Hypermethylation status around exon 1 of XIAP-associated factor 1 (XAF1) in human gastric andcolon cancer cell lines

Abstract

Background and Aims XIAP-associated factor 1 (XAF1) hasrecently been shown to negatively regulate the caspase-inhibitingactivity of XIAP (X-linked IAP), thus, XAF1 may play a pivotal rolein cancer cell apoptosis. The XAF1 mRNA is expressed at low orundetectable levels in various cancer cell lines, suggesting that thegene silencing in cancer cells is important for malignant cell survival.The aim of this study is to examine the role of promoter and exon 1hypermethylation in the transcriptional silencing of XAF1. Methods (1) We treated the gastric cancer cell lines AGS, MKN-45, BCG823 and colon cancer cell lines SW1116, HCT 15, colo 205with 5¢-AZA-2¢-deoxycytidine (5¢-AZA), then the mRNA levels weredetected by RT-PCR. (2) Genomic DNAs were isolated from thesecell lines and human normal samples, the methylation status wasdetermined by bisulfite DNA sequencing analysis for 8 CpG siteslocated around exon 1 area (nucleotides -40 to 194).Results The XAF1 mRNA were detected in all six cell lines. Therelative mRNA level ranked HCT 15 < AGS < SW1116,BCG 823 <Colo 205 < MKN-45. 5¢-AZA treatment significantly upregulated thegene expression of XAF1 in all of the cancer cell lines with the excep-tion of Colo205. HCT 15 and AGS were most sentitive to 5¢-AZAtreatment suggesting that they have the highest methylation status. Bybisulfite DNA sequencing method,we showed that normal blood celldo not display XAF1 methylation. In contrast, all cancer cell linesshowed different levels of hypermethylation of exon 1 except Colo205. Among the 5 cell lines, HCT 15 and AGS displayed highest levelof hypermethylation.Conclusions Results from our study showed that the exon 1 hyper-methylation status of XAF1 coincidences with the gene expressionsuggesting that the aberrant exon 1 methylation contributes at leastin part to the epigenetic silencing of XAF1, which may be importantfor malignant cell survival.