designed to determine if transducing a Bcl-XL gene, a potent anti- apoptotic gene, to the ... infection, Ad-Bcl-XL group demonstrated significantly lower number.
ADENOVIRAL VECTORS: CLINICAL APPLICATIONS with adenovectors based on alternative serotypes show the ability of these vectors to efficiently transduce cells within the inner ear. Alternative serotyped adenovectors were evaluated that have low pre-existing neutralizing antibody titers in the human population. Interestingly, Ad35 based vectors effectively deliver GFP to both hair cells and the spiral ganglion. In conclusion, both Ad5 based vectors and Ad35 based vectors were found to be well tolerated in the inner ear of the mouse and can form the basis of developing flexible drug delivery systems for the inner ear. Douglas E Brough and Chi Hsu are employees of GenVec Inc.
833. Combined Manganese Superoxide Dismutase Gene Therapy and 5-FU Chemotherapy for Colorectal Cancer Yanhong Zhang,1,2 Lingfeng He,1 Songqing He,2 Jian Wu,2 Xinyuan Liu.1,4 1 Biochemistry and Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai, China; 2Department of Internal Medicine, Transplant Research Program, University of California, Davis Medical Center, Sacramento, CA; 3Xinyuan Institute of Medicine and Biotechnology, School of Life Science, Zhejiang Sci-Tech University, Hangzhou, Zhejing, China. BACKGROUND: Colorectal cancer (CRC) is one of the most common tumors worldwide, with approximately 1 million new cases diagnosed in 2000, and accounting for over 500,000 deaths. Novel agents that target specific molecular pathways are needed. A replication-competent adenoviral transduction of manganese superoxide dismutase (MnSOD) inhibited growth of colorectal tumor cells in culture and tumor xenografts in a mouse model (Cancer Research; 2006 in press). Fluorouracil (5-FU) is the most widely used anticancer agent for gastrointestinal cancers, and it is commonly used as adjuvant chemotherapy for colorectal cancer. The aim of this study is to assess the effect of MnSOD gene therapy combined with chemotherapy, on tumor growth and tumor-free survival. METHODS: Nude mice were divided into four groups: PBS control, ZD55-MnSOD injection, 5-FU injection, combined ZD55-MnSOD with 5-FU injection. 5-FU was administered intraperitoneally every other day after four times injection with ZD55-MnSOD. Cell cycles were assayed by FACS. DNA fragmentation, transition electronic microscope and TUNEL assay were used to determine the extent of apoptosis. RESULTS: There was a remarkable decrease in cell viability after colorectal tumor cells were treated with ZD55MnSOD at various MOI and 5-FU from 24 hours to 72 hours. Enhanced apoptosis in tumor cells receiving the dual therapy was evident as determined by DNA fragmentation analysis, Hoechst 33258 staining, electron microcopy, and a TUNEL assay. Growth of tumor xenografts was significantly inhibited in nude mice 2 months after intratumoral injection of ZD55-MnSOD. The tumor volume decreased significantly in mice after receiving combined injection of ZD55-MnSOD and 5-FU, was much lower than that in mice receiving injections of ZD55-MnSOD or 5-FU alone (80.98±31.76 vs. 792.11±69.70, 1050±435.39 mm 3). Moreover, the combined injection of ZD55-MnSOD with 5-FU markedly improved the animal survival, which was better than ZD55-MnSOD or 5-FU alone (66.7% vs. 50% and 33.3%). CONCLUSION: Both ZD55MnSOD and 5-FU are shown to induce the apoptosis in colorectal tumor cells, while 5-FU potentiated the proapoptotic effect induced by ZD55-MnSOD. It appears that a caspase-dependent pathway is involved in the apoptotic onset in colorectal tumor cells caused by 5-FU or ZD55-MnSOD. Thus, dual adenoviral MnSOD gene therapy plus 5-FU chemotherapy achieved synergetic action in suppressing the tumor growth.
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834. Protective Effect of Bcl-XL Gene Expression on the Radiation-Induced Acute Intestinal Injuries Kazuaki Uchimoto,1 Fumikazu Koyama,1 Kazuo Ohashi,1 Kenichi Ohtsuki,1 Hirofumi Hamada,2 Takeo Nomi,1 Hisao Fujii,1 Tomohide Mukogawa,1 Yoshiyuki Nakajima.1 1 Department of Surgery, Nara Medical University, Kashihara, Nara, Japan; 2Department of Molecular Medicine, Sapporo Medical University, Sapporo, Hokkaido, Japan. [Background] Intestinal injuries represent a major dose-limiting side effect of radiation treatment for many malignancies. Recent studies have demonstrated that mitochondrial damages occurred in the mucosal epithelial cells and subsequent release of cytochrome c from the damaged mitochondria resulting in apoptotic changes, are mainly involved in this type of injuries. The present study was designed to determine if transducing a Bcl-XL gene, a potent antiapoptotic gene, to the epithelial cells could provide protective effects on the radiation-induced acute injuries of the small intestine. [Methods] C57Bl/6 mice were used in this study. Adenoviral vectors containing the human Bcl-XL gene (Ad-Bcl-XL) or β-galactosidase gene (Ad-LacZ) driven by CAG promoter were generated. In order to increase the transduction efficiency to the mucosal epithelium, especially to the intestinal crypts, we first washed the intraluminal space of the small intestine with PBS and digested mucus components with pronase. Adenoviral vector infections were performed by injecting 2x10(8) pfu adenoviral vectors into 2cm portion of the pronase-treated small intestine that had been transiently clamped on both side. These clamps were removed 30 min after the vector infusion. Transduction efficiencies of the mucosal epithelial cells were examined by X-gal staining 24 hours after Ad-LacZ infection. Radiation-induced acute injuries of the small intestine were introduced by whole body irradiation (15 Gy) performed 24 hours after the adenoviral vector infection. Six hours after the radiation, apoptotic epithelial cells were visualized by TUNEL assay. Seventy-two hours after the radiation, morphological appearance of the small intestines was assessed by histological examination. [Results] With the method for adenoviral vector infection developed herein, successful transfection could be achieved to the intestinal mucosa determined by X-gal staining. When we irradiated to the mice after the vector infection, Ad-Bcl-XL group demonstrated significantly lower number of radiation-induced apoptotic mucosal epithelial cells compared with the other two control groups examined 6 hours after the irradiation (P
