Surgery, radiation therapy, and chemotherapy have been applied to the curative therapy of 50% of cancer patients in the United States during the past 100 years. It is clear that the chemotherapeutic agents used to develop curative therapy for leukemias, lymphomas, gestational malignancy, and testicular cancer are not as active in the more numerous epithelial neoplasms, perhaps because of the complexity of genetic change in these latter neoplasms.

OBJECTIVE To investigate the efficiency of the cytosine deaminase adenoviral/5-fluorocytosine system on prostate cancer cell lines. METHODS We used cell culture, infectivity and sensitivity tests, to observe bystander effect by animal tests. RESULTS Established prostate cancer cell lines are eventually infectible by adenoviral vector. The ratio of vector/cell at which infection occurs depends on the specific cell line. The peak of expression of the transferred cytosine deaminase gene occurred in cells at different time, but persisted beyond 11 days. These prostate cell lines are sensitized to 5-fluorocytosine by infection with adenoviral vector carrying the cytosine deaminase gene. Only 5% of the LNCap and 10% of the RM-1 cells were infected and produced 100% cell death. In the animal test, there was significant inhibition of tumor growth at a ratio of 400 vector particles/cell with the systematic treatment of 5-fluorocytosine. CONCLUSIONS Adenoviral vector carrying a cytosine deaminase transcription unit can sensitize prostate cancer cell lines to 5-fluorocytosine. The system can significantly inhibit the growth of prostatic tumors in mice.

Ad.CMV-CD is a replication incompetent adenoviral vector carrying a cytomegalovirus (CMV)-driven transcription unit of the cytosine deaminase (CD) gene. The CD transcription unit in this vector catalyzes the deamination of the nontoxic pro-drug, 5-fluorocytosine (5-FC), thus converting it to the cytotoxic drug 5-fluorouracil (5-FU). This adenoviral vector prodrug activation system has been proposed for use in selectively sensitizing breast cancer cells, which may contaminate collections of autologous stem cells products from breast cancer patients, to the toxic effects of 5-FC, without damaging the reconstitutive capability of the normal hematopoietic cells. This system could conceivably kill even the nondividing breast cancer cells, because the levels of 5-FU generated by this system are 10 to 30 times that associated with systemic administration of 5-FU. The incorporation of 5-FU into mRNA at these high levels is sufficient to disrupt mRNA processing and protein synthesis so that even nondividing cells die of protein starvation. To test if the CD adenoviral vector sensitizes breast cancer cells to 5-FC, we exposed primary explants of normal human mammary epithelial cells (HMECs) and the established breast cancer cell (BCC) lines MCF-7 and MDA-MB-453 to the Ad.CMV-CD for 90 minutes. This produced a 100-fold sensitization of these epithelial cells to the effects of 48 hours of exposure to 5-FC. We next tested the selectivity of this system for BCC. When peripheral blood mononuclear cells (PBMCs), collected from cancer patients during the recovery phase from conventional dose chemotherapy-induced myelosuppression, were exposed to the Ad.CMV-CD for 90 minutes in serum-free conditions, little or no detectable conversion of 5-FC into 5-FU was seen even after 48 hours of exposure to high doses of 5-FC. In contrast, 70% of 5-FC was converted into the cytotoxic agent 5-FU when MCF-7 breast cancer cells (BCCs) were exposed to the same Ad.CMV-CD vector followed by 5-FC for 48 hours. All of the BCC lines tested were shown to be sensitive to infection by adenoviral vectors when exposed to a recombinant adenoviral vector containing the reporter gene betagalactosidase (Ad.CMV-betagal). In contrast, less than 1% of the CD34-selected cells and their more immature subsets, such as the CD34+CD38- or CD34(+)CD33- subpopulations, were positive for infection by the Ad.CMV-betagal vector, as judged by fluorescence-activated cell sorting (FACS) analysis, when exposed to the adenoviral vector under conditions that did not commit the early hematopoietic precursor cells to maturation. When artificial mixtures of hematopoietic cells and BCCs were exposed for 90 minutes to the Ad.CMV-CD vector and to 5-FC for 10 days or more, a greater than 1 million fold reduction in the number of BCCs, as measured by colony-limiting dilution assays, was observed. To test if the conditions were damaging for the hematopoietic reconstituting cells, marrow cells collected from 5-FU-treated male donor mice were incubated with the cytosine deaminase adenoviral vector and then exposed to 5-FC either for 4 days in vitro before transplantation or for 14 days immediately after transplantation in vivo. There was no significant decrease in the reconstituting capability of the male marrow cells, as measured by their persistence in female irradiated recipients for up to 6 months after transplantation. These observations suggest that adenovirus-mediated gene transfer of the Escherichia coli cytosine deaminase gene followed by exposure to the nontoxic pro-drug 5-FC may be a potential strategy to selectively reduce the level of contaminating BCCs in collections of hematopoietic cells used for autografts in breast cancer patients.

Genetic modification for cancer treatment has involved the introduction of chemotherapy protection and sensitization genes into normal and tumor cells, respectively, for the purpose improving the outcome of conventional approaches to the treatment of solid tumor neoplasms. This paper will review the use of multidrug resistance‐1 retroviral vectors and cytosine deaminase adenoviral prodrug activation vectors for this purpose.

Objective To investigate the function of the alpha v beta 5 integrin in hematopoietic cells.Methods Tissue culture, integrin expression vectors, gene transfer, polymerase chain reaction (PCR), apoptosis analyses and cytometic analysis were made on hematopoietic cells.Results The beta 5 integrin cDNA was not expressed in hematopoietic cells following exposure to the beta 5 integrin retrovirus vector pG beta 5CHT. Unbalanced expression of the alpha v beta 3 and alpha v beta 5 integrins occurred during apoptosis induced by serum depletion and upon differentiation. The treatment of hematopoietic cells with anti-alpha v beta 5 monoclonal antibody inhibited apoptosis induced by serum depletion. inducible expression of the beta 5 integrin cDNA in the hematopoietic cell line K562 caused cellular proliferation inhibition.Conclusion The alpha v beta 5 integrin cDNA in hematopoietic cells can inhibit the proliferation of the hematopoietic cell, cause the differentiation of the hematopoietic cells and induce the apoptosis of the hematopoietic cells.

The large size of the adenoviral genome unfortunately precludes there being many unique, useful restriction sites available for in vitro manipulation, Two methods have been developed for the construction of recombinant adenoviral vectors to date: in vivo homologous recombination or direct ligation in vitro, The efficiency of either the direct ligation method or the homologous recombination method is low because of the large size of the recombinant adenoviral vectors, To circumvent these problems, we have chosen to use the cosmid vector system to facilitate the assembly of recombinant adenoviral vectors, In this paper, we demonstrate for the first time that recombinant adenoviral vectors can be efficiently constructed in vitro by the cosmid vector system. With this method, it is possible to amplify the recombinant adenoviral vector DNA sufficiently to transfect 293 cells, The cosmid adenoviral vector cloning method for in vitro construction of the full-length recombinant adenoviral vectors represented here is simple and efficient and should facilitate the development of recombinant adenoviral vectors for human gene therapy.

Current approaches to the treatment of cancer (radiation and chemotherapy) are limited by the toxicity to normal tissues and the sensitivity of tumor cells to these treatments. The use of gene delivery vector systems for the introduction of genetic elements into normal and neoplastic tissues which code for proteins that protect the normal chemotherapy-sensitive tissues and sensitize the tumor cells to radiation and chemotherapy, may contribute to an overall improvement in the outcome of cancer treatment programs. This chapter will review the clinical trials either completed or about to be initiated which are designed to accomplish these goals.

The MDR1 gene product P-glycoprotein (P-gp) extrudes several anticancer drugs including taxol and fluorescent dyes such as rhodamine (Rh123). Modulation of the level of P-gp expression has the potential of overcoming multidrug resistance. One possible approach is the retroviral transfer of the human MDR1 gene into murine and human bone marrow (BM) progenitor cells. The rationale for this approach is increased chemoprotection, which allows chemotherapy of a greater level of intensity to be delivered. In this study, flow cytometric measurement of Rh123 extrusion was used to test P-gp function in human and mouse haemopoietic progenitor cells, which had been transduced with a virus containing the human MDR1 transcription unit. Human CD34+ selected cells were analysed immediately following transduction. In two successive experiments MDR1 cDNA transduction resulted in a 7% and 11% increase of P-gp expressing Rh123 dull cells. To monitor transduction efficiency over time as well as the possibility of in vivo selection of drug-resistant BM cells in mice treated with increasing numbers of taxol cycles, the assay was also successfully applied to peripheral blood lymphocytes of mice transplanted with MDR1 transduced BM cells, demonstrating increased Rh123 efflux in transduced cells. Analysis of another fluorescence assay using fluorescein di-beta galactopyranoside as a substrate for beta-galactosidase in cells transduced with a MDR1: beta-gal activity. We conclude that the Rh123 efflux assay is a sensitive method to monitor P-gp function in MDR1 cDNA transduced cells, and may be used to enrich transduced cells via flow cytometric cell sorting for Rh123 dull cells.

Human cervical carcinoma cell lines that harbor human papilloma virus (HPV) have been reported to express HPV E6 and E7 proteins at least in the beginning stages if not at all stages of the disease. The HPV E6 and E7 proteins bind to and inactivate the products of the p53 and retinoblastoma (Rb) tumor suppressor genes, which thereby allow the cervical carcinoma cells to circumvent the action of these tumor suppressor genes. We observed that the introduction of the antisense HPV 18 E6 and E7 sequences, as well as a sense cDNA for the human wild-type Rb gene into a human cervical carcinoma cell line (HeLa), which is positive for the HPV 18 provirus, decreased the in vitro and in vivo growth rate of the transfected cells if both antisense transcripts for the HPV 18 E6 and E7 and sense transcripts for human Rb were expressed. In addition, overexpression of a complementary DNA (cDNA) for the Rb messenger RNA was sufficient to slow the proliferation of HeLa cells, and the level of Rb cDNA expression was correlated with the degree to which the rate of growth of the tumor was slowed. The results of our experiments show that the presence of HPV E6 and E7 proteins and the resultant inactivation of Rb in cervical carcinoma cells contributes to the neoplastic phenotype even in highly evolved cervical carcinoma cell lines such as HeLa, which have been derived from a cervical carcinoma patient at an advanced stage of the disease process. These data suggest that the HPV proteins play a role not only at the beginning of cervical cancer, but also at advanced stages of this disease. These experiments may lead to genetic approaches to the control of this disease that involve antisense sequences that downregulate the E6 and E7 genes or lead to expression of the Rb gene.

Relapse after autologous bone marrow transplantation for chronic myelogenous leukemia (CML) can be due either to the persistence of leukemia cells in systemic tissues following preparative therapy, or due to the persistence of leukemia cells in the autologous marrow used to restore marrow function after intensive therapy. To help distinguish between these two possible causes of relapse, we used safety-modified retroviruses, which contain the bacterial resistance gene NEO, to mark autologous marrow cells that had been collected from patients early in the phase of hematopoietic recovery after in vivo chemotherapy. The cells were then subjected to ex vivo CD34 selection following collection and 30% of the bone marrow were exposed to a safety-modified virus. This marrow was infused after delivery of systemic therapy, which consisted of total body irradiation (1,020 cGy), cyclophosphamide (120 mg/kg), and VP-16 (750 mg/m2). RT PCR assays specific for the bacterial NEO mRNA, which was coded for by the virus, and the bcr-abl mRNA showed that in two evaluable CML patients transplanted with marked cells, sufficient numbers of leukemia cells remained in the infused marrow to contribute to systemic relapse. In addition, both normal and leukemic cells positive for the retroviral transgenome persisted in the systemic circulation of the patients for at least 280 days posttransplant showing that the infused marrow was responsible for the return of hematopoiesis following the preparative therapy. This observation shows that it is possible to use a replication-incompetent safety-modified retrovirus in order to introduce DNA sequences into the hematopoietic cells of patients undergoing autologous bone marrow transplantation. Moreover, this data suggested that additional fractionation procedures will be necessary to reduce the probability of relapse after bone marrow transplantation in at least the advanced stages of the disease in CML patients undergoing autologous bone marrow transplantation procedures.