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1083. Pretreatment of DU145 Prostate Cancer Cells with Ceramidase Inhibitors Can Potentiate the Apoptotic Effect of AdGFPFasLTET
CANCER APOPTOSIS 1080. Synergistic Cytotoxic Effect of Antisense K-ras RNA and Interferon alpha Against Pancreatic Cancer Cells Yoshiaki Miura,1 Koichi Suzuki,1 Kazuteru Hatanaka,1 Kimiko Yoshida,1 Shumpei Ohnami,1 Yukio Kitade,2 Teruhiko Yoshida,1 Kazunori Aoki.3 1 Genetics Division, National Cancer Center Research Institute, Tokyo, Japan; 2Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan; 3Section for Studies on Host-Immune Response, National Cancer Center Research Institute, Tokyo, Japan. Adenocarcinoma of the pancreas is highly aggressive and is one of the most difficult cancers to treat at present. Interferon alpha (IFNalpha) proteins are cytokines with multiple biological activities that include antiviral activity, regulation of cell proliferation and differentiation of immunomodulation, and IFN-alpha is used worldwide for the treatment of a variety of cancers. Growth inhibitory effect of IFN-alpha protein has been documented in pancreatic cancer even if it is not satisfactory in clinical trials. In this study, we first investigated the antiproliferative effect of IFN-alpha gene transfer in pancreatic cancer cells, and found that IFN-alpha gene expression significantly suppressed cell growth and effectively induce cell death in pancreatic cancer. It is known that IFN-inducible 2’, 5’-oligoadenylate synthetase (2-5AS)/RNase L pathway, which induces apoptosis of cells, is activated with double strand RNA. Pancreatic cancer has a characteristically high incidence of K-ras point mutation and we previously reported that the expression of antisense K-ras RNA significantly suppressed the growth of pancreatic cancer cells. The double strand RNA formed by the binding of antisense and endogeneous K-ras RNA might enhance the IFNalpha-induced activation of 2-5AS/RNase L pathway in pancreatic cancer cells. Thus, next we investigated whether combination of antisense K-ras with IFN-alpha gene transfer could effectively inhibit the growth of pancreatic cancer. The antisense K-ras expression significantly enhanced the IFN-induced cell death (1.3 - 4 fold), and effectively suppress the subcutaneous growth of pancreatic cancer cells in mice. This study suggests that the combination of IFNalpha and antisense K-ras is a promising gene therapy strategy for pancreatic cancer.
1081. In Vivo Gene Therapy of Human Bladder Cancer with PTEN Suppresses Tumor Growth, Down-Regulates Ohosphorylated Akt, and Increases Sensitivity to Doxorubicin Motoyoshi Tanaka,1 Hirotsugu Uemura,1 Yoshihiko Hirao,1 H. Barton Grossman.2 1 Urology, Nara Medical University, Kashihara, Nara, Japan; 2 Urology, UT M.D. Anderson Cancer Center, Houston, TX, United States. The PTEN gene, located on chromosome 10, is a phosphatase in the phosphatidylinositol 3’-kinase (PI3’K) mediated signal transduction pathway. PTEN acts as a phosphatidylinositol phosphatase and appears to play a role in PI3’K signal transduction pathway. Introduction of PTEN into cells deficient in this gene inhibits the activation of Akt, a serine-threonine kinase. Akt is involved in a variety of proliferative and anti-apoptotic pathways that are dependent upon PI3’K signaling for activation. We previously showed that PTEN inhibits the growth of human bladder cancer cells in vitro and restores chemosensitivity to a doxorubicin resistant human bladder cancer cell subline. In this study, we used a PTEN adenoviral vector (Ad-MMAC) to assess the role of PTEN in the in vivo treatment of bladder cancers that differ in the PTEN pathway. Direct injection of Ad-MMAC into established subcutaneous Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
UM-UC-3 (PTEN deleted, up-regulation of phosphorylated Akt) and UM-UC-6dox (wild-type PTEN, up-regulation of phosphorylated Akt) tumors in nude mice resulted in PTEN expression, apoptosis, and significantly decreased growth compared to a control adenovirus vector without a transgene (Ad-CTR) or PBS treated tumors. UM-UC-3 tumors completely disappeared in all of mice treated with Ad-MMAC, but PBS and Ad-CTR treated UM-UC-3 tumors continued to grow rapidly. UM-UC-14 tumors (wild-type PTEN) were transiently suppressed by Ad-MMAC. This is particularly effective in cells that have genomic alterations of PTEN. Vascular endothelial growth factor (VEGF) promotes tumor growth by increasing angiogenesis. PTEN has been demonstrated to regulate VEGF and tumor induced-angiogenesis thorough the PI3’K signal pathway. We demonstrated that Ad-MMAC treatment of established UM-UC-3 tumors resulted in significant down-regulation of VEGF and decreased microvessel density (CD31 staining). Smaller decreases in microvessel density were seen in UM-UC-6dox, and the least effect was seen in UM-UC-14 tumors. The anti-angiogenic effect of PTEN therapy may have contributed to complete tumor regression seen in UM-UC-3 tumors. Failure of chemotherapy due to the development of drug resistant tumors is a significant clinical problem. We previously demonstrated that combined therapy with Ad-MMAC and doxorubicin enhanced the suppression of bladder cancer cell growth in vitro. Combination therapy with Ad-MMAC and doxorubicin improved the in vivo efficacy of PTEN gene therapy in the doxorubicin resistant cell line UM-UC-6dox. Treatment with Ad-MMAC and doxorubicin completely eradicated established UM-UC-6dox tumors in 30% of mice. UM-UC-14 tumors were transiently suppressed by this combined treatment. These data show that PTEN gene therapy can effectively treat bladder cancers that have genomic alterations in PTEN and has an anti-angiogenic effect. Furthermore, our data demonstrate that inactivating a specific target such as phosphorylated Akt with gene therapy combined with chemotherapy can provide effective treatment for drug resistant cancers such as UM-UC-6dox cells.
1082. Histone Deacetylase Inhibitors Evoke Apoptosis of Gastric Cancer Cells through the Induction of PIG3 and NOXA by the Acetylation of p53 at 320, 373 Lysine Residues Takeshi Terui,1 Minoru Takahashi,1 Ken Murakami,1 Rishu Takimoto,1 Koichi Takada,1 Junji Kato,1 Yasushi Sato,1 Tsutomu Sato,1 Tsuzuku Murakami,1 Yoshiro Niitsu.1 1 4th Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan. It has been a controversial issue whether p53 is involved in apoptosis induction of tumor cells by histone deacetylase (HDAC) inhibitor and given the p53 is indeed involved in, which genes of acetylated p53 targets are responsible for giving rise to apoptotic death. We, in the present study, first confirmed that apoptotic cell death was seen when p53 deficient cells (KATO-III) were transfected with wild type p53 and treated with sodium butyrate (SB) or trichostatin A (TSA). By western blotting, using specific antibodies, we then demonstrated that residues of 320, 373 and 382 lysine of p53 were acetylated in KATO-III cells transfectant with wild type p53 (KATO-III/p53) treated with HDAC inhibitor. However, only those KATO-III cells transfected with K320R p53 or K373R p53 but not K382R p53 became insensitive to HDAC inhibitor, as revealed by TUNEL staining, suggesting former two residues of p53 may be essential for HDAC inhibitor induced apoptosis. Further, RT-PCR demonstrated that amongst various p53 related proapoptotic genes, PIG3 and NOXA were induced by SB treatment in KATO-III/p53 cells. The induction of PIG3 and NOXA was not S417
CANCER APOPTOSIS observed in KATO-III/K320R and KATO-III/K373R cells treated with SB. Finally, we revealed that apoptosis could be evoked by SB even in cells with mutated p53 of which mutation residues were other than 320 lysine or 373 lysine and the apoptosis was suppressed by anti p53 antisense. In conclusion, it is suggested that the HDAC inhibitor can activate the p53 molecule by acetylation of 320 and 373 lysine residues, up-regulate PIG3 and NOXA and induce apoptosis in cancer cells with wild and pseudo-wild type p53 genes.
1083. Pretreatment of DU145 Prostate Cancer Cells with Ceramidase Inhibitors Can Potentiate the Apoptotic Effect of AdGFPFasLTET
Ahmed M. El-Zawahry,1 David H. Holman,1 Marc H. Hyer,2 David A. Schwartz,1 Alicja Bielawska,3 Lina M. Obeid,4 Yusuf A. Hannun,3 James S. Norris.1 1 Microbiology and Immunology, Medical University of South Carolina, Charleston, SC; 2The Burnham Institute, La Jolla, CA; 3 Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC; 4Medicine, Medical University of South Carolina, Charleston, SC. The recent availability of the human genome sequence and the continuing development of bioinformatic tools to analyze it suggest that our understanding of the causes of cancer will expand using the tools of genomics and proteomics and will reveal new signaling pathways as targets for cancer therapy. As far as cancer gene therapy is concerned, the difficulty will lie not in the choice of therapeutic genes per se but in our ability to deliver a corrective signal to every cell in the cancer for its eradication. Thus, studies on the delivery of therapeutic genes as well as studies on amplification of delivery systems are urgently required. In our DU145 model of prostate cancer, we have determined that resistance to the induction of apoptosis through the Fas receptor signaling pathway is due to overexpression of apoptotic resistance genes including cFLIPs [Hyer et al., Cancer Biology and Therapy 1(4): 405-410, 2002]. We have also determined that expression of a FasL-GFP fusion gene will overcome resistance in infected cells [Hyer et al., Molecular Therapy 2(4):348-358, 2000]. To overcome the delivery issue, we have determined that overexpression of the GFPFasL fusion protein overcomes resistance, kills the cell apoptotically, and produces apoptotic vesicles that also can signal Fas to induce apoptosis in adjacent cells (i.e. bystander activity) [Hyer et al., Cancer Gene Therapy, In Press, 2003]. However, because of expression of apoptotic resistance genes some cancers, including the DU145 model, are resistant to the apoptotic vesicles and are thus relatively insensitive to vesicle-mediated bystander activity. To overcome this, we have examined a number of different chemotherapeutic drugs and other small molecules, for their effect on apoptotic resistance mechanisms. We can show that doxorubicin (0.2 ug/ml) will decrease expression of cFLIP protein without a concomitant decrease in levels of FLIP mRNA. This decrease in protein levels may be due to proteasomal degradation or a translational block. Under these conditions, a 20% increase in sensitivity of apoptotic vesicles is observed with 0.2 ug/ml doxorubicin. However, 0.2 ug/ ml doxorubicin is itself toxic to DU145 at 48-72 hours which somewhat obscures the role of the bystander vesicles in promoting apoptosis. We have also examined LCL102, a ceramidase inhibitor, which acts by increasing intracellular ceramide levels. This molecule is highly efficient at activating cell death in DU145 cells at nontoxic doses when combined with AdGFPFasL virus at MOIs achievable in vivo. We are now studying the mechanism of this, vis-à-vis the bystander effect by examining how LCL102 modulates the mechanisms of resistance. These studies involve Mass Spectroscopic analysis of sphingolipid metabolism. Concomitant studies of proteasome function and alternative splicing of apoptosis resistance genes are underway and will be presented at the meeting. S418
(1st and 2nd Authors, Ahmed El-Zawahry and David Holman shared equally in this work).
1084. The Effect of E1A on Sensitization of Gastrointestinal Cancer Cell Lines Overexpressing HER-2/neu to Anticancer Agents Xi-Zhang Lin,1 Ni-Chin Ko,1 Jian-Chyi Chen,3 Hsiao-Sheng Liu,2 Hsiao-Sheng Liu Hu.1 1 Department of Internal Medicine, National Cheng Kung University, Tainan, Taiwan; 2Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan; 3 Department of Biotechnology, Southern Taiwan University of Technology, Tainan, Taiwan. BackgroundThe overexpression of HER-2/neu oncogene has been implicated in the development and modulation of many types of cancers. Adenovirus type 5 E1A gene is known to be able to suppress the overexpression of HER-2/neu AIM By delivering the E1A gene into the gastroenterological cancer cells to block the HER-2/ neuoverexpression, we expect to see that the E1A-transfected cancer cells will become more sensitive to the chemotherapeutic agents. Materials and methods SNU-5 cell (ATCC number CRL-5973) and AGS cell (ATCC number CRL-1739) are human gastric carcinoma and Colo 205 cell (ATCC number CCL-222) is a human colon adenocarcinoma. E1A gene driven by a CMV promoter in the plasmid pSG5 was delivered into the cells by an electroporation system. The electroporation regimen was 300 volts, 5 ms in duration. The protein expression of E1A and HER-2/neu in the transfected cells were checked by Western blotting. The cytotoxicity of pSG5 and E1A transfected cells to taxol (10 μg/ml, 15 μg/ml and 30 μg/ml) was determined by tetrazolium-based colorimetric assay. Results E1A gene was successfully delivered into all three cancer cells by electroporation. E1A expression resulted in down-regulation of HER-2/neu gene expression by Western blotting. However, only E1A-transfected AGS cell showed more sensitivity to taxol. After 24 hour treatment, the pSG5 and E1A transfected SNU-5 and Colo 205 cells showed no significant growth inhibition to taxol. The pSG5 transfected AGS cell showed 48.9 ± 5.5 %, 56.5 ± 7.4 %, and 81.8 ± 7.8 % inhibition rate at 10 μg/ml, 15 μg/ml, and 30 μg/ml of taxol, respectively. The E1A transfected AGS cell showed 61.4 ± 11.9 %, 66.9 ± 8.7 %, and 83.9 ± 5.7 % inhibition rate at 10 μg/ml, 15 μg/ml, and 30 μg/ml of taxol, respectively. Conclusion E1A gene can be successfully delivered into gastroenterological cancer cells by electroporation. The transfected E1A gene can suppress the expression of HER-2/neu. On the other hand, E1A transfection can enhance chemosensitivity in some cell lines, such as AGS cell, but the enhancement is not a universal phenomenon as expected.
1085. In Vivo and In Vitro Inhibition of Human Cancer Cell Growth Using Adenoviral Vector Expressing FHIT Gene: Mechanism Analysis Using Microarray Fumiaki Tanaka,1 Hideto Sonoda,1 Koshi Mimori,1 Takeshi Shiraishi,1 Hideshi Ishii,2 Carlo M. Croce,2 Hiroshi Inoue,1 Masaki Mori.1 1 Division of Surgical Oncology, Department of Molecular and Cellular Biology, Medical Institution of Bioregulation, Kyushu University, Beppu, Oita, Japan; 2Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, United States. The fragile histidine triad (FHIT) gene has been cloned from the chromosome FRA3B region, which is deleted in a large fraction of human tumor. Previous studies showed that the introduction of Fhit suppresses tumor cell growth. To further explore the pre-clinical cancer therapy by Fhit, we have studied a biological effect of H1299, Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
1081. In Vivo Gene Therapy of Human Bladder Cancer with PTEN Suppresses Tumor Growth, Down-Regulates Ohosphorylated Akt, and Increases Sensitivity to Doxorubicin Motoyoshi Tanaka,1 Hirotsugu Uemura,1 Yoshihiko Hirao,1 H. Barton Grossman.2 1 Urology, Nara Medical University, Kashihara, Nara, Japan; 2 Urology, UT M.D. Anderson Cancer Center, Houston, TX, United States. The PTEN gene, located on chromosome 10, is a phosphatase in the phosphatidylinositol 3’-kinase (PI3’K) mediated signal transduction pathway. PTEN acts as a phosphatidylinositol phosphatase and appears to play a role in PI3’K signal transduction pathway. Introduction of PTEN into cells deficient in this gene inhibits the activation of Akt, a serine-threonine kinase. Akt is involved in a variety of proliferative and anti-apoptotic pathways that are dependent upon PI3’K signaling for activation. We previously showed that PTEN inhibits the growth of human bladder cancer cells in vitro and restores chemosensitivity to a doxorubicin resistant human bladder cancer cell subline. In this study, we used a PTEN adenoviral vector (Ad-MMAC) to assess the role of PTEN in the in vivo treatment of bladder cancers that differ in the PTEN pathway. Direct injection of Ad-MMAC into established subcutaneous Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
UM-UC-3 (PTEN deleted, up-regulation of phosphorylated Akt) and UM-UC-6dox (wild-type PTEN, up-regulation of phosphorylated Akt) tumors in nude mice resulted in PTEN expression, apoptosis, and significantly decreased growth compared to a control adenovirus vector without a transgene (Ad-CTR) or PBS treated tumors. UM-UC-3 tumors completely disappeared in all of mice treated with Ad-MMAC, but PBS and Ad-CTR treated UM-UC-3 tumors continued to grow rapidly. UM-UC-14 tumors (wild-type PTEN) were transiently suppressed by Ad-MMAC. This is particularly effective in cells that have genomic alterations of PTEN. Vascular endothelial growth factor (VEGF) promotes tumor growth by increasing angiogenesis. PTEN has been demonstrated to regulate VEGF and tumor induced-angiogenesis thorough the PI3’K signal pathway. We demonstrated that Ad-MMAC treatment of established UM-UC-3 tumors resulted in significant down-regulation of VEGF and decreased microvessel density (CD31 staining). Smaller decreases in microvessel density were seen in UM-UC-6dox, and the least effect was seen in UM-UC-14 tumors. The anti-angiogenic effect of PTEN therapy may have contributed to complete tumor regression seen in UM-UC-3 tumors. Failure of chemotherapy due to the development of drug resistant tumors is a significant clinical problem. We previously demonstrated that combined therapy with Ad-MMAC and doxorubicin enhanced the suppression of bladder cancer cell growth in vitro. Combination therapy with Ad-MMAC and doxorubicin improved the in vivo efficacy of PTEN gene therapy in the doxorubicin resistant cell line UM-UC-6dox. Treatment with Ad-MMAC and doxorubicin completely eradicated established UM-UC-6dox tumors in 30% of mice. UM-UC-14 tumors were transiently suppressed by this combined treatment. These data show that PTEN gene therapy can effectively treat bladder cancers that have genomic alterations in PTEN and has an anti-angiogenic effect. Furthermore, our data demonstrate that inactivating a specific target such as phosphorylated Akt with gene therapy combined with chemotherapy can provide effective treatment for drug resistant cancers such as UM-UC-6dox cells.
1082. Histone Deacetylase Inhibitors Evoke Apoptosis of Gastric Cancer Cells through the Induction of PIG3 and NOXA by the Acetylation of p53 at 320, 373 Lysine Residues Takeshi Terui,1 Minoru Takahashi,1 Ken Murakami,1 Rishu Takimoto,1 Koichi Takada,1 Junji Kato,1 Yasushi Sato,1 Tsutomu Sato,1 Tsuzuku Murakami,1 Yoshiro Niitsu.1 1 4th Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan. It has been a controversial issue whether p53 is involved in apoptosis induction of tumor cells by histone deacetylase (HDAC) inhibitor and given the p53 is indeed involved in, which genes of acetylated p53 targets are responsible for giving rise to apoptotic death. We, in the present study, first confirmed that apoptotic cell death was seen when p53 deficient cells (KATO-III) were transfected with wild type p53 and treated with sodium butyrate (SB) or trichostatin A (TSA). By western blotting, using specific antibodies, we then demonstrated that residues of 320, 373 and 382 lysine of p53 were acetylated in KATO-III cells transfectant with wild type p53 (KATO-III/p53) treated with HDAC inhibitor. However, only those KATO-III cells transfected with K320R p53 or K373R p53 but not K382R p53 became insensitive to HDAC inhibitor, as revealed by TUNEL staining, suggesting former two residues of p53 may be essential for HDAC inhibitor induced apoptosis. Further, RT-PCR demonstrated that amongst various p53 related proapoptotic genes, PIG3 and NOXA were induced by SB treatment in KATO-III/p53 cells. The induction of PIG3 and NOXA was not S417
CANCER APOPTOSIS observed in KATO-III/K320R and KATO-III/K373R cells treated with SB. Finally, we revealed that apoptosis could be evoked by SB even in cells with mutated p53 of which mutation residues were other than 320 lysine or 373 lysine and the apoptosis was suppressed by anti p53 antisense. In conclusion, it is suggested that the HDAC inhibitor can activate the p53 molecule by acetylation of 320 and 373 lysine residues, up-regulate PIG3 and NOXA and induce apoptosis in cancer cells with wild and pseudo-wild type p53 genes.
1083. Pretreatment of DU145 Prostate Cancer Cells with Ceramidase Inhibitors Can Potentiate the Apoptotic Effect of AdGFPFasLTET
Ahmed M. El-Zawahry,1 David H. Holman,1 Marc H. Hyer,2 David A. Schwartz,1 Alicja Bielawska,3 Lina M. Obeid,4 Yusuf A. Hannun,3 James S. Norris.1 1 Microbiology and Immunology, Medical University of South Carolina, Charleston, SC; 2The Burnham Institute, La Jolla, CA; 3 Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC; 4Medicine, Medical University of South Carolina, Charleston, SC. The recent availability of the human genome sequence and the continuing development of bioinformatic tools to analyze it suggest that our understanding of the causes of cancer will expand using the tools of genomics and proteomics and will reveal new signaling pathways as targets for cancer therapy. As far as cancer gene therapy is concerned, the difficulty will lie not in the choice of therapeutic genes per se but in our ability to deliver a corrective signal to every cell in the cancer for its eradication. Thus, studies on the delivery of therapeutic genes as well as studies on amplification of delivery systems are urgently required. In our DU145 model of prostate cancer, we have determined that resistance to the induction of apoptosis through the Fas receptor signaling pathway is due to overexpression of apoptotic resistance genes including cFLIPs [Hyer et al., Cancer Biology and Therapy 1(4): 405-410, 2002]. We have also determined that expression of a FasL-GFP fusion gene will overcome resistance in infected cells [Hyer et al., Molecular Therapy 2(4):348-358, 2000]. To overcome the delivery issue, we have determined that overexpression of the GFPFasL fusion protein overcomes resistance, kills the cell apoptotically, and produces apoptotic vesicles that also can signal Fas to induce apoptosis in adjacent cells (i.e. bystander activity) [Hyer et al., Cancer Gene Therapy, In Press, 2003]. However, because of expression of apoptotic resistance genes some cancers, including the DU145 model, are resistant to the apoptotic vesicles and are thus relatively insensitive to vesicle-mediated bystander activity. To overcome this, we have examined a number of different chemotherapeutic drugs and other small molecules, for their effect on apoptotic resistance mechanisms. We can show that doxorubicin (0.2 ug/ml) will decrease expression of cFLIP protein without a concomitant decrease in levels of FLIP mRNA. This decrease in protein levels may be due to proteasomal degradation or a translational block. Under these conditions, a 20% increase in sensitivity of apoptotic vesicles is observed with 0.2 ug/ml doxorubicin. However, 0.2 ug/ ml doxorubicin is itself toxic to DU145 at 48-72 hours which somewhat obscures the role of the bystander vesicles in promoting apoptosis. We have also examined LCL102, a ceramidase inhibitor, which acts by increasing intracellular ceramide levels. This molecule is highly efficient at activating cell death in DU145 cells at nontoxic doses when combined with AdGFPFasL virus at MOIs achievable in vivo. We are now studying the mechanism of this, vis-à-vis the bystander effect by examining how LCL102 modulates the mechanisms of resistance. These studies involve Mass Spectroscopic analysis of sphingolipid metabolism. Concomitant studies of proteasome function and alternative splicing of apoptosis resistance genes are underway and will be presented at the meeting. S418
(1st and 2nd Authors, Ahmed El-Zawahry and David Holman shared equally in this work).
1084. The Effect of E1A on Sensitization of Gastrointestinal Cancer Cell Lines Overexpressing HER-2/neu to Anticancer Agents Xi-Zhang Lin,1 Ni-Chin Ko,1 Jian-Chyi Chen,3 Hsiao-Sheng Liu,2 Hsiao-Sheng Liu Hu.1 1 Department of Internal Medicine, National Cheng Kung University, Tainan, Taiwan; 2Department of Microbiology and Immunology, National Cheng Kung University, Tainan, Taiwan; 3 Department of Biotechnology, Southern Taiwan University of Technology, Tainan, Taiwan. BackgroundThe overexpression of HER-2/neu oncogene has been implicated in the development and modulation of many types of cancers. Adenovirus type 5 E1A gene is known to be able to suppress the overexpression of HER-2/neu AIM By delivering the E1A gene into the gastroenterological cancer cells to block the HER-2/ neuoverexpression, we expect to see that the E1A-transfected cancer cells will become more sensitive to the chemotherapeutic agents. Materials and methods SNU-5 cell (ATCC number CRL-5973) and AGS cell (ATCC number CRL-1739) are human gastric carcinoma and Colo 205 cell (ATCC number CCL-222) is a human colon adenocarcinoma. E1A gene driven by a CMV promoter in the plasmid pSG5 was delivered into the cells by an electroporation system. The electroporation regimen was 300 volts, 5 ms in duration. The protein expression of E1A and HER-2/neu in the transfected cells were checked by Western blotting. The cytotoxicity of pSG5 and E1A transfected cells to taxol (10 μg/ml, 15 μg/ml and 30 μg/ml) was determined by tetrazolium-based colorimetric assay. Results E1A gene was successfully delivered into all three cancer cells by electroporation. E1A expression resulted in down-regulation of HER-2/neu gene expression by Western blotting. However, only E1A-transfected AGS cell showed more sensitivity to taxol. After 24 hour treatment, the pSG5 and E1A transfected SNU-5 and Colo 205 cells showed no significant growth inhibition to taxol. The pSG5 transfected AGS cell showed 48.9 ± 5.5 %, 56.5 ± 7.4 %, and 81.8 ± 7.8 % inhibition rate at 10 μg/ml, 15 μg/ml, and 30 μg/ml of taxol, respectively. The E1A transfected AGS cell showed 61.4 ± 11.9 %, 66.9 ± 8.7 %, and 83.9 ± 5.7 % inhibition rate at 10 μg/ml, 15 μg/ml, and 30 μg/ml of taxol, respectively. Conclusion E1A gene can be successfully delivered into gastroenterological cancer cells by electroporation. The transfected E1A gene can suppress the expression of HER-2/neu. On the other hand, E1A transfection can enhance chemosensitivity in some cell lines, such as AGS cell, but the enhancement is not a universal phenomenon as expected.
1085. In Vivo and In Vitro Inhibition of Human Cancer Cell Growth Using Adenoviral Vector Expressing FHIT Gene: Mechanism Analysis Using Microarray Fumiaki Tanaka,1 Hideto Sonoda,1 Koshi Mimori,1 Takeshi Shiraishi,1 Hideshi Ishii,2 Carlo M. Croce,2 Hiroshi Inoue,1 Masaki Mori.1 1 Division of Surgical Oncology, Department of Molecular and Cellular Biology, Medical Institution of Bioregulation, Kyushu University, Beppu, Oita, Japan; 2Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, United States. The fragile histidine triad (FHIT) gene has been cloned from the chromosome FRA3B region, which is deleted in a large fraction of human tumor. Previous studies showed that the introduction of Fhit suppresses tumor cell growth. To further explore the pre-clinical cancer therapy by Fhit, we have studied a biological effect of H1299, Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy