Correlation between the single-site CpG methylation and expression silencing of the XAF1 gene in human gastric and colon cancers.
OBJECTIVE
X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) antagonizes the anti-caspase activity of XIAP. XAF1 messenger RNA is present in normal tissues but undetectable in various cancers and thus poses a potential tumor suppressor gene. The aim of this study was to examine the novel pattern of methylation of XAF1 in gastric and colon cancers and locate the important CpG sites for transcriptional regulation and tumor progression.


METHODS
XAF1 expression was detected by reverse-transcription polymerase chain reaction (PCR) and Western blot analysis. Four different fragments around the transcription start site of XAF1 were cloned and examined putative promoter activities by luciferase reporter assay. Each CpG site in fragment F291 was mutated by site-directed mutagenesis technique, and the change of promoter activity of this fragment was detected by luciferase reporter assay. Methylation status of XAF1 was determined by methylation-specific PCR (MSP) and bisulfite DNA sequencing PCR analysis.


RESULTS
Down-regulation of XAF1 in association with hypermethylation was detected in 3 of 4 human gastric cancer cell lines and 6 of 8 colon cancer cell lines. Of the 4 promoter fragments, F291 showed the highest promoter activity, which could be down-regulated obviously by the mutation of particular CpG sites. Moreover, aberrant hypermethylation of these important CpG sites was strongly associated with the development of gastric and colon cancers.


CONCLUSIONS
A cluster of methylated CpG sites instead of CpG islands located in the promoter area resulted in gene silencing of XAF1, and CpGs at -2nd, -1st, and +3rd positions are functionally more important in its transcriptional regulation.
Zou
Bing
B
Department of Gastroenterology, Rui-jin Hospital, Shanghai Jiao Tong University, Shanghai, China.
Chim
Chor Sang
CS
Zeng
Hui
H
Leung
Suet Yi
SY
Yang
Yi
Y
Tu
Shui Ping
SP
Lin
Marie C M
MC
Wang
Jide
J
He
Hua
H
Jiang
Shi Hu
SH
Sun
Yun Wei
YW
Yu
Li Fen
LF
Yuen
Siu Tsan
ST
Kung
Hsiang Fu
HF
Wong
Benjamin C Y
BC
D048868:Adaptor Proteins, Signal Transducing; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D051017:Apoptosis Regulatory Proteins; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism; D018899:CpG Islands; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics*; D000077209:Decitabine; D015536:Down-Regulation / Q000187:drug effects; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D000964:Antimetabolites, Antineoplastic; D051017:Apoptosis Regulatory Proteins; D004273:DNA, Neoplasm; D006877:Hydroxamic Acids; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C417551:XAF1 protein, human; C012589:trichostatin A; D000077209:Decitabine; D001374:Azacitidine