- Email: [email protected]
560. Therapeutic Effect on Bladder Cancer with a Conditionally Replicating Oncolytic Virus Derived from Herpes Simplex Virus Type II
Cancer - Immunotherapy: Novel Viral Therapeutics using OT-1 transgenic T cells which recognize the SIINFEKL epitope of ovalbumin presented by the AE17-ova cells. Data will be presented on how oncolytic virotherapy influences the generation of both antiviral and anti-tumor responses and how such immune responses can be enhanced to improve on the therapy.
558. Baculovirus-Mediated Interferon Alleviates Dimethylnitrosamine-Induced Liver Cirrhosis Symptoms in a Murine Model
Yoshimi Nishibe,1 Hiroyasu Kaneko,1 Hitoshi Suzuki,1 Takashi Abe,3 Yoshiharu Matsuura,3 HIroshi Takaku.1,2 1 Department of Life and Environmental Science, Chiba Institute of Technology, Narashino, Chiba, Japan; 2High Technology Research Center, Chiba Institute of Technology, Narashino, Chiba, Japan; 3 Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan. Chronic infection by the hepatitis C virus (HCV) is a major cause of liver cirrhosis (LC) in Japan and some southern European countries, including Italy and Spain, and affects more than 3% of the population worldwide. Infected individuals are a reservoir for new infections, as well as being at risk of developing LC and hepatocellular carcinoma. Consequently, HCV infection is the foremost reason for performing liver transplants. Antiviral therapy consisting of peginterferon alfa2a/ribavirin leads to a sustained response in only half of all patients infected with HCV genotypes 1 and 4, resulting in the need to develop new antiviral therapies. The main characteristic of LC is fibrosis. The normal liver equilibrium of collagen synthesis and decomposition by liver cell proteases is well balanced. In damaged cells, collagen is secreted to form persistent scars, and excess secreted collagen is degraded. However, this balance is disrupted by chronic hepatic damage such as fibrosis, which results from wound healing after repeated hepatic injuries. Following liver damage, inflammatory lymphatic cells infiltrate the liver parenchyma, and many hepatic cells undergo apoptosis, leading to the activation of Kupffer cells. Hepatic stellate cells (HSCs) proliferate, become activated, and secrete large amounts of extracellular matrices (such as collagen, fibronectin and elastin). Sinusoidal endothelial cells lose their fenestrations, and continual cardiac contraction of HSCs increases the blood-flow resistance in the liver sinusoid. The reduction of secreted collagen to normal levels is therefore likely to limit the progression of liver cancer and, potentially, lead to a cure for hepatitis. We recently found that baculovirus stimulates Toll-like receptor 9 which plays an important role in the activation of innate immunity. Baculovirus infection activates tumor necrosis factor (TNF)-α, interleukin (IL)-1α, and IL-1β expression in primary hepatocyte cultures,probably due to the presence of small numbers of Kupffer cells in the culture population, while adenoviral vector expression of interferon (IFN)-α and IFN-γ in the liver of rats with hepatic fibrosis has been shown to improve the symptoms of hepatic fibrosis. The wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a range of mammalian cell types in vitro but does not replicate in these cells. The current study investigated the in vivo effect of AcMNPV in the mouse model of liver cirrhosis (LC) induced by the mutagen dimethylnitrosamine (DMN). Intraperitoneal injection of AcMNPV induced an immune response, and the baculovirus was taken up by the liver and the spleen where it suppressed liver injury and fibrosis through the induction of interferons. This study presents the first evidence of the feasibility of using baculovirus to treat LC.
559. Enhancement of Antitumor Activity by a Genetically-Modified Dendritic Cell Vaccine Expressing Interleukin-15 and Its Receptor
Jason C. Steel,1 Brian J. Morrison, Charmaine A. Ramlogan, Ping Yu, Thomas A. Waldmann, John C. Morris. 1 Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD. Dendritic cells (DCs) are powerful antigen presenting cells that are capable of activating and directing naive T and B cells. This has lead to their study as potential anticancer vaccines. Tumor cells; however, have correspondingly developed a number of mechanisms to evade the immune response by suppressing DC function and reducing the effectiveness of DC antitumor vaccination. The ability of tumor cells to suppress DC function may be overcome by the cytokine interleukin-15 (IL-15). IL-15 has also been shown to induce T-cell proliferation, enhance cytolytic effector cells including natural killer (NK) and cytotoxic T-cells, and enhance stimulation of B-cells. IL-15 functions through interaction with its receptor (IL-15Ra) that presents IL-15 in trans to immune effectors cells. The efficacy of exogenously administered IL-15 may be limited by the availability of IL-15Ra. BALB-neuT transgenic mice develop breast cancers as a consequence of mammary gland-specific expression of an activated neu oncogene. We examined the antitumor effect of adenoviral-mediated gene transfer of the combination of IL-15 and IL-15Ra to augment a DC vaccine directed against the neu oncoprotein in these mice. Methods: Bone marrow-derived DCs were generated from BALB/c mice and transduced with recombinant adenoviruses expressing a non-signaling truncated neu antigen, murine IL-15 and/or its receptor, IL-15Ra. BALB-neuT mice at 10-12 weeks of age were subcutaneously vaccinated with four weekly injections of 1 x 106 DCs and followed for tumor development and immune response. Results: Mice vaccinated with DCs expressing the neu antigen, IL-15 and IL-15Ra had significantly delayed onset of mammary carcinomas with 70% of animals tumor free at 25 weeks compared to none of the unvaccinated control mice, 10% of animals treated with DC expressing the neu antigen, 30% of animals vaccinated with DCs expressing neu + IL-15, and 40% of animals vaccinated with DC expressing neu + IL-15Ra. Animals vaccinated with DCs expressing the neu antigen, IL-15 and IL-15Ra exhibited significantly greater antibody responses to the neu antigen compared to those treated with DCs expressing neu alone, or in combination with IL-15, or IL-15Ra. Sera from vaccinated mice exhibited antibody-dependant cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) against neu-expressing target cells and induced down-regulation of neu signaling in vitro. Conclusion: Antitumor vaccination with genetically-modified DCs expressing the neu antigen, IL-15 and IL-15Ra increased tumor-free survival highlighting the potential for the use of IL-15 and IL-15Ra gene transfer to augment DC anticancer vaccines.
560. Therapeutic Effect on Bladder Cancer with a Conditionally Replicating Oncolytic Virus Derived from Herpes Simplex Virus Type II
Kwanjoong Joo,1,5 Hideyuki Yamashita,1 Hongtao Li,2,3 Xiaoliu Zhang,2,3,4 Seth P. Lerner.1 1 Scott Department of Urology, Baylor College of Medicine, Houston, TX; 2Department of Pediatrics, Baylor College of Medicine, Houston, TX; 3Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 4Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX; 5Department of Urology, Sungkyunkwan University School of Medicine, Suwon, Korea. Purpose: Despite recent improvements, resistance to traditional immunotherapy using Bacillus Calmette-Guerin or chemotherapy is still common in patients with bladder cancer. We recently constructed
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Cancer - Mechanisms of Cancer Cell Killing an oncolytic virus from herpes simplex virus type II (HSV-2), which selectively targets tumor cells with an activated Ras signaling pathway. We evaluated the anti-tumor effect of this oncolytic HSV (FusOn-H2) against bladder cancer. Materials and Methods: We directly compared the anti-tumor effect of FusOn-H2 against bladder cancer xenografts with that of a first generation oncolytic virus that was derived from HSV-1 (Baco-1). For in vitro studies, we infected 5637 human bladder cancer cells and MBT-2 murine bladder cancer cells with Baco-1 or FusOn-H2, respectively, and the percentage of viable cells was calculated to determine the killing activity. For in vivo evaluation, we established bladder tumor at the orthotopic site in C3H/He mice using the murine MBT-2 cells. Baco-1 or FusOn-H2 was then instilled into the bladder through the urethra respectively. Tumor volume and weight were recorded by the end of the experiment. Animal spleens were also collected to determine if any anti-tumor immunity was elicited during virotherapy in this syngeneic bladder cancer model. Results: Infection of oncolytic HSVs in both cell lines led to progressive killing of tumor cells. By 72 hours after infection, FusOn-H2, when given at the initial dose of 0.1 plaque forming unit/cell, almost completely eradicated the tumor cells. Two instillations of the oncolytic HSVs into bladder of tumor-bearing mice completely eradicated the tumor in 81.8 % of mice. The remaining tumor in the FusOn-H2 treated group was visibly smaller than those of Baco-1 treated group, though not statistically significant (p>0.05). The results of tumor-specific CTL activity assay showed that tumor destruction by oncolytic viruses in vivo, especially by the HSV-2-based FusOn-H2, induced potent anti-tumor immune responses. Conclusions: Oncolytic virus derived from HSV-2 has potent anti-tumor activity against bladder cancer. Oncolytic effect of this virus in vivo induces tumor specific cellular immunity that further enhances the overall anti-tumor activity. Translating this novel virotherapy into the clinic could present an alternative intravesical therapy strategy for patients with bladder cancer.
Cancer – Mechanisms of Cancer Cell Killing 561. Overexpression of Mitochondrially Targeted Catalase and Manganese Superoxide Dismutase (MnSOD) in 32D cl 3 Mouse Hematopoietic Progenitor Cells Increases Antioxidant Levels and Radioresistance
Michael W. Epperly,1 Xicheng Zhang,1 Tracy M. Dixon,1 Darcy Franciola,1 J. Andres Melendez,2 Benjamin A. Greenberger,1 Paavani Komanduri,1 Joel S. Greenberger.1 1 Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 2Center for Immunology and Microbial Disease, Albany Medical School, Albany, NY. Irradiation results in increased production of superoxide which is dismuted to hydrogen peroxide and subsequently detoxified by catalase, or glutathione peroxidase (GPX). We tested whether increased mitochondrial expression of both MnSOD and catalase increased radiation resistance. A plasmid was constructed containing either the human catalase transgene or the catalase transgene attached to the mitochondrial localization sequence of MnSOD. Cells from 32D cl 3 or a 32D cl 3 subclone overexpressing MnSOD (2C6) were transfected with the catalase plasmid (32D-cat or 2C6-cat, respectively) or the plasmid containing the catalase gene containing the mitochondrial localization sequence (32D-mt-cat or 2C6mt-cat, respectively). Clones from 32D-cat and 32D-mt-cat had significantly increased catalase activity (595.7 + 15.3 or 603.3 + 3.0 µM, respectively) compared to 539.7 + 3.7 µM (p = 0.0288 or 0.0002, respectively) for parent cell line 32D cl 3. The 2C6 cells had decreased catalase activity of 205.0 + 10.0 compared to 539.7 + 3.7 (p < 0.0001) for 32D cl 3. Catalase activity was further increased in 2C6-cat and 2C6-mt-cat (333.3 + 12.7 and 467.0 + 1.0, respectively) S210
compared to 205.0 + 10.0 for 2C6 (p <0.0001). Western analysis using an anti-catalase antibody confirmed the increases in catalase. Radiation survival curves were performed by irradiating 32D cl 3, 32D-cat, 32D-mt-cat, 2C6, 2C6-cat or 2C6-mt-cat with doses ranging from 0 to 8 Gy, plating in methycellulose, incubating for 7 days, and counting colonies of greater than 50 cells. Cells from 32D-mt-cat were more resistant compared to 32D-cat with increased shoulder on the survival curve (n = 10.3 + 0.5, or 5.9 + 0.2, respectively, p = 0.0025). Both were more radioresistant than 32D cl 3 cells (n = 2.9 + 1.1, p = 0.0196 or 0.0479, respectively). Cells from 2C6 transfected with mtcatalase, but not catalase, showed increased radioresistance increasing the Do from 0.979 + 0.1Gy for 2C6 to 1.171 + 0.1 Gy for 2C6-mt-cat cells. 32D-cat and 32D-mt-cat had a significantly increased GSH levels (222.7 + 1.1 or 310.9 + 1.7 mU/mg, respectively) compared to 32D cl 3 (138.9 + 0.1 mU/mg, p < 0.0001). Cells from 2C6-cat or 2C6-mt-cat also had significantly increased GSH levels (286.6 + 2.5 or 307.5 + 1.4 mU/mg) compared to 2C6 cells (254.1 + 1.5, p < 0.0001). Similar increases in GPX activity were detected in 32D-cat and 32D-mt-cat (260.4 + 24.6 and 257.1 + 17.1 mU/mg, respectively) compared to 32D cl 3 (105.5 + 1.6 mU/mg, p = 0.0002 or < 0.0001, respectively). Thus, overexpression of both MnSOD and mt-catalase transgenes is superior to one alone for radioprotection of mouse hematopoietic progenitor cells.
562. Systemic Intravenous Manganese Superoxide Plasmid/Liposome Complex (MnSODPL) Protects Mice from Total Body Irradiation with No Increased Carcinogenesis
Joel S. Greenberger,1 Tracy M. Dixon,1 Hong Wang,2 James Schlesselman,2 Darcy Franciola,1 Michael W. Epperly.1 1 Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 2Biostatistics, University of Pittsburgh, Pittsburgh, PA. A question arising from MnSOD-PL protection of normal tissues from irradiation is the potential for increased cancer. Prevention of irradiation-induced apoptosis may allow expression of oncogenes or other genetic changes resulting in increased cancer. To determine if increased survival leads to increased cancer or decreased life span, groups of fifty female and male C57BL/6 NHsd mice were injected intravenously with 100 µg plasmid DNA in 100 µl MnSOD-PL and irradiated along with control mice to 0, 1, or 9.5 Gy whole body irradiation. Moribund mice were sacrificed and examined for gross and microscopic pathology. Female mice followed for 625 days and male mice followed for 450 days were analyzed. Female control mice irradiated to 9.5 Gy had 42% survival at 30 days with hypocellularity consistent with the hematopoietic syndrome compared to 90% for MnSOD-PL treated mice (p = 0.0013). Male control irradiated mice had 26% survival compared to 74% for MnSOD-PL treated mice (p < 0.0001). Blank plasmid injected mice showed no effect on irradiation killing. The life-saving effect of MnSOD-PL at day 30 did not come at the expense of higher mortality rates as conditional survival from day 31. Among the 9.5Gy irradiated females surviving 31 days or more, the conditional median survival time was 393 days for the MnSOD-PL-treated animals as compared to 430 days for the irradiated controls (p = 0.64). Among the 9.5-Gy irradiated males surviving 31 days or more, the conditional median survival time was 427 days for the MnSOD-PL-treated animals as compared to 248 days for the irradiated controls (p = 0.03). Female mice irradiated to 1 Gy and receiving MnSOD-PL had increased median survival of 561 days compared to control irradiated mice of 548 days (p = 0.02). Therefore, MnSOD-PL did not further exacerbate irradiation induced life shortening. The incidence of cancer among female and male irradiated and controls was 9.0 cancers per 100 animals per year for 0 Gy, 12.1 at 1 Gy, and 4.8 at 9.5 Gy. The cancer incidence rate in the MnSOD-PL treated animals was not significantly greater. Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
558. Baculovirus-Mediated Interferon Alleviates Dimethylnitrosamine-Induced Liver Cirrhosis Symptoms in a Murine Model
Yoshimi Nishibe,1 Hiroyasu Kaneko,1 Hitoshi Suzuki,1 Takashi Abe,3 Yoshiharu Matsuura,3 HIroshi Takaku.1,2 1 Department of Life and Environmental Science, Chiba Institute of Technology, Narashino, Chiba, Japan; 2High Technology Research Center, Chiba Institute of Technology, Narashino, Chiba, Japan; 3 Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan. Chronic infection by the hepatitis C virus (HCV) is a major cause of liver cirrhosis (LC) in Japan and some southern European countries, including Italy and Spain, and affects more than 3% of the population worldwide. Infected individuals are a reservoir for new infections, as well as being at risk of developing LC and hepatocellular carcinoma. Consequently, HCV infection is the foremost reason for performing liver transplants. Antiviral therapy consisting of peginterferon alfa2a/ribavirin leads to a sustained response in only half of all patients infected with HCV genotypes 1 and 4, resulting in the need to develop new antiviral therapies. The main characteristic of LC is fibrosis. The normal liver equilibrium of collagen synthesis and decomposition by liver cell proteases is well balanced. In damaged cells, collagen is secreted to form persistent scars, and excess secreted collagen is degraded. However, this balance is disrupted by chronic hepatic damage such as fibrosis, which results from wound healing after repeated hepatic injuries. Following liver damage, inflammatory lymphatic cells infiltrate the liver parenchyma, and many hepatic cells undergo apoptosis, leading to the activation of Kupffer cells. Hepatic stellate cells (HSCs) proliferate, become activated, and secrete large amounts of extracellular matrices (such as collagen, fibronectin and elastin). Sinusoidal endothelial cells lose their fenestrations, and continual cardiac contraction of HSCs increases the blood-flow resistance in the liver sinusoid. The reduction of secreted collagen to normal levels is therefore likely to limit the progression of liver cancer and, potentially, lead to a cure for hepatitis. We recently found that baculovirus stimulates Toll-like receptor 9 which plays an important role in the activation of innate immunity. Baculovirus infection activates tumor necrosis factor (TNF)-α, interleukin (IL)-1α, and IL-1β expression in primary hepatocyte cultures,probably due to the presence of small numbers of Kupffer cells in the culture population, while adenoviral vector expression of interferon (IFN)-α and IFN-γ in the liver of rats with hepatic fibrosis has been shown to improve the symptoms of hepatic fibrosis. The wild-type baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a range of mammalian cell types in vitro but does not replicate in these cells. The current study investigated the in vivo effect of AcMNPV in the mouse model of liver cirrhosis (LC) induced by the mutagen dimethylnitrosamine (DMN). Intraperitoneal injection of AcMNPV induced an immune response, and the baculovirus was taken up by the liver and the spleen where it suppressed liver injury and fibrosis through the induction of interferons. This study presents the first evidence of the feasibility of using baculovirus to treat LC.
559. Enhancement of Antitumor Activity by a Genetically-Modified Dendritic Cell Vaccine Expressing Interleukin-15 and Its Receptor
Jason C. Steel,1 Brian J. Morrison, Charmaine A. Ramlogan, Ping Yu, Thomas A. Waldmann, John C. Morris. 1 Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD. Dendritic cells (DCs) are powerful antigen presenting cells that are capable of activating and directing naive T and B cells. This has lead to their study as potential anticancer vaccines. Tumor cells; however, have correspondingly developed a number of mechanisms to evade the immune response by suppressing DC function and reducing the effectiveness of DC antitumor vaccination. The ability of tumor cells to suppress DC function may be overcome by the cytokine interleukin-15 (IL-15). IL-15 has also been shown to induce T-cell proliferation, enhance cytolytic effector cells including natural killer (NK) and cytotoxic T-cells, and enhance stimulation of B-cells. IL-15 functions through interaction with its receptor (IL-15Ra) that presents IL-15 in trans to immune effectors cells. The efficacy of exogenously administered IL-15 may be limited by the availability of IL-15Ra. BALB-neuT transgenic mice develop breast cancers as a consequence of mammary gland-specific expression of an activated neu oncogene. We examined the antitumor effect of adenoviral-mediated gene transfer of the combination of IL-15 and IL-15Ra to augment a DC vaccine directed against the neu oncoprotein in these mice. Methods: Bone marrow-derived DCs were generated from BALB/c mice and transduced with recombinant adenoviruses expressing a non-signaling truncated neu antigen, murine IL-15 and/or its receptor, IL-15Ra. BALB-neuT mice at 10-12 weeks of age were subcutaneously vaccinated with four weekly injections of 1 x 106 DCs and followed for tumor development and immune response. Results: Mice vaccinated with DCs expressing the neu antigen, IL-15 and IL-15Ra had significantly delayed onset of mammary carcinomas with 70% of animals tumor free at 25 weeks compared to none of the unvaccinated control mice, 10% of animals treated with DC expressing the neu antigen, 30% of animals vaccinated with DCs expressing neu + IL-15, and 40% of animals vaccinated with DC expressing neu + IL-15Ra. Animals vaccinated with DCs expressing the neu antigen, IL-15 and IL-15Ra exhibited significantly greater antibody responses to the neu antigen compared to those treated with DCs expressing neu alone, or in combination with IL-15, or IL-15Ra. Sera from vaccinated mice exhibited antibody-dependant cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) against neu-expressing target cells and induced down-regulation of neu signaling in vitro. Conclusion: Antitumor vaccination with genetically-modified DCs expressing the neu antigen, IL-15 and IL-15Ra increased tumor-free survival highlighting the potential for the use of IL-15 and IL-15Ra gene transfer to augment DC anticancer vaccines.
560. Therapeutic Effect on Bladder Cancer with a Conditionally Replicating Oncolytic Virus Derived from Herpes Simplex Virus Type II
Kwanjoong Joo,1,5 Hideyuki Yamashita,1 Hongtao Li,2,3 Xiaoliu Zhang,2,3,4 Seth P. Lerner.1 1 Scott Department of Urology, Baylor College of Medicine, Houston, TX; 2Department of Pediatrics, Baylor College of Medicine, Houston, TX; 3Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 4Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX; 5Department of Urology, Sungkyunkwan University School of Medicine, Suwon, Korea. Purpose: Despite recent improvements, resistance to traditional immunotherapy using Bacillus Calmette-Guerin or chemotherapy is still common in patients with bladder cancer. We recently constructed
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
S209
Cancer - Mechanisms of Cancer Cell Killing an oncolytic virus from herpes simplex virus type II (HSV-2), which selectively targets tumor cells with an activated Ras signaling pathway. We evaluated the anti-tumor effect of this oncolytic HSV (FusOn-H2) against bladder cancer. Materials and Methods: We directly compared the anti-tumor effect of FusOn-H2 against bladder cancer xenografts with that of a first generation oncolytic virus that was derived from HSV-1 (Baco-1). For in vitro studies, we infected 5637 human bladder cancer cells and MBT-2 murine bladder cancer cells with Baco-1 or FusOn-H2, respectively, and the percentage of viable cells was calculated to determine the killing activity. For in vivo evaluation, we established bladder tumor at the orthotopic site in C3H/He mice using the murine MBT-2 cells. Baco-1 or FusOn-H2 was then instilled into the bladder through the urethra respectively. Tumor volume and weight were recorded by the end of the experiment. Animal spleens were also collected to determine if any anti-tumor immunity was elicited during virotherapy in this syngeneic bladder cancer model. Results: Infection of oncolytic HSVs in both cell lines led to progressive killing of tumor cells. By 72 hours after infection, FusOn-H2, when given at the initial dose of 0.1 plaque forming unit/cell, almost completely eradicated the tumor cells. Two instillations of the oncolytic HSVs into bladder of tumor-bearing mice completely eradicated the tumor in 81.8 % of mice. The remaining tumor in the FusOn-H2 treated group was visibly smaller than those of Baco-1 treated group, though not statistically significant (p>0.05). The results of tumor-specific CTL activity assay showed that tumor destruction by oncolytic viruses in vivo, especially by the HSV-2-based FusOn-H2, induced potent anti-tumor immune responses. Conclusions: Oncolytic virus derived from HSV-2 has potent anti-tumor activity against bladder cancer. Oncolytic effect of this virus in vivo induces tumor specific cellular immunity that further enhances the overall anti-tumor activity. Translating this novel virotherapy into the clinic could present an alternative intravesical therapy strategy for patients with bladder cancer.
Cancer – Mechanisms of Cancer Cell Killing 561. Overexpression of Mitochondrially Targeted Catalase and Manganese Superoxide Dismutase (MnSOD) in 32D cl 3 Mouse Hematopoietic Progenitor Cells Increases Antioxidant Levels and Radioresistance
Michael W. Epperly,1 Xicheng Zhang,1 Tracy M. Dixon,1 Darcy Franciola,1 J. Andres Melendez,2 Benjamin A. Greenberger,1 Paavani Komanduri,1 Joel S. Greenberger.1 1 Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 2Center for Immunology and Microbial Disease, Albany Medical School, Albany, NY. Irradiation results in increased production of superoxide which is dismuted to hydrogen peroxide and subsequently detoxified by catalase, or glutathione peroxidase (GPX). We tested whether increased mitochondrial expression of both MnSOD and catalase increased radiation resistance. A plasmid was constructed containing either the human catalase transgene or the catalase transgene attached to the mitochondrial localization sequence of MnSOD. Cells from 32D cl 3 or a 32D cl 3 subclone overexpressing MnSOD (2C6) were transfected with the catalase plasmid (32D-cat or 2C6-cat, respectively) or the plasmid containing the catalase gene containing the mitochondrial localization sequence (32D-mt-cat or 2C6mt-cat, respectively). Clones from 32D-cat and 32D-mt-cat had significantly increased catalase activity (595.7 + 15.3 or 603.3 + 3.0 µM, respectively) compared to 539.7 + 3.7 µM (p = 0.0288 or 0.0002, respectively) for parent cell line 32D cl 3. The 2C6 cells had decreased catalase activity of 205.0 + 10.0 compared to 539.7 + 3.7 (p < 0.0001) for 32D cl 3. Catalase activity was further increased in 2C6-cat and 2C6-mt-cat (333.3 + 12.7 and 467.0 + 1.0, respectively) S210
compared to 205.0 + 10.0 for 2C6 (p <0.0001). Western analysis using an anti-catalase antibody confirmed the increases in catalase. Radiation survival curves were performed by irradiating 32D cl 3, 32D-cat, 32D-mt-cat, 2C6, 2C6-cat or 2C6-mt-cat with doses ranging from 0 to 8 Gy, plating in methycellulose, incubating for 7 days, and counting colonies of greater than 50 cells. Cells from 32D-mt-cat were more resistant compared to 32D-cat with increased shoulder on the survival curve (n = 10.3 + 0.5, or 5.9 + 0.2, respectively, p = 0.0025). Both were more radioresistant than 32D cl 3 cells (n = 2.9 + 1.1, p = 0.0196 or 0.0479, respectively). Cells from 2C6 transfected with mtcatalase, but not catalase, showed increased radioresistance increasing the Do from 0.979 + 0.1Gy for 2C6 to 1.171 + 0.1 Gy for 2C6-mt-cat cells. 32D-cat and 32D-mt-cat had a significantly increased GSH levels (222.7 + 1.1 or 310.9 + 1.7 mU/mg, respectively) compared to 32D cl 3 (138.9 + 0.1 mU/mg, p < 0.0001). Cells from 2C6-cat or 2C6-mt-cat also had significantly increased GSH levels (286.6 + 2.5 or 307.5 + 1.4 mU/mg) compared to 2C6 cells (254.1 + 1.5, p < 0.0001). Similar increases in GPX activity were detected in 32D-cat and 32D-mt-cat (260.4 + 24.6 and 257.1 + 17.1 mU/mg, respectively) compared to 32D cl 3 (105.5 + 1.6 mU/mg, p = 0.0002 or < 0.0001, respectively). Thus, overexpression of both MnSOD and mt-catalase transgenes is superior to one alone for radioprotection of mouse hematopoietic progenitor cells.
562. Systemic Intravenous Manganese Superoxide Plasmid/Liposome Complex (MnSODPL) Protects Mice from Total Body Irradiation with No Increased Carcinogenesis
Joel S. Greenberger,1 Tracy M. Dixon,1 Hong Wang,2 James Schlesselman,2 Darcy Franciola,1 Michael W. Epperly.1 1 Radiation Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA; 2Biostatistics, University of Pittsburgh, Pittsburgh, PA. A question arising from MnSOD-PL protection of normal tissues from irradiation is the potential for increased cancer. Prevention of irradiation-induced apoptosis may allow expression of oncogenes or other genetic changes resulting in increased cancer. To determine if increased survival leads to increased cancer or decreased life span, groups of fifty female and male C57BL/6 NHsd mice were injected intravenously with 100 µg plasmid DNA in 100 µl MnSOD-PL and irradiated along with control mice to 0, 1, or 9.5 Gy whole body irradiation. Moribund mice were sacrificed and examined for gross and microscopic pathology. Female mice followed for 625 days and male mice followed for 450 days were analyzed. Female control mice irradiated to 9.5 Gy had 42% survival at 30 days with hypocellularity consistent with the hematopoietic syndrome compared to 90% for MnSOD-PL treated mice (p = 0.0013). Male control irradiated mice had 26% survival compared to 74% for MnSOD-PL treated mice (p < 0.0001). Blank plasmid injected mice showed no effect on irradiation killing. The life-saving effect of MnSOD-PL at day 30 did not come at the expense of higher mortality rates as conditional survival from day 31. Among the 9.5Gy irradiated females surviving 31 days or more, the conditional median survival time was 393 days for the MnSOD-PL-treated animals as compared to 430 days for the irradiated controls (p = 0.64). Among the 9.5-Gy irradiated males surviving 31 days or more, the conditional median survival time was 427 days for the MnSOD-PL-treated animals as compared to 248 days for the irradiated controls (p = 0.03). Female mice irradiated to 1 Gy and receiving MnSOD-PL had increased median survival of 561 days compared to control irradiated mice of 548 days (p = 0.02). Therefore, MnSOD-PL did not further exacerbate irradiation induced life shortening. The incidence of cancer among female and male irradiated and controls was 9.0 cancers per 100 animals per year for 0 Gy, 12.1 at 1 Gy, and 4.8 at 9.5 Gy. The cancer incidence rate in the MnSOD-PL treated animals was not significantly greater. Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy