Abstract
1493
Objectives: One of the primary causes for therapeutic failure in cancer therapy is resistance to radiation and chemotherapy. Deficient activation of caspases and DNA-repair mechanisms play a critical role in radioresistance and chemoresistance of tumor cells and leukemias. In this study, we analyzed the role of the nonhomologous end joining (NHEJ) DNA-double-strand-break-repair (DNA-DSB-repair) mechnanism, which is predominant for DNA-DSB-repair in mammalian cells, in deficient activation of caspases in radioresistant leukemia cells after treatment with yttrium-90 [Y-90], and gamma-irradiation. Methods: Leukemia cells (HL-60) radioresistant to Y-90 or to gamma-irradiation, NHEJ DNA-DSB repair proficient and deficient leukemia cells (Nalm6) were treated with [Y-90] (8.8, 26.3 and 87.6 MBq / 100 ml over 24h) and gamma-irradiation (1 Gy, 3 Gy, 10 Gy). After 24, 48 and 72h cell death and apoptosis was measured by flowcytometry analyses, activation of caspases by Western Blot analyses and DNA-damage by the Comet-Assay. Results: Radioresistant leukemia cells and NHEJ DNA-DSB-repair proficient leukemia cells were apoptosis-resistant and were deficient in activation of caspase-8, caspase-9, caspase-3, and cleavage of PARP after treatment with [Y-90] and gamma-irradiation. Inhibition of NHEJ DNA-DSB-repair restored activation of caspase-3, caspase-8, caspase-9, cleavage of PARP and apoptosis sensitivity by radiation in apoptosis- and radioresistant leukemia cells, suggesting that caspase activation and cleavage of PARP depends on NHEJ DNA-DSB-repair. In addition, inhibition of caspase activation by the broad range caspase inhibitor zVAD-fmk prevented gamma- and [Y-90]-radiation-induced caspase activation and apoptosis but could not prevented gamma- and beta-irradiaton-induced DNA-double-strand-breaks (DNA-DSBs), indicating that induction of DNA-DSBs is independent of caspase activation. However, caspase activation depends on induction of DNA-DSBs left unrepaired by NHEJ. Conclusions: We conclude that DNA-DSBs left unrepaired by NHEJ might be the initiator for activation of caspases by radiation in cancer cells. Failure in activation of caspases in radio- and apoptosis-resistant cancer cells depends on loss of DNA-DSBs and is due to higher rates of NHEJ DNA-DSB-repair.
- Society of Nuclear Medicine, Inc.