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Carcinogenesis Advance Access published online on March 11, 2009
Carcinogenesis, doi:10.1093/carcin/bgp060
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Incorporation of 5-chlorocytosine into mammalian DNA results in heritable gene silencing and altered cytosine methylation patterns

Victoria Valinluck Lao1,2, Jason L. Herring1, Cherine H. Kim1, Agus Darwanto1, Ubaldo Soto1 and Lawrence C. Sowers1

1 Department of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, California 92350
2 Current address: Department of Surgery, University of Washington, Seattle, WA 98195

Correspondences should be addressed to lsowers{at}llu.edu.

Cytosine methylation patterns are essential for the proper control of gene expression in higher vertebrates. Although alterations in methylation patterns are frequently observed in human tumors, neither the mechanisms for establishing methylation patterns during normal development nor the mechanisms leading to pathological alterations of methylation patterns are currently known. While epidemiological studies have implicated inflammation in cancer etiology, a mechanistic link has yet to be established. Investigations of inflammation-mediated DNA damage may have provided important new insights. Our in vitro studies revealed that the inflammation-mediated DNA damage product, 5-chlorocytosine, could direct fraudulent methylation of previously unmethylated CpG sites. The purpose of this study was to recapitulate our in vitro findings by introducing 5-chlorocytosine residues into the DNA of replicating mammalian cells and to examine its impact on gene expression and cytosine methylation patterns. CHO-K1 cells hemizygous for the HPRT gene were electroporated with the triphosphates of cytosine (dCTP), 5-methylcytosine (MedCTP) and 5-chlorocytosine (CldCTP) then selected with 6-thioguanine for silencing of the HPRT gene. Both modified nucleotides, MedCTP and CldCTP, but not unmodified dCTP, silenced HPRT gene expression. Subsequent bisulfite pyrosequencing of CpG sites within the HPRT promoter region of the selected cells confirmed hypermethylation, although global methylation levels as measured by GC/MS did not change. Modified nucleotide-induced gene silencing could be reversed with 5-aza-2'-deoxycytidine (DAC) indicating an epigenetic rather than mutagenic alteration. These results provide further evidence that the inflammation damage product 5-chlorocytosine could be a link between inflammation and cancer development.

Key Words: DNA damage • cytosine methylation • epigenetic alterations • cancer development • inflammation

This study was funded by grants from the National Institutes of Health

Received October 21, 2008; revised February 22, 2009; accepted March 7, 2009.


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