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CANCER APOPTOSIS 1088 Radiation Prior Vaccinia-p53 Infection Significantly Reduces Survival of Cultured Glioma Cells 1089 VEGF Promoter-Based Conditionally Replicative Adenoviruses Are Useful for the Treatment of Lung Cancer Tatyana M Timiryasova,1 Bing Chen,1 Istvan Fodor.1 Center for Molecular Biology and Gene Therapy, Loma Linda University, Loma Linda, CA Koichi Takayama,1 Junji Uchino,1 Akiko Ikegami,1 Paul N Reynolds,2 Yasuo Adachi,3 Lioudmila Kaliberova,3 Victor N Krasnykh,3 Yoichi Nakanishi,1 David T Curiel.3 Research Institute for Diseases of the Chest, Kyushu University, Fukuoka, Fukuoka, Japan; 2Chest Clinic, Royal Adelaide Hospital, Adelaide, Australia, Australia; 3Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, University of Alabama at Birmingham, Birmingham, AL Previously we reported that that gamma-radiation treatment of established C6 glioma xenografts in nude mice followed by vaccinia virus mediated p53 treatment resulted in prolonged p53 transgene expression and significant tumor growth inhibition (Timiryasova et al., 2001) In the present study, we analyze the effect and mechanism of combination treatment of cultured C6 (p53+) and 9L (p53-) glioma cells using replication-competent and replication-deficient recombinant vaccinia viruses First, we compared the effect of two alternative treatments on cell survival in which radiation (6 Gy) was applied either prior or after virus infection Little, if any, difference was observed between radiation alone and combination of virus infection followed by radiation, for both C6 and 9L cell lines However, when radiation was applied prior to virus infection the combination treatment significantly reduced cell survival, especially 9L cells, compared to radiation alone Thus, of two compared strategies radiation prior virus infection was more effective in reduction of cell survival High level of p53 protein expression (1.5 μg/ml) mediated by VV-p53 (but not by L15 control virus), was measured by ELISA in infected cells (MOI =5) on days and post-infection Then, we analyzed replication-deficient virus strains generated by psoralen and UV inactivation (PUV) Single modality treatment of C6 glioma cells with PUV-inactivated VV-p53 virus or radiation caused significant decrease of surviving cells compared with PUVinactivated L-15 control virus However, little, if any, difference was observed between radiation and combination treatments of C6 cells Results were different using 9L glioma cells, which are more resistant to radiation compared to C6 cells Combined treatment by radiation followed by PUV-inactivated L-15, and especially VVp53, resulted in drastic reduction of cell viability, compared to single modality treatments Furthermore, early and late stages of apoptosis/ necrosis of 9L glioma cells were analyzed by flow cytometry of Annexin-V-stained cells Data showed that radiation and PUVinactivated VV-p53 virus infection caused significant decrease in percentage of live cells (17.2%) as compared to other treatments and control (61.6 - 98.3%) Apoptotic events were observed in 37.2% of cells, while the percentage of apoptotic cells in of other treatment groups were in the range 0.7-7.8% Maximal level of PUVVV-p53 mediated p53 was measured on day post-infection Thus, low-dose radiation of cells prior vaccinia virus infection is a promising strategy for p53-mediated therapy of glioma cells Reference: T.M Timiryasova et al Cancer Gene Therapy 8, (Suppl 2), S20, PD-67, 2001 [The view, opinion, and/or findings contained in this report are those of the author(s) and should not be construed as a position, policy, decision or endorsement of the federal government or the National Medical Technology Testbed, Inc Supported in part by an award from the U.S Army and NMTB, Inc.] Treatment of advanced lung cancer is one of the major challenges in current medicine because of the high morbidity and mortality of the disease Advanced stage lung cancer is refractory to conventional therapies and has an extremely poor prognosis Thus, new therapeutic approaches are needed Lung tumor formation depends on angiogenesis in which the VEGF produced by cancer cells plays a pivotal role Neutralizing VEGF with a soluble VEGF receptor suppresses tumor growth, however, the anti-cancer effect with this therapy is weakened after the intratumoral vascular network is completed In this study, we turned the expression of VEGF by tumors to therapeutic advantage using a conditionally replicationcompetent adenovirus (CRAd) in which the expression of E1 is controlled by the human VEGF promoter This virus achieved good levels of viral replication in lung cancer cells and induced a substantial anti-cancer effect in vitro and in vivo As a further enhancement the cancer cell killing effect was improved with tropism modification of the virus to express the knob domain of Ad3, which improved infectivity for cancer cells These VEGF promoter-based CRAds also showed a significant cell killing effect for various types of cancer lines other than lung cancer Conversely, the VEGF promoter has low activity in normal tissues, and the CRAd caused no damage to normal bronchial epithelial cells Since tumor-associated angiogenesis via VEGF signaling is common in many types of cancers, these CRAds may be applicable to a wide range of tumors We also investigated the synergistic anticancer effect by co-infection of CRAd and non-replicative AdCMVTK containing HSV tyrosine kinase gene In the co-existence of CRAd, AdCMVTK replicated with E1 protein supplied from CRAd, and produced much TK protein The pre-established tumor formed on the nude mice disappeared by coinjection of the both viruses intratumorally While each of the viruses showed only partial suppression of the tumor growth We concluded that VEGF promoter-based CRAds have the potential to be an effective strategy for cancer treatment 1090 VEGF Promoter-Based Conditionally Replicative Adenoviruses Are Useful for the Treatment of Malignant Pleural Mesothelioma Koichi Takayama,1 Lioudmila Kaliberova,2 Akiko Ikegami,1 Junji Uchino,1 Yasuo Adachi,2 Yoichi Nakanishi,1 David T Curiel.2 Research Institute for Diseases of the Chest, Kyushu University, Fukuoka, Fukuoka, Japan; 2Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, University of Alabama at Birmingham, Birmingham, AL Malignant pleural mesothelioma, MPM is an asbestos-related malignancy characterized by rapidly progressive and diffusely local growth, late metastases, and poor prognosis Chemotherapy or radiation therapy, alone or in combination, is not effective, and the majority of patients with MPM die within years regardless of treatment Therefore, new therapeutic approaches are needed The tumor formation of malingnant mesothelioma depends on angiogenesis in which the VEGF produced by cancer cells plays a pivotal role In our previous work, we developed a conditionally replication-competent adenovirus (CRAd) in which the expression S420 Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy CANCER APOPTOSIS of E1 is controlled by the human VEGF promoter, AdVEGFE1 This virus achieved good levels of viral replication in lung cancer cells and induced a substantial anti-cancer effect in vitro and in vivo Since many MPM tumors are kown to produce VEGF, VEGF promoter based CRAd has a potential to suppress a VEGF expressing MPM tumor growth Six MPM cell lines tested showed the significant VEGF mRNA and protein expression The cancer cell killing effect by AdVEGFE1 was roughly correlated with the extent of VEGF mRNA expression and the results of luciferase assay using AdVEGFLuci We also investigated the in vivo anticancer effect of AdVEGFE1 to MPM tumor established in the pleural space of the nude mice In this experiment, only two MPM cell lines were selected by the tumor forming ability after the screening of all six MPM cell lines When these intact MPM cells were premixed with infected cells by AdVEGFE1 at the ratio of 10%, tumor formation was suppressed significantly The results suggest the VEGF promoter based CRAd is the promising strategy for the MPM tumors 1091 Gene Therapy-Based Intracellular Cytotoxicity Using the Shigatoxin 1A1 Gene Katsutoshi Nakayama,1 Robert G Pergolizzi,1 Yadi Tan,1 Neil R Hackett,1 Ronald G Crystal.1 Weill Medical College of Cornell University, New York, NY The focus of this study is to develop a gene therapy-based intracellular cytotoxic agent that can be used to induce tumor cells to undergo cell death Shiga toxins, produced by Shigella dysenteriae (Stx) or by Escherichia coli (Stx1, Stx2, Stx2c and Stx2e), function to disrupt ribosome function and thus inhibit protein synthesis Shiga toxin type1 subunit A1 (Stx1A1), a fragment of Stx1 containing its enzymatic activity, acts as a specific N-glycosidase, cleaving a single adenine residue from 28S ribosomal RNA, resulting in the inhibition of protein synthesis and induction of apoptosis Although secreted Stx1can function to bind to cells and thus is risky to use as a cytotoxic agent, Stx1A1 has the potential to be an effective therapeutic cytoxic agent for cancer, in that it cannot enter cells, and thus can only function to destroy the cell to which the gene is delivered To develop Stx1A1 as a gene therapy-based intracellular cytotoxic agent, we modified the Stx1A1 gene so the protein product could not be secreted and cause systemic toxic effects Since Stx is cytotoxic to almost all cell types, we expected that cells infected with Stx1A1 should be killed However, RT-PCR on RNA from HeLa cells transfected with Stx1A1 cDNA revealed unexpected splicing secondary to the mammalian cells recognizing a cryptic “intron” irrelevant to the bacteria The cryptic splicing was corrected by modifying the sequence with silent site-directed mutations to produce “Stxm1.” Transfection with plasmid Stxm1 revealed the expected phenotype, with cytotoxicity induced in HeLa, NIH3T3, CT26 and 293 cells Based on these data we hypothesized that expression of Stxm1 in hepatocytes should induce liver damage To study the expression of Stxm1 plasmid in vivo, we used hydrodynamic injection of plasmid via the mouse tail vein Analysis of liver from Balb/c mice injected with Stxm1 showed no increase in apoptosis or cell death but revealed another unexpected splicing event, detected by RT-PCR and confirmed by sequencing Consequently, a second set of silent, site-directed mutations were introduced into Stxm1 to circumvent this cryptic splicing to create Stxm2 In vivo administration of Stxm2 in Balb/c mice showed expression of Stx mRNA with the correct size and sequence There was obvious damage to liver detected by histology compared to a null plasmid and elevated serum levels of liver enzymes compared to a null plasmid or untreated mice If this obligate intracellular toxin can be targeted to specific cells by an appropriate choice of vector and promoter, it should be useful for application to reduction of tumor burden and possibly to tissue remodeling in vivo Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright ® The American Society of Gene Therapy Dr Crystal has equity in, is a consultant to, and receives sponsored research funds from, GenVec, Inc., Gaithersburg, Maryland, a publicly-traded biotechnology company 1092 Adenoviral P53-Gene Transfer Increases the Response of HT-29 Colorectal Cancer Cells to 5-Fluorouracil Thomas Heinicke,1 Tilman Sauerbruch,1 Wolfgang H Caselmann.1 Internal Medicine I, Universtiy of Bonn, Bonn, Germany Introduction: Mutations in the p53 gene can lead to chemoresistance of malignant tumors In metastases from colorectal cancer (CRC) p53 mutations are found in about 60% of the cases 5fluorouracil (5-FU) is the most important drug for the treatment of metastatic CRC Despite recent improvements, overall survival in stage IV CRC is only 16 months Aim: In this study we investigated whether adenovirus mediated expresion of wild-type p53 in HT-29 CRC-cells which lack functional P53 can increase the response to 5FU Material and Methods: HT-29 cells were transduced with a wild-type p53 encoding adenoviral vector (Adp53) at a multiplicity of infection (moi) of 200 As a control an adenoviral vector (AdGFP) encoding the Green-Fluorescent-Protein (GFP) was used On day two 5-FU was added to the culture medium at a concentration of 20 μM or 50 μM Cell growth was determined for seven days afterwards In addition, the occurance of apoptosis was assessed using flow cytometry after propidium iodine staining Results: 5FU suppresses the growth of HT-29 cells by 90% compared to untreated controls in both concentrations used Cell counts dropped to 20% of the starting number only if wild-type p53 was transduced prior to 5-FU treatment Adp53 transduced cells without 5-FU treatment continued to grow after a lag period On day eight cells treated with Adp53 and 5-FU had a 80% reduced cell number when compared to untransduced cells Flow cytometry after propidium iodine staining showed an increase in apoptosis in Adp53 and 5-FU treated cells compared to either untransduced or AdGFP transduced cells Conclusion: The response of HT-29 CRC cells which lack a functional P53 to 5-FU can be increased efficiently by adenoviral gene transfer of wild-type p53 An increase in apoptosis appears to be an important mechanism for this effect Animal studies in nude mice are necessary to test whether this synergism can be obtained also in vivo 1093 Vaccinia Virus Mediated Expression of Human APC Induces Apoptosis in Colon Cancer Cells Sarah Umphress,1 Tatyana Timiryasova,1 Takeshi Arakawa, Istvan Fodor,1 William Langridge.1 Center for Molecular Biology and Gene Therapy, Loma Linda University, School of Medicine, Loma Linda, CA, United States Mutations in the adenomatous polyposis coli (apc) gene disrupt homeostatic pathways controlling enterocyte cell growth, division, and death resulting in adenoma formation leading ultimately to colon carcinoma A recombinant vaccinia virus containing the human apc gene (rVV-APC) was constructed and its effect on cultured colon cancer cells analyzed Recombinant vaccinia virus mediated transfer of the apc gene into human SW480, T84 and HCT116 adenocarcinoma cells containing an altered, deleted or full lengthapc gene respectively, resulted in synthesis of full length APC protein detected by immunoblot analysis Significant decreases in viability of the virus infected SW480, T84 and HCT116 cells were detected by TTC assay The decrease in cell viability correlated with increased levels of apoptotosis measured by cytochemistry, DNA fragmentation analysis, TUNEL assay and flow cytometry Increases in cell apoptosis were independent of the presence of endogenous APC The experimental results show that recombinant vaccinia S421 ... When these intact MPM cells were premixed with infected cells by AdVEGFE1 at the ratio of 10%, tumor formation was suppressed significantly The results suggest the VEGF promoter based CRAd is the. .. effect of AdVEGFE1 to MPM tumor established in the pleural space of the nude mice In this experiment, only two MPM cell lines were selected by the tumor forming ability after the screening of all... expression The cancer cell killing effect by AdVEGFE1 was roughly correlated with the extent of VEGF mRNA expression and the results of luciferase assay using AdVEGFLuci We also investigated the in