Methylation of the human papillomavirus-18 L1 gene: A biomarker of neoplastic progression?

Abstract

Epigenetic transcriptional regulation plays an important role in the life cycle of human papillomaviruses (HPVs) and the carcinogenic progression of anogenital HPV associated lesions. We performed a study designed to assess the methylation status of the HPV-18 genome, specifically of the late L1 gene, the adjacent long control region (LCR), and part of the E6 oncogene in cervical specimens with a range of pathological diagnoses. In asymptomatic infections and infections with precancerous (precursor) lesions, HPV-18 DNA was mostly unmethylated, with the exception of four samples where hypermethylation of L1 was detected. In contrast, L1 sequences were strongly methylated in all cervical carcinomas, while the LCR and E6 remained unmethylated. HeLa cells, derived from a cervical adenocarcinoma, contain chromosomally integrated HPV-18 genomes. We found that L1 is hypermethylated in these cells, while the LCR and E6 are unmethylated. Treatment of HeLa cells with the methylation inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) led to the expected reduction of L1 methylation. After removal of 5-Aza-CdR, L1 methylation resumed and exceeded pretreatment levels. Unexpectedly, the LCR and E6 also became methylated under these conditions, albeit at lower levels than L1. We hypothesize that L1 is preferentially methylated after integration of the HPV genome into the cellular DNA, possibly since linearization prohibits its normal transcription, while the enhancer and promoter may be protected from methylation by transcription factors. Since our data suggest that HPV-18 L1 methylation can only be detected in carcinomas, except in some few precancerous lesions and asymptomatic infections, L1 methylation may constitute a powerful molecular marker for detecting this important step of neoplastic progression.

Human papillomavirus‐16 DNA methylation patterns support a causal association of the virus with oral squamous cell carcinomas

“…[21][22][23] The resulting chromatin changes constitute a major molecular pathway that regulates cellular gene expression during embryogenesis and differentiation. In the biology of most viruses DNA methylation does not appear to play a role, but previous studies from our group [24][25][26][27][28] and others [34][35][36] have shown that HPV-16 and 18 are among those few viruses that are strongly affected by this mechanism.…”

“…in the same position of different HPV genomes either CpGs or meCpGs. [26][27][28] To approach a satisfactory analysis of such mixtures of molecules, we have developed a standardized protocol that we applied here to DNA preparations from oral carcinomas. We treat DNA samples with bisulfite, which converts cytosines into uracils but does not affect methyl cytosine.…”

“…When we targeted this approach in past studies to HPV-16 and 18 genomes in cervical and anal lesions, we observed a complex diversity of methylation patterns of HPV genomes both in the comparison of different clinical samples and even within individual clinical samples. [24][25][26][27][28] Among these different patterns, hypermethylation of the HPV-16 and 18 L1 gene and of the HPV-16 long control region (LCR) was common in carcinomas and high-grade lesions, but rare or absent in asymptomatic infections or low-grade lesions, likely a consequence of genomic recombination and aberrant transcription. Here we report that we observed the same meCpG patterns in HPV-16 DNA in oral carcinomas.…”

Viral epigenomes in human tumorigenesis

“…In this context, for instance, transverse studies throughout the carcinogenic stages and the search for viral and cellular biomarkers are very important (Turan et al, 2006;Fernandez et al, 2009). Results from such studies, aided by highthroughput technologies, will enable us to adapt customized therapies against virus-associated cancer types.…”

“…In HPV16 and HPV18 types, the LCR and E6 sequences were commonly found to be unmethylated independent of the stage of neoplastic progression, although the L1 region was densely methylated (Badal et al, 2003(Badal et al, , 2004Turan et al, 2006Turan et al, , 2007Hublarova et al, 2009).…”