BCR/ABL Modifies the Kinetics and Fidelity of DNA Double-Strand Breaks Repair in Leukemia Cells.

Clinical observations and experimental findings indicated that BCR/ABL stimulates genomic instability leading to mutations and chromosomal abnormalities. The accumulation of genetic errors is believed to be responsible for the transition from a relatively benign CML chronic phase (CML-CP) to the aggressive blast crisis phase (CML-BC) and the resistance to imatinib mesylate. BCR/ABL- positive leukemia cells accumulate an excess of potentially lethal DNA double-strand breaks (DSBs) caused by reactive oxygen species (ROS) or genotoxic treatment. However, BCR/ABL tyrosine kinase facilitates the repair of DSBs and promotes survival. Therefore, the infidelity of DSBs repair processes may contribute to genomic instability in leukemia cells exposed to elevated numbers of spontaneous and/or induced DSBs. To test this hypothesis DSBs repair efficiency and fidelity was examined and compared in parental and BCR/ABL-transformed cells. Nuclear foci detected by γ-H2AX (the form of H2AX histone that is quickly phosphorylated on Serine 139 by ATM, ATR and/or DNA-PKcs kinases on megabase-length fragments near DSB sites) immunofluorescence served as indicators of DSBs. We found that BCR/ABL-positive leukemia cells acquire more DSBs after γ-irradiation in comparison to normal cells. Homologous recombination (HR) and non-homologous end-joining (NHEJ) represent two major mechanisms of DSBs repair in mammalian cells. HR and NHEJ reaction sites in the nuclei can be visualized by double-immunofluorescence detecting co-localization of γ-H2AX foci with RAD51 or Ku70, respectively. NHEJ and HR appear to work in a time-dependent fashion (NHEJ followed by HR) and be more active in BCR/ABL-transformed cells in comparison to normal counterparts. Time-dependent engagements of NHEJ and HR mechanisms in repair of DSBs after γ-irradiation are accompanied by elevated accumulation of Ku70 and RAD51 proteins in cell lysates obtained from BCR/ABL cells. Specific DSBs repair assays confirmed that BCR/ABL leukemia cells in comparison to normal cells displayed enhanced capability of HR and NHEJ. However, analysis of DSBs repair products revealed that the repair mechanisms were less faithful in former cells generating large deletions and point mutations during NHEJ and HR, respectively. In summary, BCR/ABL leukemia cells display facilitated, but unfaithful HR and NHEJ, which may contribute to accumulation of genetic errors in surviving leukemia cells leading to malignant disease progression.