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 hyperand 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.Human papillomavirus type 16 (HPV-16) and related HPV types are carcinogenic, and persistent HPV, infection is a prerequisite in the etiology of most or even all cervical cancers (22, 38, 52). Most women become infected by HPVs, and while some of these infections progress malignantly, most remain subclinical or lead only to precursor lesions. The factors that determine these outcomes are poorly understood. Transformation by HPVs depends on the oncoproteins E6 and E7, whose transcription is modulated by numerous transcription factors and epigenetic mechanisms (5, 18). Tumor progression may result from stimulated oncoprotein expression through transcriptional induction by steroids (10, 17), by deletion of transcriptional silencers (25), or by integration of HPV genomes into cellular DNA (3,40,43).cis-responsive elements that regulate E6 and E7 oncogene transcription are spread throughout the long control region (LCR) of HPV-16, an 850-bp segment between the L1 and E6 ...

Human papillomaviruses (HPVs) infect epithelia, including the simple and the squamous epithelia of the cervix, where they can cause cancer and precursor lesions. The molecular events leading from asymptomatic HPV infections to neoplasia are poorly understood. There is evidence that progression is modulated by transcriptional mechanisms that control HPV gene expression. Here, we report the frequent methylation of HPV-18 genomes in cell culture and in situ. DNA methylation is generally known to lead to transcriptional repression due to chromatin changes. We investigated two cell lines derived from cervical cancers, namely, C4-1, which contains one HPV-18 genome, and different clones of HeLa, with 50 HPV-18 genomes. By restriction cleavage, we detected strong methylation of the L1 gene and absence of methylation of parts of the long control region (LCR). A 3-kb segment of the HPV-18 genomes downstream of the oncogenes was deleted in both cell lines. Bisulfite sequencing showed that in C4-1 cells and two HeLa clones, 18 of the 19 CpG residues in the 1.2-kb terminal part of the L1 gene were methylated, whereas a third HeLa clone had only eight methylated CpG groups, indicating changes of the methylation pattern after the establishment of the HeLa cell line. In the same four clones, none of the 12 CpG residues that overlapped with the enhancer and promoter was methylated. In six HPV-18 containing cancers and five smears from asymptomatic patients, most of the CpG residues in the L1 gene were methylated. There was complete or partial methylation, respectively, of the HPV enhancer in three of the cancers, and lack of methylation in the remaining eight samples. The promoter sequences were methylated in three of the six cancers and four of the six smears, and unmethylated elsewhere. Our data show that epithelial cells efficiently target HPV-18 genomes for DNA methylation, which may affect late and early gene transcription.

We conclude that methylation of the E6 gene promoter in HPV-16 genome is a predictive biomarker for cervical cancer progression by regulating the expression of the E6 oncogene.

The INK4a/ARF locus encodes p14(ARF) which plays an important role in the p53 pathway. Interestingly, methylation of the INK4a/ARF locus is a common event in carcinogenesis. In this study we analyzed the effect of epigenetic alteration on the p14(ARF) promoter and its direct link to the expression of the p14(ARF) mRNA and protein. The osteosarcoma cell line U2OS was used as a model to study the regulation of the ARF promoter by DNA methylation. Treatment of U2OS cells with the demethylating agent 5-aza- 2'-deoxycytidine (5-aza-CdR) showed a marked induction of the p14(ARF) mRNA and protein. A novel quantitative method described here, using restriction enzyme digestion followed by real time PCR, allowed the analysis of the level of methylation over a defined region of DNA on the p14(ARF) promoter. The change in the methylation level of the promoter closely corresponded to the increase in the transcription of p14(ARF) mRNA and protein. Upon removal of 5-aza-CdR the methylation pattern on the p14(ARF) promoter was re-laid with a concomitant decrease in the levels of p14(ARF) mRNA and protein. The increase in the levels of p14(ARF) was concomitant with an induction of G(1)-G(2) cell cycle arrest and an induction of p21 protein. No increase in the levels of p53 was observed. However, induction of p14(ARF) upon treatment with 5-aza-CdR led to the sequestering of MDM2 to the nucleolus. Additionally, we could show a dependency between the demethylation of the p14(ARF) promoter, the induction of p14(ARF) mRNA and protein and the effect of 5-aza-CdR on cell cycle.

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