MDR-1 Gene and BCRP are involved in the resistance to imatinib in K-562 cell line

LARDO M, GARGALLO P, BIANCHINI M, LUCERO G, KOZINER B, LARRIPA I, LAZAROWSKI A

Orlando (Florida)

Congreso; American Society of Hematology; 2006

Drug resistance to imatinib in chronic myeloid leukemia (CML) was been linked to BCR/ABL gene amplification or mutations in its tyrosine kinase domain of BCR/ABL gene (Ph). However the role of the ABC-transporters in the multidrug resistant phenotype still remains to be clarified. Objective: To study the functional expression of multidrug resistance gene-1 (MDR-1) and breast cancer resistant protein (BCRP), in imatinib pharmacoresistant K562 Ph+ cells. Materials and methods: K562 Ph+ cultured cells (ATCC cat. n. CCL-2439) were immunophenotyped by flow cytometry,mRNA transcripts of BCR/ABL and MDR-1 were amplified by RT-PCR and P-glycoprotein (P-gp), the product of MDR-1 gene and BCRP, were investigated by inmunocytochemistry , using specific monoclonal antibodies (P-gp: clones C494 and C219; BCRP: clone BXP-21). Total efflux activity of Rhodamine-123 (Rho-123) and its inhibition by Cyclosporin A (CyA) or 4ºC, were evaluated using flow cytometry. The apoptotic effect in K-562 cells after 24hs and 72hs of treatment with imatinib (2µmol.L-1) with or without CyA (3ugm.L-1), was evaluated by staining the cells with 1 mL of a mixture of acridine orange (100 mg.mL-1) and ethidium bromide (100 mg.mL-1) in PBS. Results: The inmunophenotype was: HLA-DR–,CD45++,CD11c–/+,CD13+,CD14–,CD15–/+, CD16– ,CD33+,CD34– CD117–/+;CD56–,CD61–/+;CD2–,CD3–,CD4–,CD7–,CD8–,CD10+,CD19–,glyA+. MDR-1 and BCR/ABL transcripts were highly expressed, and P-gp and BCRP immunostaining were also positive. Rho-123 efflux was inhibited by CyA (3 mg/mL) or 4ºC. Increased apoptotic effect (from 7, 21% to 20,79%) at 24hs and (11% to 53%) at 72hs was observed with combined imatinib+CyA treatment compared with imatinib alone.(p<0.01).Conclusions. Our results suggest that P-gp and BCRP might play an active role in the pharmacoresistance to imatinib treatment in K562 Ph+ cells. Combined treatment with Imatinib + CyA (or other more specific inhibitors for P-gp and BCRP) could increase the apoptotic effect in BCR/ABL over-expressing refractory cells.