| Nbs1, a member of the DNA repair/checkpoint activation complex Mre11/Rad50/Nbs1, is phosphorylated by ATM in response to the presence of DNA double strand breaks (DSBs) resulting in the activation of the S phase checkpoint. Here we show that the BCR/ABL oncogenic fusion tyrosine kinase stimulates the expression of Nbs1. This effect is dependent on the kinase activity of BCR/ABL protecting Nbs1 from caspase-dependent, but not proteasome-dependent degradation, and on c-Myc transactivation. Western analyses and immunofluoresence studies showed enhanced phosphorylation of Nbs1 on Serine 343 (5343) in mitomycin C (MMC)-treated BCR/ABL leukemia cells (patient cells and cell lines) in comparison to normal counterparts. Enhanced levels of Nbs1 phosphorylated on 5343 were also detected in MMC-treated tumor cells expressing BCR/ABL-related fusion kinases such as: TEL/JAK, BCR/FGFR1, NPM/ALK, TEL/ABL, TEL/PDGF/3R, and TEL/TRKC. Downregulation of Nbs1 in BCR/ABL positive cells using siRNA caused increased sensitivity to MMC. Specifically, the expression of the Nbs1-S343A phosphorylation-less mutant also decreased resistance in BCR/ABL cells to MMC. The radioresistant DNA synthesis (RDS) assay showed that MMC-treated CML patient cells and BCR/ABL transformed cell lines displayed an inhibition of DNA synthesis associated with transient accumulation of the cells in S phase. Expression of Nbs1-S343A mutant caused a significant increase in the DNA synthesis and reduced accumulation of BCR/ABL leukemia cells in S phase after MMC treatment, whereas cells transfected with the empty construct or wild-type Nbs1 displayed inhibition of DNA synthesis and S phase accumulation. Altogether, we hypothesize that enhanced phosphorylation of Nbs1 on 5343 leads to increased resistance to genotoxic agents in BCR/ABL cells by prolonging the S phase checkpoint and allowing longer time for the repair of excessive DNA damage. |
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