Multi-Drug Resistance Phenotype in Myeloid Leukemia Cells Confers Radiation Resistance Via DNA Damage Sensor Uncoupling.

Abstract 4243

We have found that a stable multi-drug resistant (MDR) variant of the myeloid leukemia line, HL60, called RV+, which was selected for drug resistance in the presence of vincristine, is clonogenically cross-resistant to gamma radiation (RV+ D_o= 0.81 Gy, HL60 D_o = 0.49 Gy). At equal doses, these two different cell lines incurred equivalent DNA double strand breaks (DSBs) upon irradiation as measured by pulse-field electrophoresis, and showed nearly identical bulk DSB repair capacity and efficiency, suggesting that the resistence phenotype is not due to a reduction in DSBs. Interestingly, an early and proximal marker of DSBs, phosphorylation of the histone variant H2AX, is up to seven-fold lower in RV+ cells, despite equivalent bulk DNA damage. This discrepancy between physical damage and proximal signaling is accompanied by dramatically attenuated early and late G2/M checkpoint responses in RV+ cells, which require nearly 10 times the radiation dose of HL60 cells (2.0 Gy compared to 0.2 Gy) to elicit a full early G2/M check, and up to four times the dose (4.0 Gy compared to 1.0 Gy) for an equivalent late G2/M response. Consistent with decreased checkpoint stringency, up to eight time as many RV+ cells were found to be entering mitosis after radiation, with 62.7% unresolved DSBs as measured by micronuclei formation. Although HL60 had a comparable percentage of cells with unresolved breaks (72.6%), the total number of cells entering mitosis with intact nuclei was proportionally much lower. Moreover, in the same dose range, RV+ cells have a marked decrease in radiation-induced apoptosis compared to HL60, which has been shown to be an important clonogenic predictor in hematopoietic cells. As RV+ cells are 10-fold more sensitive to cytarabine-induced apoptosis, RV+ cell insensitivity is likely not due to a global anti-apoptotic phenotype. Increased clonogenic survival suggests that RV+ have partially decoupled sensing of extant DNA damage from checkpoint and apoptotic responses, possibly through downregulation of phospho-H2AX or its cognate upstream kinases. Current research seeks to determine a causal relationship between these unique radioresistance phenomena.