Investigation of Radiation Induced Cell Death Mechanisms

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 various cell death mechanisms induced by ionizing radiation (IR). 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 K-RAS) to bilateral flanks of nude mice. Tissue cultures were irradiated by using a Gammacell 40 irradiator with a Cs137 source. Mice bearing xenograft tumors were treated with radiation by using an orthovoltage X-ray machine. Cell survival was analyzed with several commonly accepted methods. Morphology changes before and after radiation were analyzed by phase contrast or fluorescent microscopy as well as transmission electron microscopy (TEM). Western blotting and real-time PCR were used to monitor various cell death and DNA damage response markers at both the translation and transcription levels. While apoptotic cell death was observed in WT MEFs after radiation, the intrinsic cell death pathway turned out to be non-essential. MEFs derived from Bax/Bak double knockout animals (no intrinsic apoptotic pathway) were killed by radiation as effectively as WT MEFs. Autophagy activation was induced after radiation. Pharmacologic inhibition of autophagy by 3-methyladenine (3MA) resulted in higher survival after radiation suggesting a cytotoxic role of autophagy following IR. 3MA, however, also causes dose dependent cell cycle arrest that could provide an alternative explanation to this phenomenon. To get a definitive answer, we generated a stable ATG5 knockdown mutant which cannot undergo autophagy. It was found that not only this defect did not rescue cell death following IR, but slightly more acute cell death was observed. Thus autophagy appeared to play a cytoprotective role following radiation. We then compared IR with certain alkylating agents known to kill cells by both apoptosis and poly(ADP)-ribose polymerase (PARP) mediated necrosis pathways. PARP-/- cells were modestly resistant to these alkylating agents such as cyclophosphamide and MNNG, as PARP has been shown to be required for this process. In contrast, PARP-/- cells had an increased sensitivity to radiation and underwent both apoptosis and necrosis 96h after treatment. Cancer cell death in response to DNA damaging agents such as IR reflects a complex regulation between apoptosis, autophagy and necrosis-inducing intracellular pathways. PARP plays a different role in mediating cell death following ionizing radiation versus DNA alkylating agents.