Carcinogenesis Advance Access originally published online on January 19, 2008
Carcinogenesis 2008 29(6):1267-1275; doi:10.1093/carcin/bgn012
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Epigenetic silencing of O6-methylguanine DNA methyltransferase gene in NiS-transformed cells
1 Faculty of Preventive Medicine, School of Public Health, Sun Yat-Sen University, 74 Zhongshan Road 2, Guangzhou 510080, People's Republic of China
2 Department of toxicology Shenzhen Center for Disease Control and Prevention, 21 Tianbei Road 1, Shenzhen 518020, People's Republic of China
3 Institute for Chemical Carcinogenesis, Guangzhou Medical College, 195 Dongfeng Xi Road, Guangzhou 510182, People's Republic of China
* To whom correspondence should be addressed. Tel/Fax: +86 755 25639066; Email: zxzhuang2007{at}126.com
Correspondence may also be addressed to Wen Chen. Tel: +86 20 87330599; Fax: +86 20 87330446; Email: wenchen1107{at}163.com
Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. To uncover the molecular mechanisms of nickel sulfide (NiS)-induced cell transformation, we investigated epigenetic alterations in a set of DNA repair genes. The silencing of the O6-methylguanine DNA methyltransferase (MGMT) gene locus and upregulation of DNA methyltransferase 1 (DNMT1) expression was specifically detected in NiS-transformed human bronchial epithelial (16HBE) cells. In addition, we noted epigenetic alterations including DNA hypermethylation, reduced histone H4 acetylation and a decrease in the ratio of Lys-9 acetylated/methylated histone H3 at the MGMT CpG island in NiS-transformed 16HBE cells. Meanwhile, we identified concurrent binding of methyl-CpG-binding protein 2, methylated DNA-binding domain protein 2 and DNMT1 to the CpG island of the MGMT promoter, demonstrating that these components collaborate to maintain MGMT methylation in NiS-transformed cells. Moreover, depletion of DNMT1 by introduction of a small hairpin RNA construct into NiS-transformed cells resulted in a 30% inhibition of cell proliferation and led to increased MGMT gene expression by reversion of the epigenetic modifications at the MGMT promoter region. MGMT suppression and hypermethylation at the CpG island of the MGMT promoter occurred 6 days after NiS treatment, indicating that epigenetic modifications of MGMT might be an early event in tumorigenesis. Taken together, these observations demonstrate that epigenetic silencing of MGMT is associated with DNA hypermethylation, histone modifications and DNMT1 upregulation, which contribute to NiS-induced malignant transformation.
Abbreviations: ACTB, β-actin; ChIP, chromatin immunoprecipitation; DAC, 5-aza-2-deoxycytidine; DNMT1, DNA methyltransferase 1; H3K9ac, histone H3 Lys-9 acetylation; H3K9me2, histone H3 Lys-9 methylation; H4ac, histone H4 acetylation; MBD, methylated DNA-binding domain protein; MeCP2, methyl-CpG-binding protein 2; MGMT, O6-methylguanine DNA methyltransferase; mRNA, messenger RNA; Ni, nickel; NiS, nickel sulfide; NSTC, nickel sulfide-transformed cell; PCR, polymerase chain reaction; Q-PCR, quantitative polymerase chain reaction; TSA, trichostatin A; 16HBE, human bronchial epithelial
Received September 20, 2007; revised December 4, 2007; accepted December 27, 2007.