Preliminary Study On Influence of Imatinib-Induced Apoptosis in Chronic Myeloid Leukemia Cells Co-Culture with Fibronectin Adhesion.

Abstract 4264

The bone marrow microenvironment supports growth, differentiation and proliferation of normal hematopoietic cells and can also contribute to malignant growth. Recently,it is considered that except for the point mutant of BCR-ABL kinase contribute to imatinib-resistant therapy for patients with chronic myeloid leukemia(CML), environment-mediated drug resistance (EM-DR) is a potential factor in imatinib resistance. Our previous studies found that Integrin, focal adhesion kinase(FAK), RhoA(a small GTPase) are important adhesion molecules,and related to imatinib resistance. But how and what they crosstalk with each other is still open to debate.

In order to simulated bone marrow microenvironment, we used the major components of bone marrow microenvironment- Fibronectin (Fn) co-cultured with human leukemia K562 cells.and then K562 cells were inoculated with Fn, collagen-coated plate(Co) and suspended cultures as control(mask) group,and then treated with 0.4μM,0.8μM,1.6μM,3.2μM,6.4μM imatinib for 24h,48h and 72h, detected cell apoptosis and proliferation by MTT and AnnexinV-PI assay, examined p-FAK and Rho-GTP by Wersten Blotting and Pull down-Wersten Blotting. The data showed that compared to the Co and mask groups, the cells growth inhibition and apoptosis in Fn co-culture group was significantly reduced. The protein expression of p-FAK and Rho-GTP was higher in the Fn group,and in time-dependent manner. When K562 cells in Fn group were transfected with 150nM siRNA-RhoA for 48h, there was no significant difference compared with the Co and mask groups. Furthermore, the above groups treated with anti-integrin monoclonal antibody (anti-CD29 mAb), we found that p-FAK was significant lower compared with without anti-CD29 mAb in the Fn group; but there was no significant difference of Rho-GTP compard with without anti-CD29 mAb in Fn group. These results indicate that Fn adhesion co-culture could reduce imatinib-induced cell growth inhibition and apoptosis, and this mechanism may be correlated to Rho-GTP activity,and anti-integrin monoclonal antibody could not completely block the integrin binding to Fn on K562 cells, or there was other pathway activated RhoA. The mechanism of EM-DR is complex,and which is well worth us to speculate and study.