Dominant Negative Effect of Kinase-Inactive Bcr-Abl on Cell Survival and DNA Damage Response Pathways in Imatinib-Treated Cells.

Abstract

Imatinib inhibits cell proliferation and induces cell death in BCR-ABL positive leukemic cells in the absence of exogenic growth factors in vitro. In this study, we investigated the effects of physiological growth factors on cell survival and DNA damage response in Imatinib treated Bcr-Abl positive and Bcr-Abl negative cells from murine and human origin. To this end, we used 32D and 32D/bcr-abl, BaF3 and BaF3/bcr-abl, and M07 and M07bcr-abl cell lines. All bcr-abl positive cell lines were highly sensitive to Imatinib in the absence of growth factors. mIL3 protected Bcr-Abl transformed murine cell lines from Imatinib-induced cell death but was not capable to restore the Imatinib-dependent blockade of the p53 response to cisplatinum or gamma irradiation in Bcr-Abl positive cells. Despite prolonged pre-incubation with mIL3, Imatinib led to a transient inhibition of PI3K-, MAPK-, and STAT5-pathways selectively in Bcr-Abl positive murine cells. Importantly, this transient blockade of survival pathways by Imatinib was completely abolished after simultaneous siRNA-mediated suppression of Bcr-Abl synthesis. In contrast to the murine cell lines, Bcr-Abl positive human megakaryocytic M07 cells can not be rescued from Imatinib-induced cell death. Imatinib treated cells died even when pre-incubated simultaneously with a growth factor mix consisting of hGM-CSF, hSCF, and hIL3. Suppression of Bcr-Abl synthesis by RNAi using a breakpoint-specific siRNA selectively inhibited Bcr-Abl-dependent growth of M07/bcr-abl cells to an extent comparable to that observed with Imatinib. Importantly, growth factor stimulation enabled survival of M07/bcr-abl cells after suppression of Bcr-Abl synthesis by siRNA but not after Imatinib-mediated inhibition of Bcr-Abl kinase activity.In summary, our results indicate a dominant negative effect of kinase-inactive Bcr-Abl both on survival and on DNA damage response pathways.