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Journal of Virology, June 2003, p. 6227-6234, Vol. 77, No. 11
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.11.6227-6234.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

CpG Methylation of Human Papillomavirus Type 16 DNA in Cervical Cancer Cell Lines and in Clinical Specimens: Genomic Hypomethylation Correlates with Carcinogenic Progression

Vinay Badal,1 Linda S. H. Chuang,1 Eileen Hwee-Hong Tan,1 Sushma Badal,2 Luisa L. Villa,3 Cosette M. Wheeler,4 Benjamin F. L. Li,1 and Hans-Ulrich Bernard2,5*

Laboratory for DNA Repair and DNA Methylation in Chemical Carcinogenesis,1 Laboratory for Papillomavirus Biology, Institute of MolecularCell Biology, Singapore 117609, Singapore,2 Department of Molecular Biology and Biochemistry, University of California—Irvine, Irvine, California 92697-3900,5 Ludwig Institute for Cancer Research, Sao Paulo, Brazil,3 Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, New Mexico 87131-62764

Received 29 January 2003/ Accepted 14 March 2003

Infection with genital human papillomaviruses (HPVs) is the primary cause of cervical cancer. The infection is widespread, and little is known about the secondary factors associated with progression from subclinical infection to invasive carcinoma. Here we report that HPV genomes are efficiently targeted in vivo by CpG methylation, a well-known mechanism of transcriptional repression. Indeed, it has been shown previously that in vitro-methylated HPV type 16 (HPV-16) DNA is transcriptionally repressed after transfection into cell cultures. By using a scan with the restriction enzyme McrBC, we observed a conserved profile of CpG hyper- and hypomethylation throughout the HPV-16 genomes of the tumor-derived cell lines SiHa and CaSki. Methylation is particularly high in genomic segments overlying the late genes, while the long control region (LCR) and the oncogenes are unmethylated in the single HPV-16 copy in SiHa cells. In 81 patients from two different cohorts, the LCR and the E6 gene of HPV-16 DNA were found to be hypermethylated in 52% of asymptomatic smears, 21.7% of precursor lesions, and 6.1% of invasive carcinomas. This suggests that neoplastic transformation may be suppressed by CpG methylation, while demethylation occurs as the cause of or concomitant with neoplastic progression. These prevalences of hyper- and hypomethylation also indicate that CpG methylation plays an important role in the papillomavirus life cycle, which takes place in asymptomatic infections and precursor lesions but not in carcinomas. Bisulfite modification revealed that in most of the HPV-16 genomes of CaSki cells and of asymptomatic patients, all 11 CpG dinucleotides that overlap with the enhancer and the promoter were methylated, while in SiHa cells and cervical lesions, the same 11 or a subset of CpGs remained unmethylated. Our report introduces papillomaviruses as models to study the mechanism of CpG methylation, opens research on the importance of this mechanism during the viral life cycle, and provides a marker relevant for the etiology and diagnosis of cervical cancer.

* Corresponding author. Mailing address: Department of Molecular Biology and Biochemistry, University of California—Irvine, Irvine, CA 92697-3900. Phone: (949) 824-5162. Fax: (949) 824-8551. E-mail: hbernard{at}uci.edu.

Journal of Virology, June 2003, p. 6227-6234, Vol. 77, No. 11
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.11.6227-6234.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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