The Establishment Of Proteasome Inhibitor Bortezomib Resistant Leukemia Cell Lines And Study On Its Drug Resistance Mechanism

Objectives To study the mechanism of proteasome inhibitor-resistance and its relationship to multi-drug resistance. Methods To establish bortezomib resistant cell lines JurkatBs from Jurkat cell line, low-dose induction and high-dose repeated selection were used .The biological characteristics of Jurkat and JurkatBs were observed. It was also observed if there are multi-drug resistance appearances .Genes associated to multi-drug resistance were detected ; the expression and function of P-gp were analyzed. mRNA exprssion levels and mutation of protesome beta 5 subunit (PSMB5) at which chymotrypsin-like activity is sited were detected.The chymotrypsin-like activities and inhibition of the activities after treated with bortezomib were assayed. IκB-αlevel in Jurkat and JurkatB5 cells after treatment of bortezomib were also examined. Results Bortezomib resistant JurkatB1, JurkatB2, JurkatB3, JurkatB4 and JurkatB5 cell lines were established. There are no significant differences in celluar biology of cell doubling times, colony forming rates, cell cycle distribution between Jurkat and JurkatBs cells. The effects in cytotoxicity,cell cycle arrestment,apoptosis inducing of bortezomib decreased in JurkatBs cells. No cross-resistance was showed and no significant effect of drug efflux was found in JurkatBs cells. PSMB5 gene was amplified in bortezomib resistant cell lines except for JurkatB2 , which correspond well to the increase of chymotrypsin-like activity and i(14q) karyotype. Point mutation of the PSMB5 gene G322A ( corresponding amino acid substitution of A108T) was found, which resulted in the decreased inhibition rates of chymotrypsin-like activity after treated with bortezomib in JurkatBs cells . The resulst of immunoblotting of IκB-αindicate a up-regulation of NF-κB activity in resistant JurkatB5 cells after treated with bortezomib. There was no overexpression of MDR1 gene and the expression of P-gp was negative. MRP, LRP genes were significantly overexpressed in JurkatB1 cells and JurkatB5 cells, especially in JurkatB1.Conclusions Amplification of PSMB5 gene followed by increase of chymotrypsin-like activity , the A108T substitution of PSMB5 resulting in a decreased binding affinity of bortezomib to the chymotrypsin-like site and up-regulation of NF-κB activity after treated with bortezomib are the important mechanisms of bortezomib resistance. So the mechanism of bortezomib resistance is different from that of multi-drug resistance, but the relationship between overexpressions of MRP and LRP gene and bortezomib resistance should be further studied.