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. 2003 Jun;77(11):6227-34.
doi: 10.1128/jvi.77.11.6227-6234.2003.

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, Eileen Hwee-Hong Tan, Sushma Badal, Luisa L Villa, Cosette M Wheeler, Benjamin F L Li, Hans-Ulrich Bernard
Affiliations

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 et al. J Virol. 2003 Jun.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
HpaII/MspI cleavage identifies CpG methylation of SiHa and CaSki DNA as a likely source of transcriptional repression. (A) Distribution of CpGs in the LCR (positions 7154 to 7906 and 1 to 96) and in the E6 gene of HPV-16 (positions 97 to 559) and positions of two HpaII/MspI sites, one (position 57) overlapping with elements of the E6 promoter P97 and the other located in the 3′ part of the E6 gene (position 502). (B) Reverse transcription-PCR confirms that similar amounts of E6 and E7 transcripts are generated by SiHa and CaSki cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase, served as the cellular control transcript. (C) Genomic PCR confirms the large excess of HPV-16 DNA in CaSki cells. (D) Chromosomal DNA of SiHa and CaSki cells was not cut (CT, control) or was cleaved with one of the two enzymes (H, HpaII; M, MspI) and PCR amplified to generate the amplicons P2, P5, and P11. None of the three amplicons could be generated after cleavage with either of the two enzymes in the case of SiHa cells, indicating lack of any methylated CCGG sequences. In the case of CaSki cells, most of the DNA was resistant to HpaII digestion but was readily cleaved by MspI, indicating methylation of these two sites in most of the 500 HPV-16 copies in CaSki cells.
FIG. 2.
FIG. 2.
McrBC cleavage of segments of the HPV-16 genomes in SiHa and CaSki cells and three clinical samples detects patterns of hyper- and hypomethylation. (A) Genomic map of HPV-16 (7,906 bp), with genes E6, E7, E1, E2, E5, L2, and L1, the LCR, and the relative locations of amplicons G1 to G8 indicated. (B) Cleavage of amplicons G1 to G8 by McrBC (right lane of each pair of samples) indicates methylation, compared with uncleaved controls (left lane of each pair). The cleavage pattern indicates hypermethylation throughout most HPV-16 genomes in DNA from CaSki cells and tumor 6, hypermethylation of the late genes in the single HPV-16 genome of SiHa cells, and hypo- or no methylation in tumor 4 and a CIN I lesion.
FIG. 3.
FIG. 3.
McrBC cleavage identifies hypomethylation of HPV-16 DNA in cervical cancers and hypermethylation in asymptomatic smears. Amplicon P4 includes the genomic segment from positions 7850 to 559 with the E6 promoter elements, the E6 oncogene, and cis-responsive elements within E6 (left side of figure). The upper panel at right shows two typical McrBC-resistant, i.e., unmethylated, amplicons detected in DNA from invasive cancers, and the lower panel shows two typical McrBC-sensitive and methylated amplicons from asymptomatic cervical smears.
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References

    1. Apt, D., T. Chong, Y. Liu, and H.-U. Bernard. 1993. Nuclear factor I and epithelial cell-specific transcription of human papillomavirus type 16. J. Virol. 67:4455-4463. - PMC - PubMed
    1. Baker, C. C., and C. Calef. 1995. Maps of papillomavirus transcripts, part III-A, p. 3-19. In G. Myers, H.-U. Bernard, H. Delius, C. Baker, J. Icenogle, A. Halpern, and C. Wheeler (ed.), Human papillomaviruses 1995 compendium. Los Alamos National Laboratory, Los Alamos, N.Mex.
    1. Baker, C. C., W. C. Phelps, V. Lindgren, M. J. Braun, M. J. Gonda, and P. M. Howley. 1987. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J. Virol. 61:962-971. - PMC - PubMed
    1. Bauer-Hofmann, R., C. Borghouts, E. Auvinen, E. Bourda, F. Rosl, and A. Alonso. 1996. Genomic cloning and characterization of the nonoccupied allele corresponding to the integration site of human papillomavirus type 16 DNA in the cervical cancer cell line SiHa. Virology 217:33-41. - PubMed
    1. Bernard, H.-U. 2000. Regulation of human papillomavirus gene expression by the factor CDP, nucleosomes and the nuclear matrix. Papillomavirus Rep. 11:73-80.

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