Methoxyamine potentiates iododeoxyuridine-induced radiosensitization not through increasing ionizing radiation-induced apoptosis and necrosis but through enhancing senescence

Proc Amer Assoc Cancer Res, Volume 46, 2005 1665 We previously reported that methoxyamine (MX, an inhibitor of base excision repair) potentiates iododeoxyuridine (IUdR)-induced radiosensitization. In this study, we investigated the mechanism of enhanced cell death. Human colorectal carcinoma RKO cells were exposed to IUdR (3 μM) and/or MX (3 mM) for 48 h before ionizing radiation (IR, 5Gy). Necrotic cell death was evidenced within the second 24h post-IR and continually increased up to 96 h, with the control/IR group demonstrating the highest necrosis and the IUdR/MX/IR group the lowest. Apoptotic cell death also occurred during the second 24 h post-IR and up to 96h, with the control/IR and MX/IR groups showing the highest extent of apoptosis and the IUdR/MX/IR group the lowest. The cells of IUdR/MX/IR group were enlarged dramatically but were alive for up to 8 days post-IR with senescence-associated beta-galactosidase activity being detectable from day 5 post-IR. Cell cycle profile showed that the pretreatment of IUdR/MX led to an increased G1 population and a decreased S population before IR and a retarded early cell cycle response after IR, as well as an increased tetraploid G1 population at the later times post-IR. Western blotting analyses demonstrated that compared to other treatment groups, the IUdR/MX/IR group showed an enhanced increase in p53, p21, p27, p16 and hypo-phospho-pRb as well as a decrease in cdc25A, pcdc2(Y15), cyclin B1 and cyclin A before and after IR, indicating a stringent G1-S checkpoint. However, pCHK1 (S317) and pCHK2 (T68) were not detectable with 48 h IUdR/MX treatment, suggesting that these DNA damage signaling pathways were not appreciably activated before IR. pCHK1(S317) and pCHK2(T68) were significantly increased in all groups at 1 h post-IR, with enhanced pCHK2 while reduced pCHK1 found in the IUdR/MX/IR group compared to other treatment groups. Interestingly, anti-apoptosis pAkt(S473) was up-regulated in the IUdR/MX/IR group before and after IR. γH2AX reappeared at 24h post-IR and sustained thereafter in all groups except the IUdR/MX/IR group, although γH2AX was increased in all groups early after IR (1-3 h post-IR). These results suggest that pretreatment of IUdR/MX before IR evoked mechanism(s) that altered DNA damage responses, and rendered cell resistance to early necrotic and apoptotic cell death, but enhanced cell susceptibility to later senescence. It appears that loss of clonogenic ability is more decisive than the extent of early necrosis and apoptosis in the ultimate outcome of the combined treatment of IUdR/MX/IR. Supported by NIH grant CA84578 to TJK.