Regulation of hMSH2 and hMLH1 expression in the human colon cancer cell line SW1116 by DNA methyltransferase 1.
Aberrant DNA methylation is now recognized as an important epigenetic alteration occurring early in human cancer. To directly study the role of DNA methyltransferase 1 (DNMT1) in the regulation of expression of tumor-related genes in human colon cancer cells, we stably transfected expression constructs containing sense or antisense DNMT1 into the human colon cancer cell line, SW1116. The expression level of mismatch repair genes (MMR), human mut-L homologue 1 (hMLH1) and human Mut S homologue 2 (hMSH2), was monitored by real-time RT-PCR. The methylation status of hMLH1 and hMSH2 promoters was determined by bisulfite modification and methylation-specific PCR (MSP). The protein levels of DNMT1, hMSH2 and hMLH1 were determined by Western analysis. The results show that DNMT1 protein expression was increased or decreased in transfected cell lines containing sense or antisense DNMT1 constructs, respectively. In cells expressing the sense DNMT1 construct, the expression of hMLH1 and hMSH2 was down-regulated through hypermethylation of their respective promoters. Furthermore, antisense DNMT1 expression induced promoter demethylation and up-regulated transcription of hMSH2 (P<0.05) and hMLH1 (P=0.064) in SW1116 cells.
Shanghai Second Medical University Renji Hospital, Shanghai Institute of Digestive Disease, 145 Shandong Zhong Road, Shanghai 200001, China. jingyuanfang@yahoo.com
Jing Yuan
JY
Fang
Rong
R
Lu
Judy A
JA
Mikovits
Zhong Hua
ZH
Cheng
Hong Yin
HY
Zhu
Ying Xuan
YX
Chen
D048868:Adaptor Proteins, Signal Transducing; D020137:Base Pair Mismatch; D002352:Carrier Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000502:physiology*; D019175:DNA Methylation; D004260:DNA Repair; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000235:genetics*; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein