Cervical carcinogenesis is driven by deregulated E6/E7 expression in dividing cells. A potential deregulating mechanism is methylation of E2 binding sites in the viral long control region, thereby prohibiting HPVE2-mediated transcription regulation. Here the frequency of HPV16E2BS methylation in cervical lesions (SCC, n=29; CIN3, n=17) and scrapes (controls, n=17; CIN3, n=21) was investigated. Three E2BSs were amplified using methylation independent PCR followed by specific detection of methylated CpGs using the Luminex® xMAP™ system. The frequency of E2BS1, E2BS3 and E2BS4 methylation was significantly higher in SCC compared to CIN3, i.e. 93% vs. 21% (p<0.01), 90% vs. 47% (p<0.01) and 69% vs. 5% (p<0.01), respectively and ranged from 6 to 15% in controls. In scrapings of women with CIN3 methylation ranged from 24 to 33%. In conclusion, we showed that the MIP-Luminex system is a highly sensitive method for methylation analysis. HPV16 E2BSs methylation appeared highly frequent in SCC, with particularly E2BS3 methylation occurring proportional to severity of cervical disease.

Abbreviations: CIN, cervical intraepithelial neoplasia; HFK, human foreskin keratinocytes; hrHPV, high-risk human papillomavirus; LZRS, empty vector; MIP, methylation independent PCR; (q)MSP, (quantitative) methylation specific PCR; SCC, squamous cell carcinoma; SFM, serum free mediumHigh-risk human papillomavirus (hrHPV)-induced immortalization and malignant transformation are accompanied by DNA methylation of host genes. To determine when methylation is established during cell immortalization and whether it is hrHPV-type dependent, DNA methylation was studied in a large panel of HPVE6E7-immortalized keratinocyte cell lines. These cell lines displayed different growth behaviors, i.e., continuous growth versus crisis period prior to immortalization, reflecting differential immortalization capacities of the 7 HPV-types (16/18/31/33/45/66/70) studied. In this study, cells were monitored for hypermethylation of 14 host genes (APC, CADM1, CYGB, FAM19A4, hTERT, mir124-1, mir124-2, mir124-3, MAL, PHACTR3, PRDM14, RASSF1A, ROBO3, and SFRP2) at 4 different stages during immortalization. A significant increase in overall methylation levels was seen with progression through each stage of immortalization. At stage 1 (pre-immortalization), a significant increase in methylation of hTERT, mir124-2, and PRDM14 was already apparent, which continued over time. Methylation of ROBO3 was significantly increased at stage 2 (early immortal), followed by CYGB (stage 3) and FAM19A4, MAL, PHACTR3, and SFRP2 (stage 4). Methylation patterns were mostly growth behavior independent. Yet, hTERT methylation levels were significantly increased in cells that just escaped from crisis. Bisulfite sequencing of hTERT confirmed increased methylation in immortal cells compared to controls, with the transcription core and known repressor sites remaining largely unmethylated. In conclusion, HPVinduced immortalization is associated with a sequential and progressive increase in promoter methylation of a subset of genes, which is mostly independent of the viral immortalization capacity.

bEpidemiological studies identified 12 high-risk HPV (hrHPV) types and 8 probable/possible hrHPV types that display different cancer risks. Functional studies on transforming properties of hrHPV are mainly limited to HPV16 and -18, which induce immortalization of human foreskin keratinocytes (HFKs) by successive bypass of two proliferative life span barriers, senescence and crisis. Here, we systematically compared the in vitro immortalization capacities, as well as influences on p53, pRb, hTERT, growth behavior, and differentiation capacity, of nine hrHPV types (HPV16, -18, -31, -33, -35, -45, -51, -52, and -59), and two probable hrHPV types (HPV66 and -70). By retroviral transduction, the respective E6/E7 coding sequences were expressed in HFKs from two or three independent donors. Reduced p53 levels and low-level hTERT expression in early-passage cells, as seen in HPV16-, -31-, -33-, and -35-, and to a lesser extent HPV18-transduced HFKs, was associated with continuous growth and an increased immortalization capacity. Less frequent immortalization by HPV45 and -51 and immortalization by HPV66 and -70 was preceded by an intervening period of strongly reduced growth (crisis) without prior increase in hTERT expression. Immortalization by HPV59 was also preceded by a period crisis, despite the onset of low hTERT expression at early passage. HPV52 triggered an extended life span but failed to induce immortality. Variations in p53 and pRb levels were not correlated with differences in alternative E6/E7 mRNA splicing in all hrHPV-transduced HFKs. On collagen rafts, transductants showed disturbed differentiation reminiscent of precancerous lesions. In conclusion, in vitro oncogenic capacities differ between the established hrHPV types, and both some established and probable hrHPV types display weak or moderate immortalization potential. C ervical cancer, the third most common cancer among women worldwide, is caused by a persistent infection with certain types of human papillomavirus (HPV) (1, 2). Most HPV infections are transient and are cleared within 1 to 2 years. However, a fraction of infections eventually give rise to either squamous cell carcinoma (SCC) or adenocarcinoma (AdCA) of the cervix. SCCs develop from so-called cervical intraepithelial neoplasia (CIN) precursor lesions. Low-grade CIN lesions (CIN1) mostly reflect a productive infection in which viral replication and virion production are linked to the differentiation program of the epithelium. High-grade CIN lesions (CIN2/3) mainly represent transforming infections and are characterized by deregulated expression of the early viral E6 and E7 genes in the proliferating basal cells of the epithelium (3, 4).HPV types belonging to the alpha (␣) genus can infect the cervical mucosa (5) and are classified into low-risk (lrHPV) and high-risk (hrHPV) HPV based on their association with malignancy (6). Infections with lrHPV types, e.g., HPV type 6 (HPV6) and HPV11, are associated with benign warts or low-grade lesions, whereas infections with hrHPV can give rise ...