techniques which will be useful in the present invention. Among these techniques are those described in Nicolau et al, 1987, Nabel et al, 1990, and Gao et al, 1991. The inventors have had particular success with liposomes comprising DC-Chol. More particularly, the inventors have had success with liposomes comprising DC-Chol and DOPE which have been prepared following the teaching of Gao et al, 1991, in the manner described in the Preferred Embodiments Section The inventors also anticipate utility for liposomes comprised of DOTMA, such as those which are available commercially under the trademark Lipofectin™, from Vical, Ine , in San Diego, California Liposomes may be introduced into contact with cells to be transfected by a variety of methods In cell culture, the liposomes can simply be dispersed in the cell culture solution For application in vivo, liposomes are typically injected Intravenous injection allow liposome-mediated transfer of DNA complex to the liposomes to, for example, the liver and the spleen In order to allow transfection of DNA into cells which are not accessible through intravenous injection, it is possible to directly inject the liposome-DNA complexes into a specific location in an animal's body For example, Nabel et al. teach injection via a catheter into the arterial wall In another example, the inventors have used intraperitoneal injection to allow for gene transfer into mice The present invention also contemplates compositions comprising a liposomal complex This liposomal complex will comprise a lipid component and a DNA segment encoding a new-suppressing gene The new-suppressing gene employed in the liposomal complex can be, for example, an LT gene or an El A gene Liposomal complexes comprising LT mutants may have certain advantages These advantages may be particularly distinct when the LT gene encodes non-transforming LT mutant, such as Kl An El A gene encoding either the El A 12S or El A 13S gene product, or both, may be complexed with a lipid to form the liposomal complex The lipid employed to make the liposomal complex can be any of the above- discussed lipids. In particular, DOTMA, DOPE, and/or DC-Chol may form all or part ofthe liposomal complex The inventors have had particular success with complexes comprising DC-Chol In a preferred embodiment, the lipid will comprise DC-Chol and DOPE While any ratio of DC-Chol to DOPE is anticipated to have utility, it is anticipated that those comprising a ratio of DC-Chol DOPE between 1 20 and 20.1 will be particularly advantageous The inventors have found that liposomes prepared from a ratio of DC-Chol DOPE of about 1 10 to about 1 5 have been useful. The 12S El A, 13S EIA, and LT gene products are capable of suppressing new gene expression, it is proposed that one may employ any product, or two or more together, in the practice of the invention Of course, in that the 12S and 13S products are derived from essentially the same gene sequences, and are merely the result of differential splicing, where the El A gene itself is employed it will be most convenient to simply use the wild type EIA gene directly However, it is contemplated that certain regions of either the EIA or the LT gene may be employed exclusively without employing the entire wild type El A or LT gene respectively. It is proposed that it will ultimately be preferable to employ the smallest region needed to suppress the new gene so that one is not introducing unnecessary DNA into cells which receive either an El A or LT gene construct This may especially be true with regards to the rather large, 708 amino acid, LT protein Techniques well known to those of skill in the art, such as the use of restriction enzymes, will allow for the generation of small regions of El A and LT The ability of these regions to inhibit new can easily be determined by the assays reported in the Examples In certain embodiments ofthe invention, the liposome may be complexed with a hemagglutinating virus (HVJ) This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al. , 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991) In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1 In that such expression constructs have been successfully employed in transfer and expression of nucleic acid m vitro and in vivo, then they are applicable for the present invention Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase (v) Other Non-viral vectors In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo (see below), as in the treatment of certain disease states As described above, delivery may be via viral infection where the expression construct is encapsidated in an infectious viral particle Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention These include calcium phosphate precipitation (Graham and Van Der Eb, 1973, Chen and Okayama, 1987, Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986, Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982, Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity icroprojectiles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987, Wu and Wu, 1988) Some of these techniques may be successfully adapted for in vivo or ex vivo use Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites In certain embodiments, the nucleic acid encoding the gene may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed In one embodiment ofthe invention, the expression construct may simply consist of naked recombinant DNA or plasmids Transfer ofthe construct may be performed by any ofthe methods mentioned above which physically or chemically permeabilize the cell membrane This is particularly applicable for transfer permeabilize the cell membrane This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well Dubensky et al (1984) successfully injected polyomavirus DNA in the form of CaPO precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection Benvenisty and Neshif ( 1986) also demonstrated that direct intraperitoneal injection of CaPO precipitated plasmids results in expression ofthe transfected genes It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product Another embodiment ofthe invention for transferring a naked DNA expression construct into cells may involve particle bombardment This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987) Several devices for accelerating small particles have been developed One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990) The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads. Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al, 1990; Zelenin et al, 1991). This may require surgical exposure ofthe tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present invention Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage ofthe selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because ofthe cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993). Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993; Perales et al, 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085). In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake ofthe insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a particular gene also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes. For example, epidermal growth factor (EGF) may be used as the receptor for mediated delivery of a nucleic acid encoding a gene in many tumor cells that exhibit upregulation of EGF receptor. Mannose can be used to target the mannose receptor on liver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties. In certain embodiments, gene transfer may more easily be performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells, in vitro, and then the return ofthe modified cells back into an animal This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues Anderson et al, U S Patent 5,399,346, and incorporated herein in its entirety, disclose ex vivo therapeutic methods The Examples which follow set forth studies wherein the inventors demonstrate the ability ofthe EIA gene to suppress new gene expression (Example I), new gene- mediated tumorigenicity (Example II), new gene-mediated metastasis (Example III), to suppress c-erbB-2/neu expression in human ovarian carcinoma (Example IV), and gene therapy with EIA (Example VIII). Examples V and VI demonstrate suppression of new with LT antigen While these studies are believed to be exemplary ofthe invention, it will be appreciated by those of skill in the art that many modifications and alterations may be made in these embodiments without departing from the spirit and scope ofthe invention EXAMPLE I Transcriptional Repression of the neu Protooncogene bv Adenovirus 5 EIA Gene Products This Example relates to studies conducted by the inventors which demonstrate that the adenovirus E 1 A 12S and 13S products are effective in repressing the transcriptional activity ofthe new promoter In particular, it is demonstrated that the conserved region 2 (CR2) ofthe EIA proteins are required for repression Moreover, these studies indicated that a c/s-acting DNA element in the upstream region ofthe new promoter is responsible for the trans inhibition ofthe promoter by the El A gene products 1. MATERIALS AND METHODS a Plasmids The recombinants used in this study have been described pEl A (Chang et al, 1989 Hearing et al, 1985) is a plasmid expressing only the El A region gene, pEl Al 2S and pElAl 3 S (Hearing et al, 1985) express 12S EIA protein and 13S E1A protein, respectively, pElA-dl343 (Hearing et al, 1985) contains a 2-base-pair (bp) frameshift deletion in the El A coding sequences (adenovirus nucleotide sequence positions 621 and 622), pElA-dl346 (Hearing et al, 1985) contains an in-frame deletion of nucleotides 859-907 (48 bp), resulting in the deletion of 16 amino acids inside the CR2 ofthe EIA proteins, pElApr contains only the EIA promoter (-499 to +113 relative to the El A cap site), pE2A-CAT (Chung et al, 1989) is a reporter plasmid containing E2 early promoter fused with the chloramphenicol acetyltransferase (CAT) reporter gene, pRSV-CAT is a reporter plasmid containing the CAT gene under the control ofthe Rous sarcoma virus (RSV) long terminal repeat (LTR); pElB, pE2, and pE3 are plasmids expressing ElB, E2, and E3 genes, respectively pnewEcoRl -CAT contains the 2 2-kilobase (kb) rat new promoter and upstream sequences linked to the CAT gene The deletion mutant ofthe new promoter used in this study are described in the legends to FIG 3 and 4 A pRSV-β-gal contains the RSV LTR linked to β-galactosidase gene used as an internal control for transfection efficiency b Cell Cultures Cell cultures were performed as described (Hung et al. , 1989, Matin, et al. , 1984) The Rat-1 and SK-BR-3 cells were grown in dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum and fetal calf serum, respectively c. DNA Transfection All transfections were carried out with the calcium phosphate precipitation technique of Graham and Van der EB as modified by Anderson et al (Hung et al, 1989, Anderson et al, 1979; Ausubel et al, 1987) In each transfection, 8 x IO 5 Rat-1 cells or 2 x IO 6 SK-BR-3 cells (2 x 10 cm dishes) were seeded 24 hr before transfection. Total transfection DNA was kept constant (maximum, 30 μg) among different samples in the same experiment by adding approximate amounts of carrier DNA (pSP64). d CAT Assays Cell extracts were prepared 40 hr after transfection Portions of cell lysates were assayed for β-galactosidase activity from the cotransfected pRSV-β-gal plasmid. All CAT assays (Gorman et al, 1982) were normalized to the internal transfection efficiency control. The CAT assay monitors acetylation of [ 14 C] chloramphenicol in cell extracts, [ 14 C]-chloramphenicol and its products are separated by thin-layer chromatography (TLC) and visualized by autoradiography. Individual spots on TLC paper were cut, their radioactivities were assayed by liquid scintillation spectrometry, and the relative CAT activities were calculated accordingly. Each experiment has been reproducibly repeated at least three times and a representative of several studies is shown e Immunoblot SK-BR-3 cell lysates were made 40 hr after transfection and immunoblots were performed as described (Matin et al. , 1984) The mAB-3 monoclonal antibody against the human new gene product — pi 85 protein — was purchased from Oncogene Science 2. RESULTS a Transcriptional Repression of new by the Adenovirus 5(AD5) El A Products A DNA segment of 2.2 kb containing the new promoter and upstream sequences was fused with the CAT expression vector to generate the pnewEcoRl-CAT plasmid In transient-expression assays using Rat-1 cells (FIG. IA), a cotransfection of pnewEcoRl-CAT with pEl A, a plasmid expressing the El A gene, led to a significant decrease of CAT activity Cotransfection with pSP64, a plasmid vector, had no effect on CAT activity To rule out the possibility that decreased transcription from new promoters could be due to the titration of cellular transcription factors by the cotransfected EIA promoter, a deletion mutant, pEl Apr, which contains only the EIA promoter, was cotransfected with pnewEcoRl-CAT No effect on CAT activity was observed A reporter plasmid containing the CAT gene under the control ofthe RSV LTR was not EIA responsive, indicating that decreased CAT expression was not due to a general decrease of transcription by El A In parallel studies, stimulation of transcription from the E2A transcription unit by the EIA products was assayed by cotransfecting pElA and pE2A-CAT (CAT gene driven by E2 early promoter) The results showed that repression of new and transactivation of E2A promoter occur in the same range of pEl A concentration To see if other adenovirus early genes can repress the new promoter, plasmids expressing the early genes of adenovirus individually were cotransfected with pnewEcoRl-CAT (FIG IB) No change in CAT activity was observed with ElB, E2, or E3 alone, indicating the among these early genes of adenovirus, only the EIA gene could function as a repressor ofthe new promoter b Repression of new Is El A Concentration Dependent and Requires the El A Conserved Region 2 To further study the interactions of EIA genes products with the new promoter, increasing amounts of pElA were cotransfected with pnewEcoRl-CAT in ratios of 1 1, 2 1, 3 1, and 4 1 (FIG 2A) Inhibition ofthe gene expression directed by the new promoter was found to be dependent on pEl A concentration, and 50% repression could be observed at as low as a 1 1 ratio of pElA pnewEcoRl-CAT The Ad5 EIA gene produces two major spliced products, the 12S and 13S mRNAs, that encode proteins 243 and 289 amino acids long, respectively (Moran et al, 1987) To determine which EIA gene product was responsible for the observed repression, the same studies were performed with recombinant plasmids expressing either 12S or 13S EIA gene product (pElA-12S and pEl A-13S) As shown in FIG 2B and C, both the 12S and 13S products were effective at repressing new transcription in a concentration-dependent manner The EIA gene products contain three highly conserved regions, CRI, CR2, and CR3 (Moran et al, 1987, Van Dam et al, 1989) CRI and CR2 exist in the 12S and 13S, whereas CR3 is unique to the 13S product Since 12S itself can repress new efficiently, the inventors reasoned that the CR3 is dispensable for transcriptional repression of new by EIA To further localize whether the CRI or the CR2 in the El A protein was required for efficient repression of new, parallel studies were performed using deletion mutants pElAdl343 and pElAd!346 (Hearing et al, 1985). The pElAdl343 mutant contains a 2-bp deletion in the El A coding sequence, resulting in a frame shift in all three conserved regions ofthe El A products and leaving only the N-terminal 40 amino acids intact. No effect on CAT activity was observed when pEl Ad 1343 mutant was cotransfected with pnewEcoRl-CAT The pElAd!346 mutant containing an in-frame deletion, which removed 16 amino acids within the CR2 but reserved the CRI, failed to express new transcription (FIG. 2D). The inventors concluded that the CR2 of EIA gene products is required for efficient transcriptional repression of new (FIG. 2E) c. Localization of Target DNA Element in the new Promoter Responding to EIA Repression. To localize the DNA element in the new promoter that mediates the transcriptional repression by the EIA products, a series of 5' deletion constructs containing portions of the new promoter linked to a functional CAT gene were cotransfected with pElA into Rat-1 cells (FIG. 3 A) The transient expression ofthe CAT gene driven by each of these promoter fragments after transfection with control plasmid vector pSP64 or with pEl A in a ratio of 1.2 is shown in FIG. 3B Only the pnewXhoI-CAT containing the smallest promoter fragment was not repressed by El A Clearly the activity of a site withm the Stu l-Xho I restriction fragment is sensitive to EIA repression This Stu l-Xho I fragment is sensitive to El A repression This Stu I- Xho I fragment is located between -198 and -59 with respect to the transcriptional start site of new The inventors concluded that the target DNA element responding to EIA repression resides inside this 139-bp Stu l-Xho I fragment d Evidence for the Involvement of Trans- Acting Factor(s) To examine whether this repression by the El A products is a trans-acting process, the inventors attempted to remove the repression by cotransfecting a third recombinant, pSP64/Stu-Xho, containing only the Stu l-Xho I restriction fragment cloned in pSP64 Increasing amounts of pSP64/Stu-Xho, in cotransfections in which transcription of pnewEcoRl-CAT was repressed by pElA, relieved the repression of new transcription in a concentration-dependent manner (FIG 4A) In contrast, no derepression was observed when pSP64/RI-Xba containing the EcoRl-XBA I restriction fragment cloned in pSP64 was cotransfected The derepression was effective at a 4 1 ratio of pSP64/Stu-Xho pnewEcoRl-CAT (FIG 4 A, lane 6), indicating that the Stw I-.A7JO I fragment can efficiently compete with the new promoter for the transcription factor(s) involved in the repression of new by El A These results confirm that the target for the El A repression in the neu promoter is a cis DNA element within the Stw l-Xho I fragment of this promoter Furthermore, this repression of transcription may involve an interaction between the DNA element and either the El A products or some cellular transcription factors(s) interacting with or induced by the EIA products e Repression of Human new Expression in SK-BR-3 Cells Comparison ofthe Stw l-Xho I fragment of rat new promoter sequence with its counterpart sequence in human new promoter (Tal et al , 1982) reveals >86% homology It was suspected by the inventors that the human neu gene might also be repressed by EIA at transcriptional level by way of similar mechanisms If this is the case, cotransfection ofthe Stw l-Xho I fragment of rat new promoter might be able to relieve the repression of human new incurred by El A To test this possibility, cotransfection studies were carried out by using as recipient cells human breast cancer cell line Sk-Br-3, which is known to overexpress human neu mRNA and pi 85 proteins (Kraus et al, 1987) Immunoblotting studies with SK-BR-3 cell lysates showed that the expression of human new gene products, the pi 85 protein, was reduced by introduction of EIA (FIG 4B, compare lane 1 with lane 4) Cotransfection of pSP64/Rl-Xba plasmids with pElA at a 4 1 ratio was ineffective in removing the repression of pi 85 expression by El A, whereas cotransfection of pSP64/Stu-Xho with pEl A at the same ratio relieved the repression by El A It is known that the maximum efficiency of transient transfection can reach only 50%) (Chen et l, 1988), the other 50% of nontransfected Sk-Br-3 cells should still produce high levels of pi 85 proteins, which can result in high background in the EiA- mediated repression of pi 85 Therefore, the repression effect on the endogenous neu- encoded pi 85 by transiently transfected EIA in the immunoblotting assay was not as dramatic as that observed in CAT assays However, the small difference was detected reproducibly The best interpretation ofthe results is that EIA can repress human new promoter at transcriptional level by targeting at the cis-acting DNA element in human new promoter corresponding to the Stw l-Xho I fragment of rat new promoter f The Sequence TGGAATG is an Important Site for the ElA-Mediated Repression EIA has been reported to repress enhancer mediated transcription activation of simian virus 40 (Borrell et al, 1984), polyomavirus (Velcich et al, 1986), immunoglobulin heavy chain (Hen et al, 1985), and insulin genes (Stein et al, 1987) Comparison ofthe enhancer sequences of these genes reveals a consensus sequence which is likely to be the core sequence ofthe El A-responding element AAA (G)TGGTTT(G) However, there has been no experimental evidence to support this notion A sequence, TGGAATG, that matches the consensus sequence has been fund in the Stw \-Xho 1 ElA-responding element ofthe rat new promoter An identical sequence also exists in the corresponding region ofthe human new promoter (Tal et al, 1987). It is therefore conceivable that the sequence TGGAATG may be an important target sequence for the El A-induced repression To investigate this possibility, a 20-mer oligonucleotide from the rat new promoter containing the sequence TGGAATG was synthesized (FIG 5) This oligonucleotide efficiently competed with the new promoter for the transcriptional factors(s) involved in the repression of new by El A, resulting in a derepression effect (FIG 5, lane 2), whereas a 22-mer random nonhomologous oligonucleotide had no derepression effect (FIG 5, lane 3) These data provide experimental evidence that the 20-mer oligonucleotide harbors a critical sequence required for the El A-induced inhibition Since the sequence TGGAATG within this 20-mer oligonucleotide resembles the consensus sequence in the enhancer sequences of other genes that can be repressed by El A, it is likely that this 7-bp sequence is the critical sequence that is mediating the El A effect 3. DISCUSSION The foregoing results show that in a cotransfection system, the EIA gene products repressed the new expression at the transcriptional level It is further demonstrated that the repressive effect on new expression is lost in El A products when part ofthe CR2 (amino acids 120-136) is deleted Notably, a structure motif in this deleted part ofthe adenoviral EIA CR2 region is shared among the papovaviral large tumor antigens, the v- and c-myc oncoproteins, the E7 transforming proteins of human papilloma viruses, and the yeast mitotic regulator DCD25 gene product (Figge et al, 1988) This region encoding the shared motif is also required by EIA, simian virus 40 large tumor antigen, and human papilloma viruses 16 E7 for their specific binding to the human retinoblastoma gene product, RB protein (Whyte et al, 1988; Whyte et al, 1989). These studies further elucidate the oligonucleotide sequence mediating El A- induced repression in the upstream region of new promoter. The sequence TGGAATG is perfectly conserved between rat and human new promoter, which is indicative of functional importance. In addition, this sequence matches the consensus sequence of other genes that can also be repressed by El A at transcriptional level. Taken together, these findings suggest that there may be common mechanisms involved in this type of El A- mediated repression. It has been proposed that EIA may form a complex with cellular transcription factor(s) and thereby modulate the specific binding ofthe transcription factor(s) to enhancer elements that are important for transcription (Mitchell et al, 1989). Identification ofthe defined DNA sequences responsible for the El A-mediated inhibition of new transcription will allow us to identify the transcription factor(s) involved in this process. The new protooncogene is notably amplified in patients with metastatic breast cancer. Expression of the EIA gene can inhibit experimental metastasis of ras oncogene-transformed rat embryo cells. Here, it is shown that new transcription can be repressed by El A products in an established rat embryo fibroblast cell line, Rat-1 Furthermore, the inventors have found that in SK-BR-3 human breast cancer cells expression of the pi 85 protein, the human new gene product, was reduced by introduction of El A gene. The derepression effect observed in the cotransfection experiment with the Stu \-Xho 1 fragment has demonstrated that this reduction of pi 85 proteins is likely due to the similar transcriptional repression mechanisms. EXAMPLE II Adenovirus-5 EIA Gene Products Act as a Transformation Suppressor of Neu Oncogene In Example I, transcription ofthe new protooncogene was shown to be strongly repressed by adenovirus-5 EIA gene products through the use of a transient transfection assay In the present Example, the E 1 A gene has been stably introduced into the new-transformed B 104-1-1 cells, to demonstrate that El A-mediated new repression can suppress new-mediated transforming activity In these studies, cells that expressed EIA products possessed reduced transforming and tumorigenic activity, as evidenced using standard assays for each These results demonstrated that EIA gene products can act negatively to suppress the transforming phenotype ofthe new oncogene, and is believed to be the first example of a gene, i e , the El A gene, that can act in one setting as a transforming oncogene, and in another as a transforming suppressor gene The B 104-1-1 cell line, an NIH3T3 transfectant that has approximately 10-20 copies of mutation-activated genomic new oncogene has been shown to be highly transforming and tumorigenic (Bargmann et al, 1986, Stern et al, 1986) For the present studies, B 104- 1-1 cells and control NIH3T3 cells were transfected with either EIA plasmids expressing adenovirus-5 EIA gene, (pElA), or a derivative plasmid containing only the El A promoter without the El A coding sequence (pEl Apr) Cells were cotransfected with pSV2neo plasmids carrying a neomycin resistant marker gene (Southern et al, 1982) The transfections were carried out with the modified calcium phosphate precipitation procedure of Chen and Okayama (1988) In each transfection, 5X10 5 B 104-1-1 cells or NIH3T3 cells (2 X 10 cm dishes) were seeded 24 h before transfection The cells were transfected with either 10 μg ofthe El A expressing pElA plasmid DNA or its derivative pEl Apr plasmid DNA, along with 1 μg of pSV2-neo plasmid DNA (Southern et al, 1982) Approximately 14 h post-transfection, cells were washed and cultured in fresh medium for 24 h and split at a 1 10 ratio. The cells were then grown in selection medium containing 500 μg/ml of G418 for 2-3 weeks and individual G418 resistant colonies were cloned using cloning rings and expanded to mass culture Three kinds of stable transfectants were thus established ( 1 ) B-E 1 A transfectants: B 104-1-1 transfectants harboring the El A gene, (2) B-El Apr transfectants: B 104-1-1 transfectants containing EIA promoter sequence, which is used as a control cell line in this study, and (3) N-El A transfectants NEH3T3 cells transfected with the E 1 A gene Cells cultures were performed as described previously (Hung et al, 1989; Matin et al, 1989). The B104-1-1 cell line and NIH3T3 cell line were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%> calf serum in a humidified atmosphere at 5% CO 2 at 37°C The B-El A transfectants and N-El A transfectants were grown under the same condition with addition of G418 (500 μg/ml) into the culture media. FIG 6 shows the molecular characterization ofthe representative stable transfectants used in this study, employing both Southern blot and immunoblot analyses Southern blot analyses were performed essentially by published techniques as previously described (Zhang et al, 1989) Genomic DNAs extracted from cultured cells were digested overnight at 37°C with a 2-fold excess of a restriction endonuclease (either EcoRI, Sstl, or BamHI). Ten μg of each sample were then resolved by electrophoresis on a 1% agarose gel and transferred to Nytran membrane (Schleicher & Schuell, Keen, NH) using a 10 x SSC (1 5 m NaCl, 0.15 M sodium citrate) The blotted DNA were hybridized under high stringent conditions (68°C) with [ 32 P] radioactive probe (1-5 x IO 8 CPM μg "1 ) labeled by using Random Primed DNA Labeling Kit (Boehringer Mannheim Biochemicals, Indianapolis, IN) The blots were washed twice for 15 min each in 2 x SSC, 0 1%> SDS at room temperature, and then twice for 30 min each in 0 1 x SSC, 0 1% SDS at 68°C with constant agitation The filters were dried at room temperature and then exposed to Kodak X-OMAT™ AR film at -80°C for 1 to 3 days Immunoblot analysis were performed basically by published techniques (Towbin et al, 1979) as previously described (Matin et al, 1990) Confluent cells growing in 10 cm plates were lysed with RIP A-B buffer (20 mM sodium phosphate, pH 7 4, 150 mM NaCl, 5mM EDTA, 1% Triton, 10 μg/ml Aprotinin, 2mM PMSF, 10 μg/ml Leupeptin and 4mM iodoacetic acid) and then centrifuged at 10 x g for 20 min at 4°C The protein concentration ofthe supernatants was determined by Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA) 50μg of each sample were subjected to SDS polyacrylamide gel electrophoresis (10%) and transferred to nitrocellulose The nitrocellulose filters were treated with 3% nonfat dry milk in TPBS buffer (0 05% Tween-20, 138 mM NaCl, 2 7 mM KCl, 4 3 mM Na2HPO4 7H 2 O and 1 4 mM KH 2 PO4) for 1 h at room temperature, followed by an overnight incubation at 4°C with primary monoclonal antibodies M73 against the El A proteins or mAb-3 against the new encoded pi 85 protein (purchased from Oncogene Science Ine , Manhasset, NY ) After three 10 min washes with TPBS buffer, the nitrocellulose was then incubated for 1 h at room temperature with 1 1000 dilution of horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Bio-Rad Laboratories) The nitrocellulose filters were washed 3 times in TPBS buffer and were subjected to color developing reaction with horseradish peroxidase substrate (Kirkegaard & Perry Laboratories, Ine , Gaithersburg, MD) To assure that the exogenous EIA gene or El A promoter DNA had integrated into the genome ofthe transfectants, DNA blot analysis with the El A probe was performed and the results confirmed the integration of transfected foreign DNA (FIG 6A) Noticeably, the three B-E 1 A transfectants studied (B-E 1 A- 1 , B-E 1 A-2 and B- El A-3) acquired different copy numbers ofthe EIA gene Immunoblot detection of E1A further confirmed that the B-El A and N-El A transfectants actually produced EIA proteins and the El A protein levels in these transfectants are lower than that in the 293 cell line, an established cell line of primary human embryonal kidney transformed by adenovirus DNA (FIG 6B) To examine if expression of EIA can inhibit new expression, immunoblot analysis for the new-encoded pi 85 protein was also performed and the pi 85 proteins were virtually undetectable in all the transfectants using horse radish peroxidase detection method (FIG 6C) However, slightly higher levels of pi 85 proteins could be detected in B-El A- 3 than those in B-El A- 1 and B-El A- 2 when the more sensitive 125 I-protein-A detection method was used Since pi 85 proteins were barely detectable in B-El A transfectants, DNA blot analysis for rat new gene was conducted to make sure that the new gene was not lost As shown in FIG 6D, the incorporation of El A gene into the genome did not alter the new gene at the DNA level Among the three B-El A transfectants, B-El A-2 and B-El A-3 had levels ofthe neu gene that were comparable to those ofthe parental B 104-1-1 cell line, while B- El A-l appeared to have a lower level new gene This may be due to partial loss ofthe new gene in this line during the establishment of this transfected cell line The three B- EIA transfectants shown in FIG 6 were chosen for further transformation assay because they represented three different subtypes of B-El A transfectants (1) B-El A- 1 had fewer copies of new gene compared to B 104-1-1 and more copies of El A gene, (2) B-El A-2 retained the same level of new as B 104-1-1 and high levels of EIA gene, (3) B-E1A-3 contained the same amount of new as B 104- 1-1, but a low quantity ofthe EIA gene The transforming phenotype ofthe new-transformed cells usually includes a transformed morphology, non-contact-inhibited growth pattern, increased DNA synthesis rate, anchorage-independent growth and the ability to induce tumors in nu/nu mice To determine the effect of EIA expression on the transforming ability of new- transformed B 104- 1 - 1 cells, the B-E 1 A transfectants as well as the control cell lines were assayed for all the above mentioned transforming parameters using standard protocols The results of these studies demonstrated that the highly transformed morphology of B 104- 1 - 1 cells was essentially unchanged after pE 1 Apr transfection but was markedly altered by pElA transfection (FIG 7) The B-El A transfectants exhibit non-transformed flattened morphology and a contact-inhibited growth pattern (FIG 7) Expression of El A proteins in NIH3T3 cells did not significantly alter the monolayered morphology The results indicated that EIA gene products could specifically reverse the transforming morphology ofthe new-transformed cells DNA synthesis was also studied as a measure of cell growth, to determine whether the B-El A transfectants were actively synthesizing DNA as compared to controls These studies were conducted through the use of a [ 3 H] -thymidine incorporation assay For these studies, cells were plated in ten replica into 96 well plates at a density of 9 x 10 3 cells/well and cultured in DMEM supplemented with 10% calf serum [ H] -thymidine (lμCl) was added to each well at time points of 16, 40 and 64 h and continuously incubated at 37°C for 2 h Cells were then harvested and cellular DNA were bound to glass fiber filters Radioactivities of individual samples were counted by Scintillation counter Average cpm were calculated from ten replicate samples The rate of DNA synthesis, as indicated by [ 3 H] -thymidine incorporation, was different among the three B-E 1 A transfectants (FIG 8 A) B-E 1 A- 1 and B-E 1 A-2 displayed a much lower DNA synthesis rate, which coincided with their slower cell growth rate compared to B 104- 1 - 1 cells This E 1 A-induced decrease in [ 3 H]-thymidine incorporation was not as dramatic in the B-El A- 3 cell line possibly due to the lower level ofthe EIA proteins These data suggested that EIA proteins can inhibit the effect ofthe new oncogene on DNA synthesis and cell growth To test the influence ofthe EIA proteins on anchorage-independent growth, B 104-1-1 cells and the B-El A transfectants were assayed for their ability to grow in soft agar. The ability of B 104-1- cells, B-El A transfectants, NTH3T3 cells and N-El A transfectant to grow in soft agarose was determined as described previously (Matin et al, 1990). Cells (1 x 10 3 cells/plate) were plated in a 24 well plate in DMEM containing 10%> calf serum and 0.35%> agarose (BRL, Gaithersburg, MD) over a 0.7% agarose lower layer. The cells were incubated at 37°C for 3 weeks and the plates were stained with p-iodonitrotetrazolium violet (1 mg/ml) for 24 h at 37°C and colonies were counted. The results ofthe soft agar studies demonstrated that colony formation by the El A transfectants were strikingly reduced compared to that of B 104-1-1 and B-El A pr transfectants (FIG. 8B). It is noteworthy that the colony formation by NIH3T3 and N-El A- 1 lines did not vary significantly. The most stringent experimental test for neoplastic behavior is the ability of injected cells to form tumors in nude mice. Studies in nude mice were conducted because the examination of El A repression of new-mediated tumorigenicity in vivo was considered to be a critical test of EIA effectiveness. For conducting tumorigenicity studies, the B 104- 1 - 1 cells, B-E 1 A transfectants, NIH3T3 cells and N-E 1 A transfectant in log-phase growth were trypsinized and washed twice with phosphate buffered saline and centrifuged at 250 x g. The viable cells were then counted, and 1 x 10 5 cells in 0.1 ml of phosphate buffered saline were injected subcutaneously into both the right and left flanks of 5 to 6- week old female homozygous nu/nu (nude) mice (Harlan Sprague Dawley Co.) under sterile conditions. Tumor formation was scored at indicated days as presence or absence of a visible tumor mass. Sixteen days after injection, tumor volumes were estimated as the product of three-dimensional caliper measurements (longest surface length and width and tumor thickness). The growth of tumors was monitored for a minimum of 16 days and maximum of 2 months. When cells ofthe parental B104-1 -1 line were injected subcutaneously in nude mice, solid tumors developed by 8 days after injection, however, the same quantity of the El A transfectants did not form tumors in nude mice until 12-26 days after injection and in every case the tumors were much smaller than those from B 104-1-1 cells (FIG 9A) Although the B-E1A-1 and B-El A-2 transfectants contained comparable amounts ofthe EIA gene, the B-E1A-1 cells did not cause tumor development until a much later time This is probably due to the lower level of new gene in this line On the other hand, although both ofthe B-El A-2 and B-El A-3 transfectants contained the same level ofthe new gene as B 104-1-1, the transforming suppression effect on B- E1A-3 was not as strong as on B-E1A-2 This was likely due to the lower level of the EIA gene in B-El A- 3 Typical results of El A expression on new oncogene induced tumorigenicity are shown in the photographs in FIG s 9B and 1 1 A Evaluated 18 days after injection, animals injected with B 104-1-1 cells were found to bear huge tumors, whereas those injected with B-El A-2 transfected cells had considerably smaller tumor nodules. As expected, control animals injected with NIH3T3 cells showed no evidence of tumor formation. Previous studies of Wilms' tumor cells and human prostate carcinoma DU145 cells demonstrated that reintroduction of chromosome 1 1 to Wilm's tumor cells or restoration of RB gene to DU145 cells suppressed tumor formation but did not alter the cell morphology, growth rate or colony-forming ability (Weissman et al, 1987, Bookstine et al, 1990) These data suggest that growth rate in culture and tumorigenicity in nude mice are separable phenomena In the present study, the B- El A-l and B-E1A-2 cells exhibited slower growth rate and much weaker tumorigenic activity However, suppression of tumorigenicity cannot entirely be explained by their slower growth rate and decreased [ 3 H] -thymidine incorporation For example, the B- E1A-3 cells possessed similar [ 3 H]-thymidine incorporation and cell growth rate as B104-1-1 cells, while their tumorigenic activity was markedly suppressed as well Taken together, these results clearly demonstrate that introduction ofthe EIA gene into B 104-1-1 cells suppresses all the transforming properties ofthe new-transformed cells EXAMPLE IH Suppression of Neκ-Mediated Metastasis by EIA Gene Products Additional studies were conducted using B-El A transfectants of B 104-1-1 to demonstrate that EIA products also suppress new-mediated metastasis These studies employed B-El A transfectants (B-El A- 1 through B-El A-5) as well as the negative and positive controls, NIH/3T3 and B 104-1-1, respectively, in a cell motility, m vitro invasion and an experimental metastasis assay The metastasis studies were performed essentially as described by Wexler, 1966 Briefly, six- week-old pathogen-free female nude mice (Harland) were quarantined for 1 week and then used in the studies Seven to ten mice/experimental group were inoculated with lxl 0 5 cells/0 1 ml in PBS via the lateral tail vein at day 0 Each cell line was then assessed at two different passage numbers Mice were sacrificed at 21 days following injection and the number of lung metastases were determined by infiltration with India ink. Only those lung nodules >1 mm in diameter were counted On further examination, no extrapulmonary metastases were found Representative photographs illustrating the gross appearance ofthe lungs from these animals are shown in FIG 1 IB, whilst the quantitative data from these studies are detailed below in Table 2 TABLE 2. EXPERIMENTAL METASTASIS ASSAY Experimental metastasis Cell Line Transfected gene Frequency No of lung nodules (mean±SE) 2NIH/3T3 - 0/9 O O±O O B104-1-1 new 9/910 9±10 3 N-El A EIA 0/8 O O±O O B-neo new+ElA 7/7 9 5±7 9 B-E1A-1 new+ElA 0/8 O O±O O B-E1A-2 new+ElA 3/9 0 8±0 4 B-E1A-3 new+ElA 0/8 O O±O O B-E1A-4 new+ElA 1/7 0 1±0 4 B-E1A-5 neu+ElA 1/10 0 1±0 4 The effectiveness of El A at inhibiting new-mediated metastasis is clearly illustrated in FIG 1 IB Furthermore, this single result was found to be representative ofthe entire study None ofthe negative control mice, NIH/3T3 and EIA transfected NIH/3T3 (N-El A), exhibited metastatic lung nodules However, all ofthe positive controls (B 104-1-1 and B-neo), exhibited metastatic nodules, at a mean frequency of about 10 nodules In contrast, all o the experimental lines (B-El A- 1 through B-El A- 5) exhibited a reduced metastatic potential, with a frequency ranging from one to three (out often and nine, respectively), and a mean number of 0 1 to 0 8 nodules in those animals that were positive Note that two ofthe experimental lines, B-E1A-1 and B- El A-3, were totally free of metastases An increase in cell motility has been shown to correlate with a higher metastatic potential Therefore, a motility assay, which measures the migration ofthe tested cell to a chemo-attractant, fibronectin or hepatic sinusoidal endothelial cell conditioned media, was performed As shown in FIG 10A, all ofthe B-El A transfectants showed decreased migration rate to different chemoattractants than that of B-neo cell line, which are B 104-1-1 cells transfected with neomycin-resistant (neo r ) gene alone The N-El A cells also had a low migration rate which is comparable to that of N1H3T3 cells Another step in the metastatic process involves invasion of tissues and basement membranes In vitro invasion assays also revealed significant differences between the B-neo cells and the B-El A cell lines B-neo cells demonstrated a high rate of invasion similar to that of B 104-1-1 cells, while the B-El A transfectants failed to invade the Matrigel Injection ofthe B-neo cells and the five B-El A cell lines into the tail vein ofthe nude mice showed dramatic differences in the frequencies and number of lung nodules (FIG 10B and Table 2) Two ofthe five B-El A transfectants did not give rise to any experimental metastatic tumors and the other three B-El A lines had a very low incidence of experimental metastasis compared to that of B-neo cells (p >0 01 ) As expected, N-E 1 A cells were unable to produce any metastatic lung nodule From these results, it is evident that EIA gene products can reduce the metastatic potential of new-transformed 3T3 cells, possible by transcriptional repression of new gene expression. These results, typified by those shown in FIG 1 IB, demonstrate that EIA gene products are able to suppress not only the tumorigenic and transformation events mediated by the new gene (Example II), but are further able to suppress metastatic events that are new mediated EXAMPLE IV EIA Suppresses c-erbB-2/neu Expression Connected with Severe Malignancies in Human Ovarian Carcinoma The present example is directed to studies concerning the action of El A in repressing c-erbB-2/neu overexpression in SKOV3 ipl cells and the functions of EIA as a tumor suppressor gene in c-er£B-2/new-overexpressing human cancer cells 1 Inhibited Expression of c-er B-2/new-encoded pi 85 in ElA-expressing Ovarian Carcinoma Transfectants The EIA -expressing plasmid was cotransfected into SKOV3 ipl cells together with the pSV2-neo plasmid carrying the neomycin-resistance marker gene, thus generating the EIA -expressing ovarian carcinoma stable transfectants The G418- resistant clones were selected and expanded into cell lines, which were designated ipl EIA cell lines The same approach was used to select control cell lines, in which the pElAdl343 plasmids containing a 2-base pair frameshift deletion in the El A coding sequence and producing nonfunctional protein products were introduced into the SKOV3 ipl cells to generate the ipl Efs cell line It was possible that some ofthe stable transfectants selected by this cotransfection strategy only harbored the neomycin resistance gene but not the EIA gene Therefore, to identify those ipl EIA transfectants that integrated the EIA gene and actually produced EIA proteins, immunoblot analysis with anti-El A antibodies was performed (FIG 12 A) Two ofthe ipl EIA transfectants expressed multiple species of El A proteins as described by Harlow et al, (1985), whereas the control ipl Efs cell line, as expected, did not express EIA proteins In this manner, the inventors thus established two kinds of stable transfectants (a) ipl El A cells (i e , SKOV3 ipl ElA-expressing transfectants), which were used to test the tumor-suppressing function of EIA, and (b) ipl Efs cells (i e , SKOV3 ipl transfectants containing EIA frameshift mutants), which were used as a control cell line to make sure that the changes in transformation phenotypes (if any) in ipl EIA transfectants were not due to the selection process or to transfection ofthe plasmids and the pSV2-neo gene As shown herein, EIA proteins can repress c-erέB-2/new-encoded pi 85 expression in the c-erbB-2/neu oncogene-transformed N1H3T3 cells In addition, it is also shown herein that EIA proteins can decrease the c-erbB-2/neu mRNA level as well as c-er/>B-2/new-encoded pi 85 in c-erόB-2/new-overexpressing breast cancer cell lines To determine if the expression of EIA in ipl El A transfectants can inhibit pi 85 expression, immunoblot analysis of c-er^B-2/new-encoded pi 85 protein was performed It was found that pi 85 protein levels were dramatically decreased in both the ipl ElAl and ipl E1A2 cell lines versus the control ipl . Efs cell line (FIG 12B), which expressed an amount of c-e/ * £B-2/new-encoded pi 85 protein comparable to that ofthe parental SKOV3.ipl cell line Since pi 85 proteins were dramatically reduced in ipl EIA transfectants, DNA blot analysis ofthe c-erbB-2/neu gene was conducted to ensure that the reduction in c-er£B-2/new-encoded pi 85 protein level was not due to loss of he c-erbB-2/neu gene As shown in FIG 12C, both the ipl ElAl and ipl E1A2 cell lines contained copy numbers ofthe c-erbB-2/neu gene similar to that of ipl Efs cell line Therefore, the incorporation ofthe EIA gene into the genome of SKOV3 ipl cells did not alter the c-erbB-2/neu gene at the DNA level Furthermore, these results indicate that the EIA can repress the c-erftB-2/new-encoded pi 85 protein expression in ipl El A transfectants 2 In Vitro Suppression of SKOV3 ipl Cell Transformation bv EIA Expression Once the EIA -expressing ipl EIA lines were established, the inventors examined the effect of EIA expression on the c-erόB-2/new-overexpressing ovarian cancer cells in vitro, assessing growth properties, DNA synthesis rate, and colony formation in soft agar The growth curves of the EIA -expressing ip 1. E 1 A 1 and ipl El A2 cell lines and control ipl Efs cell line indicated that EIA expression slightly reduced the growth rate of these ovarian cancer cells versus the control cells (FIG. 13 A) Measurement of the DNA synthesis rate by [ 3 H]thymidine incoφoration assays revealed that the control ipl Efs cells had a high level of [ 3 H]thymidine incorporation that was similar to that of SKOV3.ipl cells and significantly higher than the [ 3 H]thymidine incorporation in the EIA -expressing ipl . ElAl and ipl .ElA2 cell lines (FIG 13B) FIG 13C shows that the c-er >B-2/new-overexpressing ipl.Efs cells exhibited high efficiency in forming soft agar colonies, whereas the colony-forming efficiencies of the two ip 1 E 1 A transfectants were strikingly reduced These data suggested that EI proteins can suppress the effect ofthe c-erZ>B-2/new-overexpression in ovarian cancer cells and inhibit cell growth, DNA synthesis, and anchorage-independent growth 3 EIA as a Tumor Suppressor Gene for c-er B-2/new-Overexpressing Human Ovarian Carcinoma SKOV3.ipl Cells A critical test for EIA -mediated transformation suppression function in ovarian cancer cells is the ability of EIA to suppress tumor formation in vivo Therefore, tumorigenicity assays were performed in mice that were injected s.o. with 3 x 10 5 cells from either the EIA -expressing ipl El Al and ipl El A2 cell lines or the control ipl Efs cell line (FIG 14A). Like mice given injections ofthe parental SKOV3 ipl cells, mice given injections ofthe control ipl .Efs cells formed tumors 7 days after injection and had huge tumor burdens of 3280 ± 1310 mm 3 by 80 days postinjection However, nu/nu mice given injections ofthe same number of ipl .ElAl transfectants did not form tumors until 21-30 days after injection, and their tumor burdens were only 460 ± 170 mm 3 by 80 days postinjection. The tumor-suppressing function of EIA was more dramatic in mice given injections ofthe ipl E1A2 transfectants, which did not induce tumors until 40-50 days postinjection, and 2 of 6 mice did not develop any tumor, even at 160 days postinjection The tumor size in the four mice given injections of ipl El A2 were 290 ± 220 mm 3 at 160 days postinjection Therefore, these results clearly demonstrated that El A can suppress the tumorigenic potential ofthe ovarian carcinoma SKOV3 ip 1 cells It is shown above that SKOV3 ipl cells, when compared to SK-OV-3 cells, induced a higher mortality rate and shorter survival following i p injection into nu/nu mice To determine whether EIA expression in SKOV3 ipl cells could counteract the effect of c-erbB-2/neu overexpression and reduce the mortality rate, the inventors gave mice i p injections ofthe EIA -expressing ipl ElAl and ipl El A2 cell lines and the control ipl Efs cell line Mice given injections of 1 x IO 4 ipl Efs cells developed tumor symptoms similar to those described in the previous section, one ofthe mice died of tumor as early as 19 days postinjection, and all ofthe other mice died within 75 days postinjection (FIG 14 A) However, there was a significant increase in survival for mice given injections ofthe EIA -transfected lines versus the parent SKOV3 ipl and frameshift-transfectant ipl Efs cell line (P < 0 01) (FIG 14B) The results indicated that EIA expression can reduce the mortality of mice given injections of c-erbB-2/neu- overexpressing human ovarian carcinoma cells 4 Discussion The inventors have isolated a derivative cell line termed SKOV3 ipl from the ascites that developed in mice given injections of human ovarian carcinoma SK-OV-3 cells Compared with parental SK-OV-3 cells, the SKOV3 ipl cell line expresses higher levels of c-er >B-2/new-encoded pi 85 protein and corresponding exhibits more malignant phenotypes determined by in vitro and in vivo assays This association between enhanced c-erbB-2/neu expression and more severe malignancy is very consistent with previous studies in which c-erbB-2/neu overexpression was shown to correlate with poor prognosis in ovarian cancer patients (Slamon et al, 1 89) The inventors data provided actual evidence to support those clinical studies that c-erbB-2/neu overexpression can be used as a prognostic factor for ovarian cancer patients and that c-erbB-2/neu overexpression may play an important role in the pathogenesis of certain human malignancies such as ovarian cancer Although not important to the utility ofthe claimed invention, it will be interesting to further study the molecular mechanisms and biochemical pathways involved in c-erbB-2/neu overexpression and the associated malignant phenotype The recent identification and molecular cloning ofthe ligands for the c-er&B-2/new-encoded pl85, which can increase the tyrosine phosphorylation of pi 85, will enable future direct examination of the molecular mechanisms and the biological effects of c-erbB-2/neu overexpression in human cancer and cancer metastasis (Peles et al, 1992, Holmes et al, 1992, Lupe et al, 1990, Yarden & Peles, 1991 , Huang & Huang, 1992, Dobashi et al, 1991) The adenovirus EIA gene was originally defined as a transforming oncogene that can substitute for the myo oncogene and simian virus 40 large tumor antigen gene in the ras cotransformation assay of primary embryo fibroblasts (Land et al, 1983, Ruley, 1983, Weinberg, 1985) As detailed herein, the inventors have found that EIA products can act as transformation and metastasis suppressors in the mutation- activated rat new-transformed mouse 3T3 cells In this particular example, it is further demonstrated that the EIA gene products effectively repressed c-erbB-2/neu gene expression in SKOV3 ipl ovarian carcinoma cells, suppressed transformation phenotypes in vitro, and reduced tumorigenicity and mortality rate in vivo These results indicate that the adenovirus EIA gene can function as a tumor suppressor gene for c-erόB-2/new-over expressing human cancer cells as well as inhibit transformation induced by mutation-activated new oncogene in rodent cells Since the inventors have previously demonstrated that EIA products can dramatically inhibit the c-erbB-2/neu mRNA level and c-erόB-2/new-encoded pi 85 expression in human breast cancer cell lines, and have shown that the EIA gene products can repress new gene expression at the transcriptional level by targeting at a specific DNA element in the new gene promoter, it is likely that the reduced pi 85 expression in the ipl EIA cell lines is due to transcriptional repression ofthe overexpressed c-erbB-2/neu gene, which may be one ofthe diverse molecular mechanisms that account for the tumor suppressor function of EIA in SKOV3 ipl ovarian cancer cells Interestingly, it has been shown that adenovirus EIA can render hamster cell lines more susceptible to lysis by natural killer cells and macrophages (Cook & Lewis, 1984, Sawada et al, 1985) increased sensitivity to cytotoxicity by tumor necrosis factor in transfected NIH3T3 cells (Cook et al, 1989) Therefore, it is conceivable that the tumor-suppressing function of EIA may be partly due to an increased susceptibility to cytolytic lymphoid cells and molecules Recently, E 1 A protein was shown to induce a cytotoxic response that resembles programmed cell death (apoptosis) (Rao et al, 1992), which may also contribute to the tumor-suppressing function of EIA In addition, EIA has been reported to convert three unrelated types of human cancer cells into a nontransformed state (Frisch, 1991) This suggests that EIA may also function as a tumor suppressor gene for certain human cancer cells in which c-erbB-2/neu is not overexpressed It is not yet clear whether growth signals associated with the c-er£B-2/new-encoded pi 85 protein might be activated in these human cancer cells and whether EIA might repress transforming phenotypes of these human cancer cells by blocking the signal transduction pathway associated with pi 85 protein via repressing c-erbB-2/neu expression, or EIA might suppress tumor formation through other mechanisms in certain human cancer cells Despite the potential involvement of different molecular mechanisms, these results clearly establish EIA as a tumor suppressor gene for c-ert>B-2/new-overexpressing human ovarian cancer cells and indicate that EIA is a potential therapeutic reagent for the treatment of these human cancers It has been proposed that there are cellular "El A-like" factors that may mimic the function of El A in certain cell types (Nelson et al, 1990) Many common features between EIA and c- yc suggest that the c- yc gene product may be one ofthe cellular homologue ofthe EIA protein These common features include the following EIA and c- yc share a similar structural motif (Figge & Smith, 1988, Figge et al, 1988), both EIA and c-myc can transform primary embryo fibroblasts in cooperation the ras oncogene (Land et al, 1983, Ruley, 1983); both can bind specifically to the human Rb gene product, the RB protein (Whyte et al, 1988; Rustgi et al, 1991), both can induce apoptosis in certain cell types (Rao et al, 1992, Frisch, 1991 , Nelson et al, 1990, Figge & Smith, 1988, Figge et al, 1988; Whyte et al, 1988; Rustgi et al, 1991; Evan et al, 1992), and both have been shown to block transformation of certain transformed cell lines (Frisch, 1991 , Nelson et al, 1990, Figge & Smith, 1988, Figge et al. , 1988, Whyte et al. , 1988; Rustgi et al. , 1991 , Evan et al. , 1992, Suen & Hung, 1991) In addition, the inventors have found that, similar to the EIA proteins, the c-myc gene product can repress c-erbB-2/neu gene expression at the transcription level, resulting in reversal ofthe new-induced transformed morphology in NIH3T3 cells (Wang et al. , 1991 ) Whether c-myc can suppress the malignancy of c-erbB-2/neu- overexpressing human cancer cells is an interesting issue that the inventors propose to examine EIA can inactivate the Rb tumor suppressor gene by complexing the Rb gene product, Rb protein, and by inducing RB protein phosphorylation (Whyte et al, 1988; Rustgi et al, 1991, Evan et al, 1992, Suen & Hung, 1991 , Wang et al. , 1991) Therefore, the inventors have recently examined whether RB might also regulate c-erbB-2/neu expression Similar to EIA, RB can also repress c-erbB-2/neu gene expression at the transcriptional level (Yu et al, 1992). The cw-acting elements responding to El A and RB are different but only a few base pairs away from each other It will be interesting to study further the possibility that EIA and RB might interact with each other to regulate c-erbB-2/neu transcription The EIA gene of adenovirus 2, a close sera type of adenovirus 5, was shown to reduce the metastatic potential of ras-transformed rat embryo cells (Pozzatti et al, 1988) It was hypothesized that the Ad-2 EIA gene may regulate the expression of one or more cellular genes that contribute to the metastatic phenotype and expression ofnm23, a gene associated with low metastatic potential in certain cell types that was subsequently shown to be elevated in EIA -expressing rαs-transformed rat embryo cells (Steeg et al, 1988). Although the inventors have found that EIA can repress c-erbB-2/neu gene expression and suppress the metastatic potential of c-erbB-2/neu- transformed 3T3 cell, the c-erbB-2/neu gene expression levels in the parental ras- transformed rat embryo cells and EIA -expressing /m-transformed rat embryo cells is not known. Therefore, it is not clear at this moment whether repression of c-erbB-2/neu gene expression contributes to the metastasis suppression function of EIA in røs-transformed rat embryo cells One ofthe interesting issues on the correlation between c-erbB-2/neu overexpression and poor clinical outcome in human breast and ovarian cancers is whether c-erbB-2/neu overexpression is the result of an aggressive tumor or has a causative role for aggressive tumors. The data presented here support a direct role for c-erbB-2/neu overexpression in the pathogenesis of aggressive tumors. First, comparison ofthe SK-OV-3 cell line and the derivative SKOV3.ipl cell line revealed a direct relationship between an increased c-erbB-2/neu expression level and an enhanced malignant phenotype measured by in vitro and in vivo assays. Second, c-erbB-2/neu expression in the EIA -expressing ipl .ElA cells was dramatically repressed, and, accordingly, the malignant potential of these cells was diminished. Taken together, these observations argue for a causative role of c-erbB-2/neu overexpression in the more malignant tumor pattern. Since c-erbB-2/neu- overexpressing ovarian tumors may be more malignant, more aggressive therapy might be beneficial to those ovarian cancer patients whose tumors overexpress c-erbB-2/neu- encoded pi 85. EXAMPLE V Suppression of the Neu Promoter with LT 1 Materials and Methods a Cell Culture NTH 3 T3, Bl 04-1-1 and Rat-1 cells were maintained in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM/F-12) supplemented with 10% calf serum and 100 IU/mL penicillin and 100 mg mL streptomycin Cells transfected with the drug selection plasmid, pSV2neo, were grown in the above media containing 400 mg/mL G418 b Plasmids The following plasmids have been described new deletion-CAT constructs (Suen et al, 1990), EGF receptor-CAT construct, pERCAT-9 (37), plasmid encoding activated genomic neu, cNew-104 (Hung et al, 1986) and control filler plasmid, pSV2E (Suen et al, 1990) Two LT encoding plasmids were used, pZ189 or pVU-0, both of which showed similar results Plasmids pVU-0 (Seidman et al, 1985) and mutant LT encoding plasmids, pKl and pK7 (Kalderon et al, 1984) c Stable Transfections The drug selection plasmid pSV2neo was cotransfected with plasmids encoding LT into B 104- 1-1 cells The transfected plates were trypsinized after 48 h and split into 4 plates and subsequently maintained in media containing 400 mg mL G418 After 3 weeks, colonies were isolated and established in media containing G418) d Transient Transfections and CAT Assays Cells were transfected using the modified calcium phosphate precipitation technique (Chen et al, 1987) Cells were harvested 48 h after transfection and cell extracts obtained by freeze-thawing For transfections involving LT, the protein concentration was determined using an aliquot ofthe extract Aliquots of extracts containing equal amounts of protein were used for CAT assay (Gorman et al, 1982) Transfections and CAT assays were repeated 3-4 times and representative data is shown e Immunoblotting Immunoblotting was done as described (Matin et al, 1990) Confluent cells grown in 10-cm dishes were washed and lysed with lysis buffer and 100 mg protein was loaded for electrophoresis on SDS-polyacrylamide gels followed by transfer to nitrocellulose To detect expression of pi 85, blots were incubated with anti-new antibody (c-new, Ab-3, Oncogene Science, Manhasset, NY), then reacted with secondary antibody, goat anti-mouse conjugated with horse radish peroxidase The nitrocellulose was subsequently developed with horse radish peroxidase substrate, 4-chloro- 1 -napthol and hydrogen peroxide To analyze the expression of LT antigen, blots were probed with monoclonal antibody specific for LT (SV 40 T-Ag, Ab-2, Oncogene Science) Blots were incubated with 1 mg/mL [125I]-protein A After further washing, dried blots were exposed for autoradiography f Southern Blotting Genomic DNA was harvested from cells and digested with Bam HI for Southern blotting as described (Zhang et al, 1989) Blots were hybridized using 32P-labeled rat new cDNA probe g Focus Forming Assay Focus forming assay was carried out as described (Yu et al, 1992) The cosmid clone, cNew-104 (Hung et al, 1986), contains 30 kb of activating genomic rat neu including 2 2 kb ofthe new promoter cNew-104 (0 5 mg) was cotransfected into normal fibroblasts (Rat-1 cells) with 0 1 mg ofthe drug selection plasmid, pSV2neo, and 5-10 mg plasmids encoding mutant LT (pKl) or control filler plasmid, pSV2E Cells were trypsinized and split into 4 plates 48 h after transfection Two plates were maintained in regular media while the other 2 plates were maintained in media supplemented with G418 For cells kept in regular media for 3 weeks, foci of - I l l - transformed cells appeared on a background monolayer of nontransformed cells G418 resistant colonies appeared for plates maintained in G418 media Foci and G418 resistant colonies were stained with \% crystal violet and counted To normalize for transfection efficiency, the number of foci formed for each transfection was divided by the number of G418 colonies obtained 2. RESULTS a LT Reduces New-Encoded pi 85 Levels in Cells That Overexpress pi 85 To test the effect of LT in cells that overexpress new encoded pl85, plasmids encoding LT, pZ189 (driven by the SV 40 promoter), together with pSV2neo (plasmids encoding the gene for neomycin resistance) were cotransfected into B 104-1-1 cells B 104-1-1 cells are derived from ΝIH 3T3 cells transformed by the mutation-activated genomic rat new oncogene (Shih et al, 1981, Hung et al, 1986) B 104- 1-1 cells express high levels of activated new encoded pi 85, are phenotypically transformed (Padhy et al, 1982, Shih et al. , 1981), highly tumorigenic ( Yu et al. , 1991, Hung et al. 1989) and have increased metastatic potential (Yu et al, 1991, Yu et al. 1992) The LT-transfected and G418 resistant B 104- 1 - 1 cells were cloned after 3 weeks and 2 cell lines expanded from the clones (named BTnl4 and BTnlό cell lines) were analyzed for expression of LT and pi 85 Immunoblotting of cell lysates for LT using anti-LT antibody (SV 40 T-Ag, Ab-2, Oncogene Science), showed 2 bands of molecular weights less than 111 kd indicating expression of LT in BTnl4 and BTnlό cell lines (FIG 15-B, lanes 1 and 2) The bands are probably different phosphorylated forms of LT, as reported previously (Livingston et al, 1987) A control cell line, BEn5, was generated by transfecting B 104-1-1 cells with pSV2neo and pSV2E (control plasmid similar to pZ189, containing the SV 40 promoter but lacking the LT coding region) As expected, BEn5 and ΝIH 3T3 cells do not express LT (FIG 15-B, lanes 3 and 4) The level of new encoded pi 85 in these cell lines by immunoblotting whole cell lysates with monoclonal anti-p!85 antibody (c-new Ab-3, Oncogene Science), which recognizes the carboxy terminus of pi 85 was then analyzed The control cell line, BEn5, expresses a high level of rat new encoded pi 85 (FIG 15-A, lane 3) similar to parental B 104-1-1 cells (data not shown) No pi 85 expression was detected in the negative control cells, NIH 3T3, using this antibody and detection system (FIG. 15-A, lane 4) The two cell lines expressing LT antigen (BTnl4 and BTnlό cell lines) had significantly lower levels of pi 85 expression (FIG 15-A, lanes 1 and 2) compared to BEn 5 cell line which does not express LT The expression of pi 85 in the LT transfected cells decreased by approximately 60%) to 80%) BTnl 6 cells (FIG 15-A & B, lane 1) expressed higher levels ofLT and had lower pi 85 expression, suggesting an inverse correlation between LT expression and pi 85 level To ensure that the decreased expression of pi 85 was not due to decreased copy number of rat genomic new oncogene, the level of rat new DNA in these cells was analyzed by Southern blot analysis The levels of genomic rat new oncogene in the BTnl 4 and BTnlό cell lines (FIG 15-C, lanes 1 and 2) were equivalent to that in BEn5 cell line (FIG 15-C, lane 3) The parental NIH 3T3 cells used as control does not have rat new DNA These studies show that when LT is stably expressed in cells that originally express high levels of new-encoded pi 85, there is a resulting decrease in the level of pi 85, indicating that LT, similar to c-myc and EIA, can repress new expression b LT Specifically Inhibits the New Promoter To determine whether the LT antigen inhibited rat new expression at the transcriptional level, the effect ofLT on the upstream regulatory sequences of new using transient transfection assays was examined Plasmids encoding LT antigen (pVU-0 or pZ189) (Kalderon et al, 1984) were cotransfected with plasmids encoding 2 2 kb rat new upstream regulatory sequences linked to a reporter chloramphenicol acetyl transferase (CAT) gene (pNewEcoRlCAT) (Suen et al, 1990) into ΝIH 3T3 cells The control plasmid, pSV2E, was used as a filler plasmid to adjust concentrations in cotransfections About 80% inhibition ofthe 2 2 kb new promoter activity was achieved by a 10-fold excess ofLT plasmid (FIG 16, lanes 1 & 2) The inhibitory activity ofLT was specific to new since the activity ofthe epidermal growth factor receptor regulatory sequence (pEGFrCAT) (Johnson et al, 1988) was unaffected by a similar amount ofLT (FIG 16, lanes 3 & 4) In addition, LT had a dose dependent effect on the activity ofthe regulatory sequences of new as increasing amounts ofLT led to decreased CAT activity of pnewEcoRlCAT (FIG 17) Thus, LT specifically inhibits the activity ofthe rat new promoter c Repression of New by LT is Mediated Through the Xhol-Νarl Region The region ofthe 2 2 kb new regulatory sequence that responds to LT was mapped To this end, series deletion constructs ofthe new regulatory sequence-CAT (FIG 18-A) (Suen et al, 1990) were cotransfected with plasmid encoding LT into ΝIH 3T3 cells FIG 18-B shows that the CAT activity of each ofthe new-deletion constructs and the inhibition of this activity in the presence ofLT (pVU-0 or pZ189) There was a 70%>-80% inhibition ofthe CAT activity of most ofthe new-deletion constructs except for pnewXbalCAT and pNewEcoRV2CAT In repeated studies, the inventors found less repression by LT of these two constructs Overall, the activity of all the deletion constructs, including pnewXhol CAT, were repressed by LT This indicates that repression of new by LT is mediated through the 94 base pair Xhol-Νarl region (-172 to -79, relative to first ATG) ofthe rat neu promoter S 1 protection studies have identified four transcription initiation sites in the rat new promoter Three of them, including the two major sites (at -158 and -147) are within 30 bp downstream ofthe Xhol site (Suen et al, 1990) Further deletions of nucleotides were made downstream ofthe Xhol site using Bal 31 digestion (Yanisch- Perron et al, 1985) However, this led to dramatic reduction of activity ofthe new promoter (data not shown) Thus the Xhol-Νarl region of new encompasses the minimum promoter ofthe rat new gene and LT inhibits the activity ofthe minimum promoter of new Gel-shift assays indicated that the 94 base pair Xhol-Νarl DΝA fragment specifically complexes with proteins in the nuclear extract of ΝIH 3T3 cells (FIG 19, lane 1 ) The complex, A, is detected using gels with large pore size (4 5% gels, acrylamide bisacrylamide=80 1) which have been previously shown to detect large DNA-protein complex involved in transcription initiation (Dynlacht et al, 1991), but not with gels with smaller pore size (acrylamide bisacrylamide=29.1) (data not shown) This suggests A is a large DNA-protein complex that may involve factors in the initiation or elongation complex for new transcription However, nuclear extracts from cells that express LT, BTn 14 cell line, also gave a similar DNA-protein complex profile in such gel-shift assays (FIG 19, lane 3) Thus, the presence ofLT in the nuclear extract did not affect the mobility of complex A One explanation of this is that complex A is already so large that the presence of LT (in nuclear extracts from BTn 14 cells) does not create a observable difference in the shift Indeed, complex A is found very near the top ofthe gel and is a broad band suggesting the present of multiple types of DNA-protein complexes Another possibility is that LT has indirect or subtle effects on complex A at the Xhol-Narl fragment such as a change in phosphorylation of protein factors or a change in conformation of some factors that can not be detected by gel-shift assays d A Non-transforming Mutant ofLT (Kl) is a Suppressor of New LT and Rb are known to form a protein complex (DeCaprio et al, 1988) and Rb also modulates neu expression (Yu et al, 1992), therefore, it might be expected that the LT-Rb complex is involved in repression of neu To examine this, an available mutant of LT (Kl) was utilized Kl has a single amino acid change within the region required for Rb binding (amino acids 105-1 14 ofLT) (FIG 20-A) (Kalderon et al, 984) Kl expresses mutant LT protein which is unable to complex Rb (DeCaprio et al, 1988) and Kl is defective for transformation as assayed by focus forming assay in Rat- 1 cells (Kalderon et al. , 1984, Cherington et al. , 1988) pnewXho 1 CAT together with plasmids encoding wild type (pVU-0) or mutant LT (Kl) were cotransfected into ΝIH 3T3 cells Suφrisingly, Kl represses new as effectively as wild type LT (FIG 20- B) Therefore, complex formation between LT and Rb is not required for LT-mediated neu repression Kl, unlike wild type LT, is unable to transform Rat-1 cells in focus forming assays (Kalderon et al, 1984) Therefore, the above results raises an interesting question whether Kl may function as a transformation suppressor of activated new in Rat-1 cells. To test this possibility, focus forming assays were carried out to determine the effect of stably transfecting Kl with activated genomic new The plasmid cNew-104 encodes the activated genomic new which has a single point mutation in the transmembrane domain and is driven by 2.2 kb of new upstream regulatory sequences (Hung et al, 1986) Upon introduction of cNew-104 into normal Rat-1 fibroblasts, those cells that stably express activated new are transformed and 3-4 weeks later form visible foci on a background of normal monolayer cells When Kl was cotransfected with cNew-104 into Rat-1 cells, it led to 50%> reduction in the number of foci formed by cNew-104 (FIG 20-C) Transfection of Kl only does not induce any foci Suppression of new-transforming activity with wild type LT (pVU-0) is complicated by the fact that wild type LT itself forms transformed foci in Rat-1 cells (data not shown) which makes it impossible to analyze the data Therefore, mutant LTs unable to complex with Rb that act as transformation suppressors of activated new may be the most clinically useful ofthe LT gene products 3. DISCUSSION The results of these studies show that the function ofthe rat new promoter is suppressed by the transforming viral oncoprotein, SV 40 LT antigen This activity of LT is similar to that observed for the adenovirus 5 El A and the c-myc oncoproteins, with whom LT shares a few structural and functional similarities but striking differences The inhibitory activity ofLT is apparent in the LT-transfected stable cell lines which showed an inverse correlation of new pi 85 to LT protein expression Thus, expression ofLT in cells leads to reduced expression of new encoded pi 85 in cells LT inhibits neu by repressing the activity ofthe minimum new promoter. Series deletion analysis ofthe upstream regulatory sequences of new showed that repression by LT is mediated through the 94 bp Xhol-Narl region of the new gene, which contains the minimum promoter 30 bp downstream ofthe Xhol site This result is unlike that of c-myc and EIA, since these repress new through an upstream region of the regulatory sequences of new Thus LT mediates repression of new through a different pathway compared to c-myc and EIA Therefore, these structurally related oncogenes repress the activity ofthe new promoter by acting through different regions ofthe regulatory sequences of new Although the promoter ofthe epidermal growth factor receptor and the promoter of new share some common features (Suen et al, 1 90, Johnson et al, 1988), LT did not inhibit the activity ofthe promoter of epidermal growth factor receptor Thus, LT specifically affects the promoters of certain growth factor receptors Since LT mediates repression of new through the Xhol-Narl region which contains only minimum sequence upstream ofthe two major transcription initiation sites, it is possible that LT may modulate transcription initiation or elongation from the new promoter. LT is known to interact with cellular transcription factors such as AP-2 and abrogate its function (Mitchell et al, 1987) However, examination ofthe 94 bp sequences within Xhol-Narl revealed no motif with significant homology to the AP-2 (Suen et al, 1990) EXAMPLE VI Suppression of New-Mediated Cancer with LT 1 Suppression of New-Mediated Cancer by LT in Mice The inventors are conducting ongoing studies ofthe abilities of pKl to suppress the growth and metastasis of new-overexpressing human ovarian cancer cells (SK-OV-3 cells) in female homozygous nu/nu (nude) mice SK-OV-3 cells express high levels of new and are highly metastatic in nude mice (Yu et al. 1993) These studies involve treatment of these mice with a liposomal complex liposomes comprising lipids and pKl pKl comprises DNA encoding a non-transforming mutant ofLT (Kalderon et al. 1984) Details of this study are given in VII, 2. 2 LT Suppression of New-Mediated Cancer in Humans. The results obtained using the cell lines and animal models described in this application are ofthe type widely accepted by those of skill in the art as being predictive of success in human treatment regimens Indeed, clinical trials concerning the use ofLT to suppress the expression of new in humans are contemplated However, due to precautions which are necessarily attendant to every new pharmaceutical, the compositions and methods ofthe present invention have not yet been tested in such a clinical setting Nevertheless, the results presented herein reasonably demonstrate that LT will be useful in combating cancers which exhibit new- overexpression, such as breast cancers, ovarian cancers, lung cancer, gastric cancer, oral cancers and prostate cancer One ofthe initial clinical trials to be performed involves non-transforming mutants ofLT, for example Kl These non-transforming mutants have demonstrated the ability to suppress new-mediated cancers in both cell cultures studies and in vivo animal model studies The use of such mutants avoids potential problems with transformation In these clinical studies, Kl will be introduced into the human cancer cells to suppress the production of new Among those patients who will benefit from this therapy are those whose cancer cells express high levels of new. The level of new expression in a given patient can be determined by analysis of biopsy samples of cancer tissue using routine techniques such as immunohistochemistry or western blotting These diagnostic techniques are routinely practiced and well known to those of skill in the art Targeting of cancerous tissues overexpressing new may be accomplished in any one of a variety of ways Plasmid vectors and retroviral vectors, adenovirus vectors, and other viral vectors all present means by which to target human cancers The inventors anticipate particular success for the use of liposomes to target LT genes to cancer cells In one ofthe first series of clinical phase to be performed, DNA encoding nontransforming mutants ofLT such as Kl will be complexed with liposomes in the manner described in Example VII, and this DNA/liposome complex will be injected into patients with certain forms of cancer, such as breast cancer, intravenous injection can be used to direct the Kl gene to all cells, including those which overexpress new Directly injecting the liposome complex into the proximity of a cancer can also provide for targeting ofthe complex with some forms of cancer. For example, cancers ofthe ovary can be targeted by injecting the liposome mixture directly into the peritoneal cavity of patients with ovarian cancer Of course, the potential for liposomes that are selectively taken up by a population of cancerous cells exists, and such liposomes will also be useful for targeting the LT gene. Those of skill in the art will recognize that the best treatment regimens for using LT to suppress new-mediated cancers can be straightforwardly determined This is not a question of experimentation, but rather one of optimization, which is routinely conducted in the medical arts The in vivo studies in nude mice provide a starting point from which to begin to optimize the dosage and delivery regimes The frequency of injection will initially be once a week, as was done in the mice studies However, this frequency might be optimally adjusted from one day to every two weeks to monthly, depending upon the results obtained from the initial clinical trials and the needs of a particular patient Human dosage amounts can initially be determined by extrapolating from the amount ofLT used in mice, approximately 15 μg of plasmid DNA per 50 g body weight Based on this, a 50 kg woman would require treatment with 15 mg of DNA per dose. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results ofthe initial clinical trials and the needs of a particular patient 3 Liposomal Transfection with EIA and/or LT to Suppress new-Mediated Cancers One particularly useful way to use EIA and/or LT to repress new-mediated phenotypes is via the use of liposomes for carrying the suppressor's DNA into the oncogenic cells EXAMPLE VII Preparation of Liposome/DNA Complexes and Prevention of New-Mediated Tumors with the Complexes 1 Preparation of Liposomes Catatonic liposomes which are efficient transfection reagents for both the El A and LT genes for animal cells can be prepared using the method of Gao et al (1991) Gao et al. describes a novel catatonic cholesterol derivative that can be synthesized in a single step Liposomes made of this lipid are reportedly more efficient in transfection and less toxic to treated cells than those made with the reagent Lipofectin These lipids are a mixture of DC-Chol ("3β(Ν-(Ν'Ν'-dimethylaminoethane)-carbamoyl cholesterol") and DOPE ("dioleoylphosphatidylethanolamine") The steps in producing these liposomes are as follows DC-Chol is synthesized by a simple reaction from cholesteryl chloroformate and N,N-Dimethylethylenediamine A solution of cholesteryl chloroformate (2.25g, 5 mmol in 5ml dry chloroform) is added dropwise to a solution of excess N,N- Dimethylethylenediamine (2 ml, 18 2 mmol in 3ml dry chloroform) at 0°C. Following removal ofthe solvent by evaporation, the residue is purified by recrystallization in absolute ethanol at 4°C and dried in vacuo The yield is a white powder of DC-Chol Cationic liposomes are prepared by mixing 1 2 μmol of DC-Chol and 8 0 μmol of DOPE in chloroform This mixture is then dried, vacuum desiccated, and resuspended in 1 ml sterol 20 mM HEPES buffer (pH 7 8) in a tube After 24 hours of hydration at 4°C, the dispersion is sonicated for 5-10 minutes in a sonicator form liposomes with an average diameter of 150-200 nm To prepare a liposome/DNA complex, the inventors use the following steps The DNA to be transfected is placed in DMEM F12 medium in a ratio of 15 μg DNA to 50 μl DMEM/F12 DMEM/F12 is then used to dilute the DC-Chol/DOPE liposome mixture to a ratio of 50 μl DMEZM/F 12 to 100 μl liposome The DNA dilution and the liposome dilution are then gently mixed, and incubated at 37°C for 10 minutes Following incubation, the DNA/liposome complex is ready for injection 2 In Vivo Treatment of New-Mediated Cancer Via Liposomes The inventors have shown that liposome-mediated direct gene transfer techniques can be employed to obtain EIA suppression of new- overexpressing human cancer cells in living host The protocol for this study was as follows Female nude mice (5-6 weeks old) were given intraperitoneal injections of SK- OV-3 cells (2x10*7100 μl) SK-OV-3 cells are human ovarian cancer cells that have been shown to grow within the peritoneal cavity of nude mice After five days, the mice were given intraperitoneal injections of various compounds Some mice were injected with EIA DΝA alone, some were injected with liposome/El A DΝA complex prepared in the manner described above, and some were injected with liposome/Efs (an El A frameshift mutant) DΝA complex 200 μl of a given compound was injected into a given mouse After the initial injections, injections were repeated every seven days throughout the life ofthe mouse FIG 21 shows the results of this study Mouse 1 , was injected with EIA DNA alone and developed extensive bloody ascites Mouse 1 died 65 days after the injection ofthe SK-OV-3 cells Mouse 2 was injected with liposome/Efs DNA complex Mouse 2 developed extensive bloody ascites and a large tumor and died 76 days after injection ofthe SK-OV-3 cells Mouse 3 was injected with the liposome/E 1 A DNA complex This mouse looked healthy and normal and was still alive 160 days after the injection ofthe SK-OV-3 cells These results indicate that liposome-mediated El A gene transfer can inhibit new-overexpressing human ovarian cancer cell growth Therefore, it is predictable that liposome-mediated El A or LT gene therapy may serve as a powerful therapeutic agent for HER-2 new-overexpressing human ovarian cancers by direct targeting of El A or LT at the HER-2 new-oncogene The inventors are presently testing the effects ofthe LT mutant pKl on the growth and metastasis ofthe human ovarian cancer cells SK-OV-3 in essentially the same manner as used to test the effects of EIA on these cells In these experiments, nude mice were intraperitoneally injected with 1 8 x IO 6 SK-OV-3 cells per ml of phosphate buffered saline The following week, and every week thereafter, the mice were injected with 15 μg pKl in suspension with 1 μmol liposome (DC-Chol- containing liposomes prepared as previously described) As controls, 5 mice were injected with SK-OV-3 cells and then injected with the control plasmid pGEM liposomes every week Based on the fact that previous data has shown that pKl can suppress new-induced foci and transcription from the new gene promoter, it is expected that the injected pKl will reduce tumor growth ofthe SK-OV-3 cells in the mice 3 Liposomal Transfection With EIA and/or LT to Treat Humans Based on the results ofthe in vivo animal studies described above, those of skill in the art will understand and predict the enormous potential for human treatment of new-mediated cancers with EIA and/or LT DNA complexed to liposomes Clinical studies to demonstrate these affects are contemplated One set of such studies is described in Example VII, 2 where clinical trials involving the use ofLT complexed to liposomes are described El A or any other new-suppressing gene product may be complexed with liposomes and employed in human studies in a manner similar to that described for LT These clinical trials are anticipated to show utility ofLT, EIA, and other new-suppressing gene products for the treatment of new-overexpressing cancers in humans Dosage and frequency regimes will initially be based on the data obtained from in vivo animal studies, as was described in Example VI, 2 EXAMPLE VHI Adenoviral EIA Gene Therapy of Human Cancers Expressing High Levels of PI 85 The present example provides for the introduction ofthe El A or LT gene for treatment of human cancers expressing high levels of PI 85 This may be achieved most preferably by introduction ofthe desired gene through the use of a viral vector to carry either the El A or LT sequences to efficiently infect the tumor, or pretumorous tissue These vectors will preferably be an adenoviral, a retroviral, a vaccinia viral vector or adeno-associated virus (Muro-cacho et al. , 1992) These vectors are preferred because they have been successfully used to deliver desired sequences to cells and tend to have a high infection efficiency The inventors have conducted studies showing that native adenovirus can be employed to transfer the El A gene in accordance with the invention However, a particularly preferred type of adenovirus is the group of replication-deficient adenoviruses The HER-2/new oncogene encodes a MW 185,000 epidermal growth factor receptor- related transmembrane protein (pi 85) with intrinsic tyrosine kinase activity Overexpression ofthe normal human HER-2/new protooncogene, which can also lead to higher overall tyrosine kinase activity, is a frequent event in many types of human cancers, including cancers ofthe breast, ovarian, lung, uterine cervix, stomach and colon cancer, for example Correlation between the overexpression of HER-2/new and the number of lymph node metastases in breast cancer patients and decreased survival in both breast and ovarian cancer patients has been reported The present inventors have shown in the previous examples that adenovirus 5 El A gene product can repress HER-2/new oncogene expression and suppress the tumorigenic and metastatic potential of activated rat new oncogene-transformed mouse 3T3 cells. Introduction ofthe EIA gene into the human ovarian cancer cell line SK-OV-3(i p ), which has enhanced expression of HER-2/new, resulted in reduced malignant phenotypes in vitro and in vivo Those data indicated that the El A gene can be considered as a tumor suppressor gene for HER-2/new overexpressing human cancer cells Replication-deficient adenovirus represents a gene delivery system that should be able to efficiently transfer an exogenous gene directly to tumor cells in vivo. Unlike vectors that require target cell replication for gene transfer, such as retrovirus which can only infect proliferating cells, adenovirus can transfer genes into both proliferating and non-proliferating cells. The extrachromosomal location of adenovirus in the cells (non-integration) decreases the chance of activating cellular oncogenes A high titer of adenovirus is easily produced and purified. Replication-deficient adenovirus containing EIA was constructed by E3 and ElB deletion mutant (ElB and E3 is required for adenovirus replication), control virus was constructed by additional EIA deletion mutant The present example provides for the transduction of replication-deficient adenovirus containing EIA gene [Ad E1A(+)] into human cells in vitro and in vivo Tumor suppressor gene EIA was efficiently transduced into human ovarian cancer cell SK-OV-3(i p.) cells by Ad.El A(+) in vitro and in vivo (FIG 22 and FIG 26) Up to 100%) ofthe cells can be infected at either the virus/tumor ratio >50/l or at lower ratios with multiple infections Tumor growth in vitro (FIG 23) and colony formation ability in soft agarose (FIG. 24) were greatly inhibited by Ad.El A(+) SK-OV-3(i.p ) (10 6 /mouse) was transplanted into the peritoneal cavity of nu/nu mice Five days later they received an intraperitoneal injection of viral solution (titer 2 X IO 9 PFU/ml) from either Ad E1A(+), Ad El A(-), or Just PBS for 3 days, followed by once/week for 4 5 months Clinical observation and survival rates showed that Ad El A(+) significantly prolonged the survival time ofthe mice and some mice were kept tumor free (FIG 25) Histoimmunochemical analysis indicated that Ad EIA protein was expressed in tumor tissue after gene delivery in vivo and expression of HER-2/new PI 85 protein was greatly suppressed (FIG s 28A-28C) The ovarian cancer cell line 2774, which has a very low level of expression of HER-2/new PI 85 protein, was also tested for the therapeutic effect of Ad El A(+) (FIG 27) Results showed that Ad El A(+) can not significantly prolong the survival rate ofthe 2774 cell line, indicating that Ad El A(+) specifically targets PI 85 high expression tumor cells An orthotopic human lung cancer model in nu/nu mice was used to study the effect of Ad El A(+) on tumor growth of human lung cancer cell line NCI-H820 expressing a high level of PI 85 in vivo Mouse tumor cells (5 X IO 6 ), were inoculated intratracheally Five days later, mice were treated by intratracheal instillation of viral solution (titer 2 X IO 9 PFU/ml) of Ad El A(+), Ad EIA(-), or PBS, followed by once/week i v injection treatment for 2 5 months At autopsy, more than 80% of control mice but only 20%> of treated mice had tumors as shown in Table 3 and FIG.s 29A-29C TABLE 3. THERAPEUTIC EFFECT OF Ad.ElA ON LUNG CANCER H820 IN NU/NU MICE Ad ElA(+) Ad ElA(-) PBS No mice with tumor/total (%>) 1/5 (20%) 4/5 (80%.) 5/5 (100%) Mean volume + SD (cm 3 ) 0 31 0 59 + 0 29 0 43 + 0 27 Human non-small cell lung cancer line NCI-H820 that has high expression of HER-2/new was injected intratracheally into nu/nu mice (5 X 10 6 /mouse) via a tracheotomy incision Five days later, the mice were treated once with intratracheal injection (0 1 ml) of either PBS, or Ad El A(-), Ad El A(+) (Viral titer 2 X IO 9 PFU/ml), followed by weekly i v injection treatment for 2 5 months Then, mediastinal blocks were removed and tumor volume was calculated The results indicate that Ad El A(+) can prevent the growth of human lung cancer cells implanted orthotopically in nu/nu mice From the above data, it is clear that the adenoviral gene delivery system is effective and that Ad El A(+) has a therapeutic effect on HER-2/new expressing human ovarian and lung cancer tumors EXAMPLE IX Chemosensitization of new-overexpressed cancer cells to taxol by adenovirus 5 EIA The present Example relates to HER-2/new-targeting genetic therapy by El A combined with chemotherapy in breast cancer cell lines In order to examine the effects of taxol on cell growth rat fibroblasts with various new and EIA expression were grown in varying concentrations of taxol 0 01- lOOμM The cell lysates were subsequently run through 8%> SDS-PAge and blotted with anti-pl85 and anti-El A antibodies (FIG 30A) The effects on cell growth were measured using the MTT assay as described earlier The highest inhibition of cell growth was seen in B ElAl.Hy with new down-regulated by EIA in a taxol concentration of 0 l-10uM (FIG 30B) Preparation of Adenoviruses Against Breast Cancer Cell Lines Three replication-defective adenoviral vectors were prepared in this study Ad.El A(+), an adenovirus type 5 containing EIA but lacking ElB and E3 (dl324), control vector Ad ElA(-) lacks EIA (dl 312); and Ad RS VlacZ, an adenovirus type 5 vector lacking El A, ElB, and E3, and containing the Rous sarcoma virus long terminal repeat as a promoter driving the E Coli lacZ gene (coding for the λ-gal protein) Establishment of Transduction Efficiency of Adenoviruses Against Breast Cancer Cell Lines (In Vitro) Four different breast cancer cell lines, MDA-MB-435 and MDA-MB-231, which express low level of neu, and MD-MB-453, MDA-MB-361, which express high level of neu, were treated with Ad RSV/αcZ at different virus/tumor cell (V/T) ratios Two days later, the cells were fixed and stained with X gal, which turned the lacZ gene blue The efficiency increased with higher V/T ratio Transduction efficiency of Ad.RSV/αcZ was the highest among new-overexpressing breast cancer cell lines (FIG 31 A) With a V/T ratio of 200 1, almost 100% transduction efficiency was obtained for MDA-MB-361, and MDA-MB-453 Cytopathic toxicity was observed with V/T ratios above 400 1 Low-expressing neu breast cancer cell lines were difficult to transduce due to rapid increase in the cell counts Transduction efficacy was about 20- 30%. with V/T ratio of 200 1. In a high infection protocol, which depletes FBS, infection efficiency was higher than 95% can be obtained in MDA-MB-435, with a V/T ratio of 400 1 and MDA-MB-453, with a V/T ratio of 200 1 (FIG 3 IB) The HER-2/new-overexpressing breast cancer cell line MDA-MB-453 was treated with Ad.El A(+) and Ad.El A(-) at different V/T ratios (50 1, 100 1, 150: 1, and 200 1) Our observations showed that within 2 days, there was an expression of EIA protein with dose-dependent correlation by western blot analysis with anti-El A antibody A virus/tumor cell ratio of 200 1 of Ad E1A)+) had a maximum effect on down regulating HER-2/new expression (pl85) by western blot analysis with anti-pl85 antibody (FIG. 32A) There was no down-regulation among cells that were treated with Ad El A(-) Among low expression HER-2/new breast cancer cell lines, there was no change in the low expression of HER-2/new (FIG. 32B). With maximum down-regulation of HER-2/new expression (pi 85) 2 days after giving V/T ratio of 200 1 Ad El A(+) against MDA-MB-453, we have conducted a cytoxic assay using MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide) assay. An appropriate control of Ad.El A(-) (dl312), Ad.El A(-) with Taxol, Taxol alone, and Ad E1A(+) alone (dl324) were prepared and compared with Ad E1A(+) with Taxol Taxol was given from 0 1 μM to 100 μM We showed a maximum suppression of survival fraction of MDA-MB-453 cells when both Ad.El A(+) and Taxol at concentration of 10-50 μM were given (FIG. 33A and 33C) A similar assay was conducted after giving V/T ratio of 400 : 1 Ad.E 1 A(+) against MDA-MB-435 cells. An appropriate control of Ad.El A(-). Ad.ElA(-) with Taxol, Taxol alone, and Ad.El A(+) alone were prepared and compared with Ad.El A(+) with Taxol Taxol was given from 0.1 μM to 100 μM (FIG. 33B and 33D) No Synergistic effect was seen The suppression differences between Taxol alone and Taxol plus Ad.ElA(-) were almost the same at the Taxol doses of 0.1 μM to 100 μM (FIG. 33A and 33C) Also, when treatment with 10 μM Taxol was analyzed based on a time scale at high infection (FIG. 34A) and low infection protocols (FIG. 34C), the survival fraction difference started around day 2 in the high infection protocol (FIG. 34A) and day 1 in the low infection protocols (FIG 34C)with maximum difference at day 4. Conversely there was no synergistic effect of Ad.ElA(+) with taxol on MDA- MB-435 cells (FIG. 34B and FIG 34D). However these low neu expressing cells are more sensitive to taxol alone Clonogenic assay was done to see if transformation in the above condition would be inhibited (FIG 35) Once again the synergistic effect between Taxol and EIA was observed in MDA-MB-453 cells However, breast cancer cell line with low HER-2/new expression (MDA-MB-435) did not show any synergistic effect in both MTT assay or soft agar clonogenic assay When direct cell counts were performed to check for viable cells using trypan blue 0 5%> it was again clear that there was a synergistic effect of El A and taxol (10 μM) against HER-2/new-overexpressing breast cancer cell line MDA-MB-453 on direct cell counting (FIG 36 A) However, there was no synergistic effect of Ad El A(+) V/T ratio of 400 1 and taxol on the viable cell fraction of MDA-MB-435, adenoviral vector was delivered under high infection protocol (FIG 36B) The results prove the concept of our hypothesis, namely the El A-mediated HER-2/new repression is able to sensitize the response of HER-2/new-overexpressing cancer cells to chemotherapeutic agents This phenomenon occurs only to the HER- 2/new-overexpressing cancer cells When the breast cancer cells in which HER-2/new is not overexpressed (MDA-MB-435), sensitization can not be observed either in MTT assay (FIG 33) or clonogenic assay (FIG 35) EXAMPLE X In Vivo Prevention of Breast, Lung and Ovarian Tumor Development in vivo bv Inhibiting Tyrosine Kinase Activitv of the HER-2/neu Receptor. In an initial round of in vivo trials, inventors will use a mouse model of human cancer with the histologic features and metastatic potential resembling tumors seen in humans (Katsumata et al, 1995) and treat these animals with El A or LT to examine the suppression of tumor development These studies are based on the discovery that Ad El A has tumor suppressor activity for new-overexpressing cancer cells The Examples above further show that Ad.El A inhibits the growth of new-mediated cancer cells and furthermore sensitizes new-mediated cancer cells to chemotherapeutic drugs. The current example uses of either Ad.El A or LT, in combination with chemotherapeutic drugs, to provide a useful preventive and therapeutic regimen for patients with new-overexpressing cancers. Two groups of mice of a suitable cancer model will be treated with doses of El A or LT in combination with anti cancer drugs starting at 6 weeks of age. Several combinations and concentrations of El A or LT and anti-cancer drugs will be tested. Control mice will be treated with buffer only. The effect of EIA or LT, in combination with an anticancer drug, on the development of breast tumors will be compared with the control group by examination of tumor size, pl 85 πeu tyrosine kinase activity (using IP- western blot analysis) and histopathologic examination (breast tissue will be cut and stained with hematoxylin and eosin) of breast tissue With the chemopreventive potential of EIA and LT, it is predicted that, unlike the control group of mice that develop tumors, the testing group of mice will be resistant to tumor development. Breast Cancer Model In order to obtain mice having human breast cancer, nu/nu mice may be given intraperitoneal injections of, for example, 2 x IO 6 viable new overexpressing breast cancer cells from cell line MDA-MB-361 are injected in the mammary fat pad in nude mice. Palpable solid tumors are detected 1.5 months later These mice may then be given an appropriate dosage of El A or LT using methods of delivery described above; in combination with an anti-cancer drug for 3 consecutive days, then once a week for six months. Ovarian Cancer Model In order to obtain mice having human ovarian cancer, nu/nu mice may be given intraperitoneal injections of, for example, 2 x IO 6 viable pl85-overexpressιng SKOV-3 human ovarian cancer cells Mice sacrificed 5 days post treatment exhibit tumors resulting from such treatment Five days after treatment with the pl85-overexpressing cells, mice may be separated into control and experimental groups One group of mice will be left untreated Other groups will be treated Active compounds may be supplied to a treated group in phosphate buffer saline One treated group will be treated with the buffered saline only Another treated group may receive treatment with an appropriate dosage of El A or LT A third treated group may be treated with an appropriate dosage of an anti-cancer drug alone A final group may be treated with an appropriate dosage of El A or LT in combination with an anti-cancer drug Treatments may be given using any ofthe methods described above. Mice may be examined for tumor signs and symptoms, and killed when they appear moribund Mice treated with the El A or LT plus the anti-cancer drug will be expected to have a longer survival time Small Cell Lung Cancer Model In order to obtain mice with the human lung cell cancer, nu/nu mice may be given as intratracheal injections of, for example, 2 x IO 6 viable new overexpressing cancer cells from cell line H82 Five days after inoculation, following tumor formation, mice may be separated into groups to begin treatment One group may be treated with an appropriate dosage of El A or LT alone, another with an appropriate dosage of an anti¬ cancer drug alone A third group may be treated with an appropriate dosage of El A or LT in combination with an anticancer drug for 3 consecutive days, then once a week for two months EXAMPLE XI Human Treatment with EIA or LT in Combination with Anti-cancer Drugs or Alone This example describes a protocol to facilitate the treatment of new-mediated cancer using El A or LT in combination with anti-cancer drugs A patient presenting a new-mediated cancer may be treated using the following protocol new-overexpression may be detected using the immunohistochemistry methods described below Patients may, but need not, have received previous chemo- radio- or gene therapeutic treatments Optimally the patient will exhibit adequate bone marrow function (defined as peripheral absolute granulocyte count of > 2,000/mm3 and platelet count of 100, 000/mm3, adequate liver function (bilirubin 1 5mg/dl) and adequate renal function (creatinine 1 5mg/dl) Monitoring neu Overexpression in Tumors The over-expression of new is typically monitored before, during, and after the therapy The following assay may be used to monitor new-overexpression. Sections of 3- to 4 mm thickness ofthe primary tumors and ofthe cell block preparations are cut, deparaffinized in xylene, and rehydrated in descending grades (100-70%>) of ethanol Endogenous peroxidase activity is blocked with 3% hydrogen peroxide in methanol After several washes in distilled water and phosphate-buffered saline, the sections are incubated with a 1 10 dilution of normal horse serum to minimize background staining This is followed by incubation for 1 hr at room temperature with the primary antibody (Ab-3 monoclonal antibody, Oncogene Sciences, Uniondale, NY; 1 100) The peroxidase staining procedure utilizes ABC Elite Kits (Vector Laboratories, Burlingame, CA) The immunostaining reactions are visualized using 3-amino-9-ethylcarbazole as the chromogen The sections and/or cytospin preparations are stained with toluidine blue and mounted in permount Positive and negative control immunostains are also prepared The sections are reviewed by the pathologist Two features ofthe immunoreaction will be recorded using a semi quantitative scale the relative number of positive cells (0%>, <10%>, 10-50%>, and > 50%) and the intensity ofthe reaction (0-3) The pattern of immunostaining (membranous, cytoplasmic) is recorded separately A tumor is considered new positive if any neoplastic cells show cell membrane reactivity Cytoplasmic staining is considered non-specific A breast carcinoma known for its strong positive membrane staining will be used as a positive control The quantitative measurement of new immunostaining will be performed using computerized image analysis with the SAMBA 4000 Cell Image Analysis System (Image Products International, Ine , Chantilly, VA) integrated with a Windows based software A strong staining tumor tissue section will be used as positive control The primary antibody will be replaced by an isotype-matched irrelevant antibody to set the negative control threshold, averaging the results from ten fields Protocol for the Treatment of new-Mediated Cancer A composition of the present invention is typically administered orally or parenterally in dosage unit formulations containing standard, well known non-toxic physiologically acceptable carriers, adjuvants, and vehicles as desired The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intra-arterial injection, or infusion techniques. The EIA or LT may be delivered to the patient before, after or concurrently with the other anti-cancer agents. A typical treatment course may comprise about six doses delivered over a 7 to 21 day period. Upon election by the clinician the regimen may be continued six doses every three weeks or on a less frequent (monthly, bimonthly, quarterly etc ) basis. Of course, these are only exemplary times for treatment, and the skilled practitioner will readily recognize that many other time-courses are possible. A major challenge in clinical oncology of new-mediated cancers is that tumor cells over-expressing the new-protooncogene are resistant to chemotherapeutic treatment One goal ofthe inventors' efforts has been to find ways to improve the efficacy of chemotherapy In the context ofthe present invention, El A or LT can be combined with any of a number of conventional chemotherapeutic regimens To kill new-overexpressing cancer cells using the methods and compositions described in the present invention one will generally contact a target cell with EIA or LT and at least one chemotherapeutic agent (second agent), examples of which are described above These compositions will be provided in a combined amount effective to kill or inhibit the proliferation ofthe cell This process may involve contacting the cell with EIA or LT and the second agent at the same time Alternatively, this process may involve contacting the cell with a single composition or pharmacological formulation that includes both agents or by contacting the cell with two distinct compositions or formulations at the same time, wherein one composition includes the EIA or LT and the other includes the second agent Alternatively the El A or LT administration may precede or follow the delivery ofthe second agent by intervals ranging from minutes to weeks In embodiments where the El A or LT and the second compound are applied separately, one would ensure that a significant period of time did not expire between the time of each delivery, such that the second agent and the EIA or LT would still be able to exert an advantageously combined effect on the cancer In such instances, it is contemplated that one would contact the cell with both agents within about 6 hours to one week of each other and more preferably , within 24-72 hours of each other In some situations however, it may be desirable to extend the time period for treatment significantly where several days (2, 3. 4, 5, 6, 7 or more) to several weeks (1, 2, 3, 4, 5, 6 , 7 or more) lapse between respective administrations Regional deliver) of El A or LT will be an efficient method for delivering a therapeutically effective dose to counteract the clinical disease Likewise, the chemotherapy may be directed to a particular effected region Alternatively systemic delivery of either, or both, agent may be appropriate The therapeutic composition ofthe present invention is administered to the patient directly at the site ofthe tumor This is in essence a topical treatment ofthe surface ofthe cancer The volume ofthe composition should usually be sufficient to ensure that the entire surface ofthe tumor is contacted by the El A or LT and second agent In one embodiment, administration simply entails injection of the therapeutic composition into the tumor In another embodiment, a catheter is inserted into the site ofthe tumor and the cavity may be continuously perfused for a desired period of time Clinical responses may be defined by acceptable measure For example, a complete response may be defined by the disappearance of all measurable disease for at least a month Whereas a partial response may be defined by a 50%> or greater reduction ofthe sum ofthe products of perpendicular diameters of all evaluable tumor nodules or at least 1 month with no tumor sites showing enlargement Similarly, a mixed response may be defined by a reduction ofthe product of perpendicular diameters of all measurable lesions by 50%> or greater with progression in one or more sites Of course, the above-described treatment regimes may be altered in accordance with the knowledge gained from clinical trials such as those described in Example 14 Those of skill in the art will be able to take the information disclosed in this specification and optimize treatment regimes based on the clinical trials described in the specification EXAMPLE XII Clinical Trials o the Use of EIA or LT in Combination with Anti-Cancer Drugs in Treating New-Mediated Cancer This example is concerned with the development of human treatment protocols using the El A and LT in combination with anti-cancer drugs El A or LT and anti¬ cancer drug treatment will be of use in the clinical treatment of various new- overexpressing cancers in which transformed or cancerous cells play a role Such treatment will be particularly useful tools in anti-tumor therapy, for example, in treating patients with ovarian, breast and lung cancers that are mediated by new over¬ expression and resistant to conventional chemotherapeutic regimens The various elements of conducting a clinical trial, including patient treatment and monitoring, will be known to those of skill in the art in light of the present disclosure The following information is being presented as a general guideline for use in establishing El A or LT in combinations with anti-cancer drugs in clinical trials Patients with advanced, metastatic breast and/or epithelial ovarian carcinoma chosen for clinical study will typically have failed to respond to at least one course of conventional therapy Measurable disease is not required, however the patient must have easily accessible pleural effusion and/or ascites Further the patients must carry tumors that overexpress new oncoprotein. Overexpression may be defined as grade 2 or 3 staining by immunohistochemistry as described above In an exemplary clinical protocol, patients may undergo placement of a Tenckhoff catheter, or other suitable device, in the pleural or peritoneal cavity and undergo serial sampling of pleural/peritoneal effusion Typically, one will wish to determine the absence of known loculation ofthe pleural or peritoneal cavity, creatinine levels that are below 2 mg/dl, and bilirubin levels that are below 2 mg/dl The patient should exhibit a normal coagulation profile In regard to the El A or LT and other anti-cancer drug administration, a Tenckhoff catheter, or alternative device may be placed in the pleural cavity or in the peritoneal cavity, unless such a device is already in place from prior surgery A sample of pleural or peritoneal fluid can be obtained, so that baseline cellularity, cytology, LDH, and appropriate markers in the fluid (CEA, CA15-3, CA 125, pl85) and in the cells (EIA, pi 85) may be assessed and recorded In the same procedure, El A or LT may be administered alone or in combination with the anti-cancer drug The administration may be in the pleural/peritoneal cavity, directly into the tumor, or in a systemic manner. The starting dose may be 0 5mg/kg body weight Three patients may be treated at each dose level in the absence of grade > 3 toxicity Dose escalation may be done by 100% increments (0 5mg, lmg, 2mg, 4mg) until drug related grade 2 toxicity is detected Thereafter dose escalation may proceed by 25%> increments The administered dose may be fractionated equally into two infusions, separated by six hours if the combined endotoxin levels determined for the lot of El A or LT and the lot of anti-cancer drug exceed 5EU/kg for any given patient The El A or LT and anti-cancer drug combination may be administered over a short infusion time or at a steady rate of infusion over a 7 to 21 day period The EIA or LT infusion may be administered alone or in combination with the anti-cancer drug The infusion given at any dose level will be dependent upon the toxicity achieved after each Hence, if Grade II toxicity was reached after any single infusion, or at a particular period of time for a steady rate infusion, further doses should be withheld or the steady rate infusion stopped unless toxicity improved. Increasing doses of El A or LT in combination with an anti-cancer drug will be administered to groups of patients until approximately 60%> of patients show unacceptable Grade III or IV toxicity in any category. Doses that are 2/3 of this value could be defined as the safe dose. Physical examination, tumor measurements, and laboratory tests should, of course, be performed before treatment and at intervals of about 3-4 weeks later Laboratory studies should include CBC, differential and platelet count, urinalysis, SMA-12- 100 (liver and renal function tests), coagulation profile, and any other appropriate chemistry studies to determine the extent of disease, or determine the cause of existing symptoms Also appropriate biological markers in serum should be monitored e.g. CEA, CA 15-3, pi 85 for breast cancer, and CA 125, pi 85 for ovarian cancer. To monitor disease course and evaluate the anti-tumor responses, it is contemplated that the patients should be examined for appropriate tumor markers every 4 weeks, if initially abnormal with twice weekly CBC, differential and platelet count for the 4 weeks; then, if no myelosuppression has been observed, weekly If any patient has prolonged myelosuppression, a bone marrow examination is advised to rule out the possibility of tumor invasion ofthe marrow as the cause of pancytopenia Coagulation profile shall be obtained every 4 weeks. An SMA- 12- 100 shall be performed weekly Pleural/peritoneal effusion may be sampled 72 hours after the first dose, weekly thereafter for the first two courses, then every 4 weeks until progression or off study. Cellularity, cytology, LDH, and appropriate markers in the fluid (CEA, CA15-3, CA 125, pl85) and in the cells (pi 85) may be assessed For an example of an evaluation profile, see Table 4 When measurable disease is present, tumor measurements are to be recorded every 4 weeks. Appropriate radiological studies should be repeated every 8 weeks to evaluate tumor response. Spirometry and DLCO may be repeated 4 and 8 weeks after initiation of therapy and at the time study participation ends. An urinalysis may be performed every 4 weeks. Clinical responses may be defined by acceptable measure. For example, a complete response may be defined by the disappearance of all measurable disease for at least a month Whereas a partial response may be defined by a 50%> or greater reduction ofthe sum ofthe products of perpendicular diameters of all evaluable tumor nodules or at least 1 month with no tumor sites showing enlargement. Similarly, a mixed response may be defined by a reduction ofthe product of perpendicular diameters of all measurable lesions by 50%> or greater with progression in one or more sites. TABLE 4 EVALUATIONS BEFORE AND DURING THERAPY EVALUATIO PRE- TWICE EVERY EVERY NS STUD WEEKL WEEKL 4 8 Y Y Y WEEKS WEEKS History X X Physical X X Tumor X X Measurements CBC X X 1 X Differential X X 1 X Platelet Count X X 1 X SMA12-100 X X (SGPT, Alkaline Phosphatase, Bilirubin, Alb/Total Protein) Coagulation X X Profile Serum Tumor X X 3 markers (CEA, CA15-3, CA- 125, Her-2/new) Urinalysis X X X-rays: chest X X 4 others X X Pleural/Peritone X X 5 X al Fluids: (cellularity, cytology, LDH, tumor markers, EIA, HER- 2/new) Spirometry and X X 6 X 6 DLCO For the first 4 weeks, then weekly, if no myelosuppression is observed. 2 As indicated by the patient's condition. Repeated every 4 weeks if initially abnormal. For patients with pleural effusion, chest X-rays may be performed at 72 hours after first dose, then prior to each treatment administration. Fluids may be assessed 72 hours after the first dose, weekly for the first two courses and then every 4 weeks thereafter Four and eight weeks after initiation of therapy While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spirit and scope ofthe invention More specifically, it will be apparent that certain agents which are both chemically and physically related may be substituted for the agents described herein while the same or similar results would be achieved All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept ofthe invention as defined by the appended claims REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference Adelman et al. (1983) DNA 2 183 Alberts et al , Clin. Pharmacol. Ther. , 86:737-745, 1979. Alley et al. (1988) Cancer Res., 48 589 Ayash, etal. Clin Oncol, 12 37-44, 1994 Baichwal and Sugden, In: Kucheriapati R, ed Gene transfer New York Plenum Press, pp 117-148, 1986 Bargmann et al. (1986), Nature, 319 226-230 Campisi et al. (1983), Cell, 33 357 Chang et al. (1989), J. Virol, 63 3419 Chen et al. (1988), BioTechniques, 6 632 Chen and Okayama, ?/. Cell Biol, 7 2745-2752, 1987 Cherington et al. (1988), Mol. Cell. Biol, 8 1380-1384 Coffin, In Virology. Fields et al (eds ), New York Raven Press, pp 1437-1500, 1990 Couch et al, Am. Rev. Resp. Dis., 88 394-403, 1963 Coupar et α/ , Gene, 68 1-10, 1988 Crea et al. (1978), Proc Natl. Acad. Sci USA 75 5765 DeCaprio et al. (1988), 54 275-283 Downward et al. (1984), Nature (London), 307 521 Dubensky tα/., Proc. Nat'lAcad. Sci USA, 81 7529-7533, 1984 Dynlacht et al. (1991), Cell, 66 563-576 Eichenlaub, R (1979), J. Bacteriol 138 559-566 Evan et al. ( 1992), Cell, 69 U9-US Fechheimer et α/., Proc. Nat'lAcad Sci. USA, 84 8463-8467, 1987 Ferkol et al., EASEB ., 7 1081-1091, 1993 Figge et al (1988), J. Virol , 62 1814-1818 Fraley et al, Proc. Nat'lAcad Sci. USA, 76 3348-3352, 1979 Friedmann, "Progress toward human gene therapy," Science, 244 1275-1281, 1989 Frisch, S M (1991), Proc. Natl Acad. Sci. USA, #§ 9077-9081 Gao et al, (1 91), Biochemical and Biophysical Research Communications, 779(l).280-285. Ghalie, et α/., JC//n Oncol, 12:342-6, 1994 Ghosh and Bachhawat, In: Wu G and C. Wu ed. Liver diseases, targeted diagnosis and therapy using specific receptors and ligands New York Marcel Dekker, pp 87-104, 1991 Gomez-Foix et α/., J £/o/. Chem., 267:25129-25134, 1992 Gopal, Mol. Cell Biol. , 5 : 1188- 1190, 1985. Gorman et al. (1982), Mol. Cell. Biol, 2 1044-1051 Graham and Prevec, Biotechnology, 20:363-390, 1992 Graham and van der Eb, Virology, 52 456-467, 1973 Gribskov et al. (1986), Nucl. Acids Res., 14 6745 Grunhaus and Horwitz, Seminar in Virology, 3.237-252, 1992 Gusterson, etcr/., J Clin Oncol, 10:1049-56, 1992. Haley et al. (1984), Proc. Natl. Acad. Sci. USA, 81.5134. Harland and Weintraub, J. Cell Biol, 101 :1094-1099, 1985 Harlow et al. (1985), J. Virol, 55 533) Hearing et al. (1985), Mol. Cell. Biol, 5 3214. Hen et al. (1985), Science, 230 1391 Hermonat and Muzycska, Proc. Nat'lAcad. Sci. USA, 81 6466-6470, 1984 Hersdorffer et al. , DNA Cell Biol , 9 113-723, 1990 Herz and Gerard, Proc. Nat'lAcad. Sci. USA 90 2812-2816, 1993 Horwich et al, J. Virol, 64 642-650, 1990 Houweling et al. (1980), Virology, 105 531 Hung et al , Proc. Natl. Acad. Sci. USA, 86:2545-2548, 1989. Johnson et al. (1988), J. Biol Chem., 263 5693-5699 Kalderon et al. (1984), Virology, 759:109-137 Kaneda et al, "Increased expression of DNA cointroduced with nuclear protein in adult rat liver," Science, 243 375-378, 1989 Ka ή sson et l, EMBOJ., 5 2377-2385, 1986 Kato et al, "Expression of hepatitis B virus surface antigen in adult rat liver," J. Biol Chem., 266 3361-3364, 1991 Klein et al, Nature, 327 70-73, 1987 Kraus et al (1987), EMBO , 6.605 Kyte & Doolittle, J. Mol. Biol, 157 105-132, 1982 Land et al. (1983), Science, 222 111 Le Gal La Salle et al. , Science, 259 988-990, 1993 Levrero et al , Gene, 101 195-202, 1991 Livingston et al. (1987), Mol Biol Med, 4 63-80 Mann et α/., Cell, 33.153-159, 1983 Markowitz et al. , J. Virol , 62.1 120-1124, 1988. Matin et al. ( 1989), Oncogene, 5 1 1 1 McCann, ettf/., Cancer Res, 51 3296-303, 1991. Messing et al. (1981) Third Cleveland Symposium on Macromolecules and Recombinant DNA, Editor A Walton, Elsevier, Amsterdam. Mitchell et al (1989), Science, 245 371 Moran et /. (1987), Cell, 48 177 Muller et al (1982), Nαtwre (London), 299 640 Muller et al , Cell, 54: 105-115, 1988. Muro-cacho, C A (1992), J. of Immunotherapy, 11 231-237 Muss, et al, N. Engl J Med, 330 1260-6, 1994 Νabel et al (1990), Science, 249 1285-1288 Νeedleman et al (1970), J. Mol. Biol, 48 443 Nelson et al. (1990), Proc. Natl. Acad. Sci. USA, 87 8041-8045 Nicolas & Rubenstein, In Vectors A survev of molecular cloning vectors and their uses, Rodriguez & Denhardt (eds ), Stoneham Butterworth, pp 493-513, 1988 Nicolau et al. (1987), Methods in Enzymology, 149 157-176 Nicolau and Sene, " Biochem. Biophys. Acta, 721 185-190, 1982 Padhy et al. (1982), Cell, 28 865-871 Paskind et al, Virology, 67 242-248, 1975 Perales et al, Proc. Natl. Acad. Sci. USA, 91 4086-4090, 1994 Peters, et al, J Clin Oncol, 11 1132-43, 1993 Potter etal, " Proc. Nat'lAcad. Sci. USA, 81 7161-7165, 1984 Pozzatti et al. (1988), Mol. Cell. Biol, 8 2984 Ragot etal, Nature, 361 647-650, 1993 Rao et al. (1992), Proc. Natl. Acad. Sci. USA, 89 7742-7746 Rich etal, Hum. Gene Ther., 4461-476, 1993 Ridgeway, In: Rodriguez RL, Denhardt DT, ed Vectors A survey of molecular cloning vectors and their uses Stoneham Butterworth, pp 467-492, 1988 Rippe ef α/., >/. Cell Biol, 10 689-695, 1990 Rosenfeld etal, Cell, 68 143-155, 1992 Rosenfeld et al, Science, 252 431-434, 1991 Roux et al, Proc. Natl. Acad. Sci. USA, 86 9079-9083, 1989 Ruley, H.E (1985), Nature (London), 304 602 Rustgi et al (1991), Nature, 352 541-544 Schechter et al.. Nature, 312:513-516, 1984. Schwartz et al , eds (1979), Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pgs 353-358 Senba et al (1985), Proc. Natl. Acad. Sci. USA, 82 6497 Shih et al. (1981), Nature (London), 290:261-264. Slamon et al. , (1989), 244:707-712. Slamon, et al, Science, 240 177-182, 1987 Smith et al. (1981), Adv. Appl. Math., 2 482 Smith and Rutledge, "Chemotherapy in advanced ovarian cancer, " Natl. Cancer Inst. Monogr. , 42: 141-143, 1975. Southern et al. (1982), J. Mol. Appl Genet., 1 327 Steeg et al. (1988), Cancer Res., 48 6550-6554 Stein et al. (1987), Mol. Cell. Biol, 7 1 164 Stratford-Perricaudet and Perricaudetp 51-61, In: Human Gene Transfer, Eds, O Cohen-Haguenauer and M Boiron, Editions John Libbey Eurotext, France, 1991 Stratford-Perricaudet et al, Hum. Gene Ther., 1 241-256, 1990 Suen et al (1990), Mol. Cell Biol, 10 6306-6315 Tal et fl/ (1987), Mol. Cell. Biol, 7.2597 Temin, In Gene Transfer, Kucheriapati (ed ), New York Plenum Press, pp 149-188, 1986 Toikkanen, et al. , J Clin One, 10 1044-48, 1992 Top et al, J. Infect. Dis., 124 155-160, 1971 Towbin et al. (1979), Proc. Natl. Acad. Sci., USA, 76 4350 Tur-Kaspa et al, Mol Cell Biol, 6 716-718, 1986 Tsai, et al. , JNatl Cancer Inst, 87 682-4, 1995 Van Dam et al. (1989), Oncogene, 4 1207 Varmus et al, Cell, 25 23-36, 1981 Velcich et α/. (1986), Mol. Cell. Biol, 6 4019 Vijver, et al, Mol. Cell. Biol, 1 2019-23, 1987 Wagner et al. , Science, 260 1510- 1513 , 1990 Wallich et al. (1985), Nαtwre (London), 315 301 Wang et al (1991), Mol. Cell. Biol, 11 4253-4265 Whyte et al (1988), Nαtwre (London), 334 124-129 Yan, et al, Oncogene, 6 343-345, 1991 Wu and Wu, J. Biol Chem. , 262 4429-4432, 1987 Wu and Wu, Biochemistry, 27 887-892, 1988 Yanisch-Perron et al. (1985), Gene, 33 103-109 Young et l , N Engl. J. Med. , 299: 1261-1266, 1978. Yu, et al, Proc Natl Acad Sci USA, 87 4499-503, 1990 Weinberg, R A (1985), Science, 230 110-116 Zelenin et al, FEBS Lett., 280 94-96, 1991 Zhang et al. (1989), Oncogene, 4 985-989

Claims: CLAIMS A method of inhibiting oncogene-mediated transformation of a cell, comprising contacting the cell with a new-suppressing gene product and a chemotherapeutic drug in amounts effective to inhibit the transformed phenotype The method of claim 1, wherein the oncogene-mediated transformation is neu oncogene-mediated transformation The method of claim 1 , wherein the new-suppressing gene product is introduced into the cell by the introduction of a gene encoding a new-suppressing gene product The method of claim 3, wherein the new-suppressing gene is an El A gene The method of claim 4, wherein the El A gene encodes the EIA 12S or 13S gene product The method of claim 4, wherein the El A gene encodes either the El A 12S or 13S gene product The method of claim 4, wherein the E 1 A gene encodes both the E 1 A 12S and 13 S gene products 8 The method of claim 3, wherein the new-suppressing gene is an LT gene 9 The method of claim 8, wherein the LT gene encodes an LT mutant 10 The method of claim 8, wherein the LT mutant is a nontransforming mutant 11 The method of claim 10, wherein the nontransforming mutant is Kl 12 The method of claim 1, wherein the chemotherapeutic drug is selected from the group consisting of alkylating agents, plant alkaloids, or antibiotics and other antineoplastic agents 13 The method of claim 1, wherein the chemotherapeutic is an alkylating agent 14 The method of claim 14, wherein the alkylating agent is mechlorethamine, cyclophosphamide, ifosfamide chlorambucil, melphalan, busulfan, thiotepa, carmustine, lomustine, or shreptozoin 15 The method of claim 1, wherein the chemotherapeutic agent comprises a plant alkaloid 16 The method of claim 15, wherein the plant alkaloid is vincristine, vinblastine or taxol 17 The method of claim 15, wherein the plant alkaloid is taxol 18 The method of claim 1, wherein the chemotherapeutic agent is an antibiotic 19 The method of claim 18, wherein the antibiotic is dactinomycin, daunorubicin, idarubicin, bleomycin, mitomycin, or doxorubicin 20 The method of claim 18, wherein the antibiotic is doxorubicin 21 The method of claim 1 , wherein the chemotherapeutic drug is an antineoplastic 22 The method of claim 21 , wherein the antineoplastic agent is selected from the group consisting of cisplatin, VPI 6, and TNF 23 The method of claim 1, wherein the new-suppressing gene product is introduced to the cell prior to the administration ofthe chemotherapeutic agent 24 The method of claim 1, wherein the chemotherapeutic agent is administered to the cell prior to introduction ofthe new-suppressing gene product The method of claim 1, wherein the new-suppressing gene product is introduced to the cell and the chemotherapeutic drug is administered to the cell substantially simultaneously The method of claim 1, wherein the cell is located within an animal and effective amounts ofthe new-suppressing gene product and the chemotherapeutic drug are administered to the animal The method of claim 1, wherein the chemotherapeutic drug is suitably dispersed in a pharmacologically acceptable formulation The method of claim 1, wherein the cell is contacted with a single composition for introducing an new-suppressing gene product and administering a chemotherapeutic agent The method of claim 28, wherein the composition is suitable the composition is suitably dispersed in a pharmacologically acceptable formulation The method of claim 1, wherein the a new-suppressing gene product is introduced into the cell by introduction of a new-suppressing gene using a new-suppressing gene DNA/liposome complex 31 The method of claim 30, wherein the liposome comprises DOTMA, DOPE, or DC- Chol 32 The method of claim 30, wherein the liposome comprises DC-Chol 33 The method of claim 30, wherein the liposome comprises DC-Chol and DOPE 34 The method of claim 30, wherein the DNA/liposome complex is administered by injection 35 The method of claim 1, wherein the a new-suppressing gene product is introduced into the cell by introduction of a vector containing a new-suppressing gene 36 The method of claim 35 wherein the vector is a viral vector 37 The method of claim 35 wherein the vector is an adenoviral vector 38 The method of claim 1, wherein the cell is a human cell 39 The method of claim 38, wherein the cell is a lung cancer cell A method of sensitizing a cancer cell to a chemotherapeutic drug comprising exposing the cell with an amount of a new-suppressing gene product effective to inhibit new-mediated tyrosine kinase activity in the cell The method of claim 41, wherein the new-suppressing gene product is introduced via the introduction of a gene encoding the new-suppressing gene product The method of claim 42, wherein the new- suppressing gene product is an El A gene product The method of claim 41, wherein the new-suppressing gene product is an LT gene product A method of inhibiting new-mediated cancer comprising administrating to an animal having or suspected of having cancer an effective combination of new-suppressing gene product and chemotherapeutic drug in an effective amount to inhibit the cancer The method of claim 45, wherein the animal is a mammal The method of claim 46, wherein the mammal is a human The method of claim 45 comprising introducing into the animal a therapeutically effective amount of a new-suppressing gene product and contacting the animal with a chemotherapeutic drug The method of claim 45 wherein a cancer site is contacted with a chemotherapeutic drug by administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising a chemotherapeutic drug The method of claim 45, wherein the chemotherapeutic drug is cisplatin The method of claim 45, wherein the chemotherapeutic drug is doxorubicin The method of claim 45, wherein the chemotherapeutic drug is VP16 The method of claim 45, wherein the new-suppressing gene product is introduced via the introduction of a gene encoding the new-suppressing gene product The method of claim 53, wherein the new-suppressing gene product is an EIA gene product The method of claim 53, wherein the new-suppressing gene product is an LT gene product 56 A pharmaceutical composition comprising DNA encoding a new-suppressing gene product gene and a chemotherapeutic drug 57 The composition of claim 56, wherein the chemotherapeutic drug is cisplatin, doxorubicin, VPI 6, taxol, or TNF 58 The composition of claim 56, wherein the chemotherapeutic drug is cisplatin 59 The composition of claim 56, wherein the chemotherapeutic drug is doxorubicin 60 The composition of claim 56, wherein the chemotherapeutic drug is VPI 6 61 The composition of claim 56, wherein the new-suppressing gene product is an El A gene product 62. The composition of claim 56, wherein the new-suppressing gene product is an LT gene product 63 A pharmaceutical combination comprising a gene expressing a new-suppressing gene product and a chemotherapeutic drug 64 The pharmaceutical combination of claim 63, wherein the new-suppressing gene product is an E 1 A gene product The pharmaceutical combination of claim 63, wherein the new-suppressing gene product is an LT gene product The pharmaceutical combination of claim 63, wherein the chemotherapeutic drug is cisplatin, doxorubicin, VPI 6, taxol, or TNF The pharmaceutical combination of claim 63, wherein the chemotherapeutic drug is cisplatin The pharmaceutical combination of claim 63, wherein the chemotherapeutic drug is taxol a The pharmaceutical combination of claim 63, wherein the chemotherapeutic drug is doxorubicin The pharmaceutical combination of claim 63, wherein the chemotherapeutic drug is VP16 The pharmaceutical combination of claim 63, wherein the chemotherapeutic drug is TNF 72 The pharmaceutical combination of claim 63, wherein the new-suppressing gene product gene and the chemotherapeutic drug are comprised in the same pharmaceutical composition 73 A therapeutic kit comprising in suitable container, a pharmaceutical formulation of an new-suppressing gene product, and a pharmaceutical formulation of a chemotherapeutic drug 74 The kit of claim 73, wherein the pharmaceutical formulation of an new-suppressing gene product and the pharmaceutical formulation of a chemotherapeutic drug are present in the same container 75 The kit of claim 73, wherein the pharmaceutical formulation of an new-suppressing gene product and the pharmaceutical formulation of a chemotherapeutic drug are present within distinct containers

Inventors: HUNG MIEN-CHIE, UENO NAOTO T, HUNG, MIEN-CHIE, UENO, NAOTO, T.
Applicants: HUNG MIEN CHIE, UENO NAOTO T, UNIV TEXAS, BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM, HUNG, MIEN-CHIE, UENO, NAOTO, T.
Application year: 1997
Main classification: C12N15
IPC codes: A61K9, A61K31, A61K38, A61K45, A61K48, A61P35, C12N5, C12N15