Abstract
DNA damage and defects in DNA repair pathways may severely predispose to genomic instability which is one of the major factors associated with the formation and progression of chronic myelogenous leukemia (CML). However, leukemia cells transfected by BCR/ABL seem to be better equipped to survive DNA damage generated by reactive oxygen species (ROS) and external factors through activation of double-strand breaks (DSBs) repair by homologous recombination (HR) and non-homologous end-joining (NHEJ). A genome-wide screen was performed to identify genes regulated by BCR/ABL kinase and involved in DSBs repair. Werner syndrome protein (WRN), which exhibits helicase and exonuclease activity, was upregulated in CML cells. WRN is capable of unwinding various DNA structures associated with progressing replication forks as well as promoting Holliday junctions formed as intermediates in DNA recombination. Moreover, the helicase can directly interact with a variety of proteins involved in DSBs repair including Ku complex (NHEJ), and RAD51 (HR). Lack of WRN protein in Werner syndrome is characterized by accumulation of DSBs, genomic instability and a high incidence of cancer. Here we present evidence that BCR/ABL induced the expression of WRN mRNA and protein by activation of c-MYC transcription and inhibition of caspase-dependent cleavage, respectively. Immunoprecipitation and pull-down studies indicated that WRN is phosphorylated by BCR/ABL, and that BCR/ABL SH2 domain interacts directly with phospho-Y1346 of WRN. Drug sensitivity assays performed after downregulation of WRN expression by shWRN in BCR/ABL-positive cells have demonstrated an increased sensitivity to genotoxic stress induced by cisplatin and oxidative stress caused by H2O2. Experiments using TUR90010 lymphoblast cell line established from a Werner syndrome patient (3724C>T) and transfected with BCR/ABL confirmed that WRN plays an important role in response to DNA damage in CML cells. Further studies revealed that BCR/ABL-positive leukemia cells exert an enhanced WRN-mediated helicase activity. Bone marrow cells derived from transgenic mice expressing the helicase-defective WRN mutant (K577M) and transfected with BCR/ABL display increased sensitivity to cisplatin compared to those obtained from the wild-type littermates. The role of WRN in BCR/ABL-induced DSBs repair pathways, HR and NHEJ, was examined. NHEJ activity was measured in nuclear cell lysates of BCR/ABL-positive leukemia cells using linearized double-stranded plasmid as a substrate. Removal of WRN by immunoprecipitation did not affect the efficacy of NHEJ reaction. HR was assessed using cells containing one copy of the modified gene for GFP containing a unique I-SceI restriction site with two stop codons as a recombination reporter and a truncated fragment of the GFP gene as a template for homologous repair. A HR event restores functional GFP expression. Downregulation of WRN protein by shRNA abrogated HR activity induced by BCR/ABL. Therefore BCR/ABL-dependent overexpression of WRN helicase seemed to be important for HR, but not NHEJ. Finally, an enhanced interaction between WRN and RAD51 upon DNA damage in BCR/ABL-positive cells supported that conclusion. In summary, BCR/ABL-mediated overexpression and enhanced activation of WRN helicase played an essential role in response of CML cells to elevated numbers of DBSs induced by oxidative and genotoxic stress.
p53 Modulates RPA-Dependent and RPA-Independent WRN Helicase Activity
