Methoxyamine potentiates iododeoxyuridine-induced radiosensitization by altering cell cycle kinetics and enhancing senescence.

We previously reported that methoxyamine (an inhibitor of base excision repair) potentiates iododeoxyuridine (IUdR)-induced radiosensitization in human tumor cells. In this study, we investigated the potential mechanisms of this enhanced cell death. Human colorectal carcinoma RKO cells were exposed to IUdR (3 micromol/L) and/or methoxyamine (3 mmol/L) for 48 hours before ionizing radiation (5 Gy). We found that IUdR/methoxyamine altered cell cycle kinetics and led to an increased G1 population but a decreased S population before ionizing radiation. Immediately following ionizing radiation (up to 6 hours), IUdR/methoxyamine-pretreated cells showed a stringent G1-S checkpoint but an insufficient G2-M checkpoint, whereas a prolonged G1 arrest, containing 2CG1 and 4CG1 cells, was found at later times up to 72 hours. Levels of cell cycle-specific markers [p21, p27, cyclin A, cyclin B1, and pcdc2(Y15)] and DNA damage signaling proteins [gammaH2AX, pChk1(S317), and pChk2(T68)] supported these altered cell cycle kinetics. Interestingly, we found that IUdR/methoxyamine pretreatment reduced ionizing radiation-induced apoptosis. Additionally, the extent of cell death through necrosis or autophagy seemed similar in all (IUdR +/- methoxyamine + ionizing radiation) treatment groups. However, a larger population of senescence-activated beta-galactosidase-positive cells was seen in IUdR/methoxyamine/ionizing radiation-treated cells, which was correlated with the increased activation of the senescence factors p53 and pRb. These data indicate that IUdR/methoxyamine pretreatment enhanced the effects of ionizing radiation by causing a prolonged G1 cell cycle arrest and by promoting stress-induced premature senescence. Thus, senescence, a novel ionizing radiation-induced tumor suppression pathway, may be effectively targeted by IUdR/methoxyamine pretreatment, resulting in an improved therapeutic gain for ionizing radiation.