Abstract 2513: Molecular dissection of cell death mechanisms following ionizing radiation in vitro and in vivo

Abstract

Abstract Purpose: A majority of human cancers have the ability to evade cell death with dysregulated cell death and survival mechanisms. This may have a direct impact on treatment resistance and tumor recurrence. This study aims to elucidate the roles of apoptosis, autophagy and necrosis induced by ionizing radiation (IR). Materials/Methods: Immortalized murine embryonic fibroblast (MEF) cell lines with various genetic backgrounds were used in the study. Xenograft tumors were generated by injecting tumor cells (engineered by retrovirally transfecting the above MEF cell lines with oncoprotein E1A and KRAS) to bilateral flanks of nude mice. Mice bearing xenograft tumors were treated with radiation by using an orthovoltage X-ray machine. Tissue cultures were irradiated by using a Gammacell 40 irradiator with a Cs137 source. Cell survival was analyzed with several commonly accepted methods. Morphology changes before and after treatments were analyzed by phase contrast microscopy as well as transmission electron microscopy (TEM). Western blotting and Immunohistochemistry (IHC) methods were carried out as described. Results: There was no significant difference in the level of tumor regression between xenografted tumors lacking Bax and Bak (Bax−/−Bak−/−) and WT tumors after radiation. While apoptotic cell death was observed in immortalized WT MEF cell line and xenograft tumors after irradiation, the intrinsic apoptosis pathway was not required Autophagy was found to be activated after radiation, and pharmacologic inhibition of autophagy by 3-methyladenine (3MA) resulted in higher survival fraction after radiation, suggesting a cytotoxic role of autophagy following radiation. However, there was no difference in radiation response when an essential autophagy gene ATG5 was stably knocked down, suggesting that autophagy activation may not be required for radiation induced cell death. We then investigated the role of poly(ADP)-ribose polymerase (PARP) in radiation response by using PARP-1−/− immortalized MEFs. These cells were more resistant to MAF (an active metabolite of cyclophosphamide) induced necrosis as was shown previously, but were more sensitive to radiation both in vitro and in vivo (as in xenograft). We also found that a specific PARP inhibitor ABT-888 can significantly sensitize xenograft tumors to radiation treatment. Conclusion: While apoptosis and autophagy occur after radiation, the intrinsic apoptosis and autophagic pathways is not required for tumor regression following IR. Conversely, PARP protein plays an important pro-survival role after IR, and deficiency of PARP renders cells more sensitive to radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2513. doi:10.1158/1538-7445.AM2011-2513