Study On The Expression And Function Of GSTpi In Chronic Myelogenous Leukemia Cells

GSTpi is an important member of GSTs,which is a family of dimeric enzymes encoded by the active and polymorphic genes.The functions in vivo of GSTpi protein are thought to be involved in the elimination of hazardous agents and prevent the genes from injury.The co-expression of GSTpi with a number of drug-resistance associated genes might be an important reason of the tumors resistance to chemotherapeutics and/or biotherapy reagents.Chronic myelognous leukemia(CML) is a malignant chronic myeloproliferative disorder(MPD) of the hematopoietic stem cells.Interferon-a(INF-a) is one of the effective drugs to treat CML,but some of the CML patients exhibit the drug resistance to IFN-a clinically.Three CML cell lines including KT-1/A3,KT-1/A3R and K562 with different sensitivity to IFN-a effect were used in the present study.By using the technologies of immunocytochemistry with streptavidin peroxidase and Western blotting with anti-GSTpi antibody,the different expression levels of GSTpi in KT-1/A3,KT-1/A3R and K562 cells were comparatively detected before and after IFN-a induction.The results showed that the expression level of GSTpi protein in KT-1/A3R cells was significantly higher than that in KT-1/A3 cells in the conditions of both before and after IFN-a treatment.However,there was no GSTpi protein expression in K562 cells with or without IFN-a induction.The laser immunofluorescence-scanning confocal microscopy showed that GSTpi proteins were mainly localized in cytoplasm of KT-1/A3R and KT-1/A3 cells,but also no GSTpi expression was seen in K562 cells.Based on the results mentioned above,pcDNA3.1-GSTpi recombinant expression vector was constructed and transfected into KT-1/A3 cells with little GSTpi expression and K562 cells without GSTpi expression.The expression level difference and the subcellular localization of GSTpi protein in the pcDNA3.1-GSTpi recombinant expression vector transfected cells before and after IFN-a treatment were analyzed.The results of Flow Cytometry(FCM) for the apoptosis ratio detection and Typan Blue dye exclusion test for cell viability analysis showed that the IFN-a response of KT-1/A3 cells was significantly decreased by pcDNA3.1-GSTpi recombinant expression vector transfection.However,the resistant features of K562 cells to IFN-a were not affected by pcDNA3.1-GSTpi recombinant expression vector transfection.The results of caspase-3 activity detection showed that the caspase-3 enzymatic activity of KT-1/A3 cells without pcDNA3.1-GSTpi recombinant expression vector transfection and then induced by INF-a was significantly increased,while the caspase-3 enzymatic activity of KT-1/A3 cells transfected with pcDNA3.1-GSTpi recombinant expression vector and then induced by INF-a was almost not changed, meanwhile,the caspase-3 enzymatic activity of K562 cells transfected with pcDNA3.1-GSTpi recombinant expression vector and then induced by INF-a was slightly increased.