Switch to Full-Length of XAF1 mRNA Expression in Myeloma RPMI8226 Cells by DNA Methylation Inhibitor 5-Azacytidine.

Abstract X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family that exerts antiapoptotic effects by interfering with the activities of caspase-3, -7, -9, while the XIAP-associated factor1 (XAF1), a zinc finger protein, antagonizes XIAP activities, thereby promotes apoptosis.The aberrant silencing of the XAF1 gene has recently been found in various types of cancer cells, which is suggested to be one of the potential mechanisms underlying survival advantages of malignant cells. We investigated the XAF1 expression in myeloma RPMI8226 cells. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and western blot methods were used for detecting the expressions of XAF1, XIAP, SURVIVIN, β-actin genes in RPMI8226 cells at different time points after treatment with 2.5 μM or 5 μM 5-azacytidine. The apoptosis of RPMI8226 cells was simultaneously detected using flow cytometric assay. Methylation-specific PCR was used to assay methylation status of the CpG sites in the XAF1 promoter. Compared with normal bone marrow mononuclear cells where only a full-length of XAF1 mRNA is detected, RPMI8226 cell expressed a full-length form of XAF1 transcript and a short form of XAF1 transcript. Only a full-length form of XAF1 transcript was detected after RPMI8226 cells treated with 5 μM 5-azacytidine for 120h. More percentages of RPMI8226 cells progressed to early apoptosis after 5 μM 5-azacytidine treatment for 120h. Collectively, our study suggests that epigenetic silencing of XAF1 by aberrant promoter methylation may contribute to the malignant progression of human myeloma. DNA methylation inhibitor 5-azacytidine treatment can increase myeloma RPMI8226 cell XAF1 gene expression and promote apoptosis.