ABCG2 Expression And The Study Of Methylaion In Gleevec-resistant Chronic Myeloid Leukemia Cell Line

BACKGROUD AND OBJECTIVE:Chronic myeloid leukemia (CML) is a clonal proliferative disease, with self-renewing populations of the stem and progenitor cell, the characteristic cytogenetic abnormality underlying CML is the Philadelphia chromosome (Ph) [t (9; 22) (q34; q11)], which cause the Abelson tk gene(ABL) to fuse with the breakpoint cluster region of the BCR gene. The BCR/ABL fusion protein has sustained tyrosine kinase activity, is the key factor in the pathogenesis of CML. Gleevec is widely recognized as a small molecule drugs of ideal targeted treatment for CML. But Gleevec resistance have seriously affected the treatment of CML, it is urgent to resolve this problem. Alough Gleevec resistance is mainly mediated by BCR/ALB, but some researches has shown that the membrane transporters also have close relationship with it. Studies have found that the breast cancer resistance protein (BCRP/ABCG2), a member of ATP binding cassette (ABC) transporter superfamily, may led to resistance to Gleevec. In the ABCG2 promoter region contains a large number of CpG islands, suggesting that the expression of ABCG2 is likely regulated by promoter methylation.At present, the study on the ABCG2 gene expression and regulation of methylation is less, especially in chronic myeloid leukemia. Therefore, our research analyzed chronic myeloid leukemia cell line K562 and the Gleevec-resistant chronic myeloid leukemia cell line K562/Glv, then carry out in virto experiments to investigate the relationship between the methylation status of ABCG2 promoter and its expression in cell, which provides a new target to reverse the Gleevec resistance mediated by ABCG2 gene, for the purpose of improving therapeutic effectiveness and improving tumor patients' life quality.METHODS:①We analyzed K562 and K562/Glv cells, and used SYBR Green based Real-Time PCR and flow cytometry to detect the ABCG2 mRNA and protein expression separately, and used MSP to detect ABCG2 promoter methylation status.②After exposured to the 5-azacytidine(10μmol/l) for 72 hour, we also used SYBR Green based Real-Time PCR and flow cytometry to detect the ABCG2 mRNA and protein expression separately in two cell lines, and used MSP to detect ABCG2 promoter methylation status.RESULT:①ABCG2 mRNA expression in K562/Glv cell was significantly higher than K562 cell (P<0.01); ABCG2 protein expression is low in K562 cell [(1.953±0.252)%] and K562/Glv cell [(1.383±0.259)%]; ABCG2 promoter in K562 and K562/Glv cells is complete methylation.②After exposured to the 5-azacytidine, ABCG2 promoter was completely unmethylated in K562 cell, ABCG2 mRNA and protein expression were significantly higher than before (P<0.01).③After exposured to the 5-azacytidine, ABCG2 promoter was partly non-methylated, ABCG2 mRNA and protein expression were higher than before, but no significant difference (P>0.05). ④After exposured to the 5-azacytidine, ABCG2 promoter methylation status in K562 cell was lower than K562/Glv cell (P<0.05), but ABCG2 mRNA and protein expression in K562 cell were higher than K562/Glv cell (P <0.05).CONCLUSIONS:①ABCG2 expression maybe has a certain relationship with Gleevec resistence in chronic myeloid leukemia, and ABCG2 expression is regulated by promoter methylation at least in partial.②ABCG2 protein expression is correlated with mRNA transcriptional level and the regulation of post-transcription.