[Effects of 5-Aza-2'-deoxycytidine and trichostatin A on P16, hMLH1 and MGMT genes and DNA methylation in human gastric cancer cells].
OBJECTIVE
To investigate the effects of 5-Aza-2'-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) on DNA methylation and expression of P16, hMLH1 and MGMT genes in the human gastric cancer cell line MGC-803, and to explore the mechanism of P16, hMLH1 and MGMT gene silencing in human gastric cancer cells.


METHODS
MGC-803 cells were cultured in RPMI-1640 medium and were treated with 5-Aza-dC or TSA. Methylation-specific polymerase chain reaction (MS-PCR) was used to detect the promoter methylation status of P16, hMLH1 and MGMT genes. RT-PCR was used to detect the mRNA expressions of P16, hMLH1 and MGMT.


RESULTS
Promoter hypermethylation of P16, hMLH1 and MGMT genes were detected in MGC-803 cells, and mRNA expressions of P16, hMLH1 and MGMT were absent before treatment. After treatment with 5-Aza-dC, the promoter region of the P16, hMLH1 and MGMT gene exhibited a demethylation status, and their mRNA expressions were increased. The treatment with TSA had no effects on DNA demethylation or restoration of P16 or hMLH1 expression. P16, hMLH1 and MGMT mRNA relative expression levels after treatment with a combination of 5-Aza-dC and TSA were 0.412+/-0.030, 0.397+/-0.024 and 0.553+/-0.043 respectively, which were higher than those after 5-Aza-dC treatment alone (0.221+/-0.022, 0.214+/-0.018 and 0.156+/-0.017, all P<0.05).


CONCLUSIONS
Promoter hypermethylation is a major mechanism of P16, hMLH1 and MGMT gene silencing in human gastric cancer cells. Treatment with 5-Aza-dC alone or the combination of 5-Aza-dC and TSA can reactivate the expressions of these genes.
Meng
Chun-feng
CF
Department of Surgical Oncology, The First Affiliated Hospital, China Medical University, Shenyang, China.
Zhu
Xin-jiang
XJ
Dai
Dong-qiu
DQ
Peng
Guo
G
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics; D000077209:Decitabine; D019942:Genes, p16; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D013274:Stomach Neoplasms / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics
D048868:Adaptor Proteins, Signal Transducing; D000964:Antimetabolites, Antineoplastic; D006877:Hydroxamic Acids; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; C012589:trichostatin A; D000077209:Decitabine; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D000070957:MutL Protein Homolog 1; D045643:DNA Repair Enzymes; D001374:Azacitidine