Involvement of proapoptotic genes in autophagic cell death induced by irradiation

Irradiation is widely used in anticancer therapy; however, the efficiency is limited. Most cancer cells have mutations in apoptosis that they can easily escape the apoptosis induced by irradiation. Autophagy has been known as type II programmed cell death that can be activated by irradiation, especially when apoptosis is blocked, but the underlying molecular mechanism is largely unknown. We demonstrated that proapoptotic genes PUMA and Bid are involved in the regulation of autophagic cell death. When wild-type (WT), Bax(−/−) and PUMA(−/−) HCT116 cells were exposed to irradiation, we found that, compared with WT, Bax(−/−) cells showed significantly decreased cell death because of Bax deficiency; however, surprisingly PUMA(−/−) cells showed significant increase in cell death although the proapoptotic gene PUMA was knocked out. By analyzing apoptosis via Annexin V-fluorescein isothiocyanate (FITC) assay with flow cytometry, both Bax(−/−) and PUMA(−/−) cells showed less apoptosis than WT, suggesting the existence of another type of cell death in PUMA(−/−) cells. Autophagy was then examined in three cell lines by counting the percentage of cells with punctate GFP-LC3. Although all three cell lines showed significantly increased autophagy activity after irradiation, that of PUMA(−/−) cells was much higher than the other two cell lines, which suggests that PUMA(−/−) cells may die through autophagy. This was then confirmed by the decreased cell death in PUMA(−/−) cells when autophagy was blocked by 3-MA. In addition, we also tested the responses of WT and Bid(−/−) MEFs to irradiation. Bid(−/−) MEFs but not WT died through autophagy after irradiation. These results imply the involvement of apoptosis-associated genes such as PUMA and Bid in autophagic cell death, which contributes to identifying the molecular mechanism by which autophagy drives cells to death.