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PrefaceInfection of cervical epithelium with high-risk human papillomavirus (hrHPV) might result in productive or transforming cervical intraepithelial neoplasia (CIN) lesions, the morphology of which can overlap. In transforming CIN lesions aberrations in host cell genes accumulate over time, which is necessary for ultimate progression to cancer. On the basis of (epi)genetic changes, early and advanced transforming CIN lesions can be distinguished. This paves the way for new molecular tools for cervical screening, diagnosis and management of cervical cancer precursor lesions.

The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses.

Persistent infection with a high-risk human papillomavirus (hrHPV) is generally accepted as a necessary cause of cervical cancer. However, cervical cancer is a rare complication of an hrHPV infection since most such infections are transient, not even giving rise to cervical lesions. On average, it takes 12-15 years before a persistent hrHPV infection may ultimately, via consecutive premalignant stages (ie CIN lesions), lead to an overt cervical carcinoma. This argues that HPV-induced cervical carcinogenesis is multi-step in nature. In this review, the data from hrHPV-mediated in vitro transformation studies and those obtained from analysis of clinical specimens have been merged into a cervical cancer progression model. According to this model, a crucial decision maker in the early stages following infection involves individual susceptibility for certain HPV types depending on the genetic make-up of immune surveillance determinants. Once a CIN lesion has developed, altered transcriptional regulation of the viral E6/E7 oncogenes, resulting in genomic instability and distinguishing the process of cell transformation from a productive viral infection, probably provides the subsequent important step towards malignancy. The additional ( Infections with high-risk HPV are a necessary cause of cervical cancerCervical cancer is the second most common cancer amongst women world-wide, with a mean age standardized incidence rate of up to 18.7 per 100 000 women, as found in less developed countries [1]. Based on strong epidemiological evidence, supported by basic experimental findings, there is no doubt that persistent infections with high-risk types of human papillomavirus (hrHPV) represent a necessary cause of cervical cancer [1][2][3][4].Together, the viral genes E6 and E7 are responsible for the induction as well as maintenance of the transformed phenotype of cervical cancer cells, particularly by abrogating cell-cycle control and apoptosis mechanisms. Both E6 and E7 proteins can bind to multiple cellular targets (reviewed in ref 5). The interactions that are thought to be most relevant for their transforming functions are E6 binding, via the cellular protein E6-AP, to the tumour suppressor gene product p53, and E7 binding to the retinoblastoma tumour suppressor gene product pRb and its related pocket proteins, p107 and p130 [6,7]. The first interaction results in rapid ubiquitin-dependent proteolytic degradation of p53, which prevents cells from undergoing p53-mediated apoptosis [8]. A consequence of E7-pRB interaction is interference with cell-cycle control. In combination, the E6-p53 and E7-pRB interactions seem to compromise the accuracy of mitosis. In addition, hrHPV E6 can activate the telomerelengthening enzyme telomerase independent of p53 binding, and E7 can induce abnormal centrosome duplication through a mechanism independent of inactivation of pRb and its family members (reviewed in ref 9). It is likely that these latter properties also contribute to the transforming characteristics of these viral onc...

Most of the phosphatidylethanolamine (PE) in mammalian cellsis synthesized by two pathways, the CDP-ethanolamine pathway and the phosphatidylserine (PS) decarboxylation pathway, the final steps of which operate at spatially distinct sites, the endoplasmic reticulum and mitochondria, respectively. We investigated the importance of the mitochondrial pathway for PE synthesis in mice by generating mice lacking PS decarboxylase activity. Disruption of Pisd in mice resulted in lethality between days 8 and 10 of embryonic development. H]ethanolamine was correspondingly increased in hepatocytes. We conclude that the CDPethanolamine pathway in mice cannot substitute for a lack of PS decarboxylase during development. Moreover, elimination of PE production in mitochondria causes fragmented, misshapen mitochondria, an abnormality that likely contributes to the embryonic lethality. Phosphatidylethanolamine (PE)4 is an abundant phospholipid in membranes of organisms ranging from bacteria to mammals. Mammalian cells utilize two major pathways for PE biosynthesis, the CDP-ethanolamine pathway (1) and the phosphatidylserine decarboxylation pathway (2). Most of the ethanolamine that is incorporated into PE is derived from the diet but some is generated by sphingosine-1-phosphate lyase (3). In rat liver/hepatocytes (4, 5) and hamster heart (6) the majority of PE has been reported to originate from the CDP-ethanolamine pathway. In contrast, in cultured Chinese hamster ovary cells (7-9) and baby hamster kidney cells (10), the decarboxylation of phosphatidylserine (PS) produces more than 80% of PE, even when the culture medium is supplemented with ethanolamine, an obligatory substrate of the CDP-ethanolamine pathway. Many types of mammalian cells grow and divide normally when cultured in the absence of ethanolamine, suggesting that PE production from the CDP-ethanolamine pathway might not be not essential for cell growth (10), although it is likely that the relative importance of the two pathways of PE synthesis depends on the type of cell and tissue. In Escherichia coli, in which PE comprises ϳ75% of total phospholipids, all PE is derived from PS decarboxylase. Interestingly, E. coli that have been genetically manipulated to reduce the PE content to 0.007% of total phospholipids are viable when supplemented with divalent cations such as Mg 2ϩ and Ca 2ϩ (11, 12). The two major pathways for PE synthesis in mammalian cells operate in different subcellular compartments. The final reaction of the CDPethanolamine pathway, catalyzed by CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, occurs primarily on endoplasmic reticulum (ER) and nuclear membranes (13, 14), whereas PS decarboxylase activity is restricted to the outer surface of mitochondrial inner membranes (15, 16). Thus, the potential exists for compartmentalization of PE pools originating from these two spatially segregated pathways. Indeed, in Chinese hamster ovary cells and in yeast, the majority of mitochondrial PE is synthesized within mitochondria by PS decar...

BackgroundA substantial number of microRNAs (miRNAs) is subject to epigenetic silencing in cancer. Although epigenetic silencing of tumour suppressor genes is an important feature of cervical cancer, little is known about epigenetic silencing of miRNAs. Since DNA methylation-based silencing of hsa-miR-124 occurs in various human cancers, we studied the frequency and functional effects of hsa-miR-124 methylation in cervical carcinogenesis.ResultsQuantitative MSP analysis of all 3 loci encoding the mature hsa-miR-124 (hsa-miR-124-1/-2/-3) showed methylation in cervical cancer cell lines SiHa, CaSki and HeLa as well as in late passages of human papillomavirus (HPV) type 16 or 18 immortalised keratinocytes. Treatment of SiHa cells with a demethylating agent reduced hsa-miR-124 methylation levels and induced hsa-miR-124 expression. In HPV-immortalised keratinocytes increased methylation levels were related to reduced hsa-miR-124 expression and higher mRNA expression of IGFBP7, a potential hsa-miR-124 target gene. Ectopic hsa-miR-124 expression in SiHa and CaSki cells decreased proliferation rates and migratory capacity. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 139 cervical tissue specimens showed an increasing methylation frequency from 0% in normal tissues up to 93% in cervical carcinomas. Increased methylation levels of hsa-miR-124-1 and hsa-miR-124-2 were significantly correlated with reduced hsa-miR-124 expression in cervical tissue specimens. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 43 cervical scrapes of high-risk HPV positive women was predictive of underlying high-grade lesions.ConclusionsDNA methylation-based silencing of hsa-miR-124 is functionally involved in cervical carcinogenesis and may provide a valuable marker for improved detection of cervical cancer and its high-grade precursor lesions.

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the β4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in β4 expression. We found that the expression of wild-type β4 restored the ability of the β4-deficient cells to form HDs and that distinct domains in the NH2- and COOH-terminal regions of the β4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the β4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between β4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for α6 and β4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of β4 with BP180. Nevertheless, β4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of β4 can interact with the first NH2-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of β4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of β4 with other hemidesmosomal components, e.g., BP180, is regulated.

Little is known about the alterations in microRNA (miRNA) expression patterns during the consecutive stages of cervical cancer development and their association with chromosomal instability. In this study, miRNA expression in normal cervical squamous epithelium, high-grade precancerous lesions (cervical intraepithelial neoplasia (CIN2-3)), squamous cell carcinomas (SCCs) and adenocarcinomas (AdCAs) was integrated with previously generated chromosomal profiles of the same samples. Significantly differential expression during the consecutive stages of cervical SCC development was observed for 106 miRNAs. Of these differentially expressed miRNAs, 27 showed early transiently altered expression in CIN2-3 lesions only, 46 miRNAs showed late altered expression in SCCs only and 33 showed continuously altered expression in both CIN2-3 and SCCs. Altered expression of five significantly differentially expressed miRNAs, hsa-miR-9 (1q23.2), hsa-miR-15b (3q25.32), hsa-miR-28-5p (3q27.3), hsa-miR-100 and hsa-miR-125b (both 11q24.1), was directly linked to frequent chromosomal alterations. Functional analyses were performed for hsa-miR-9, representing a potential oncogene with increased expression linked to a chromosomal gain of 1q. Hsa-miR-9 overexpression was found to increase cell viability, anchorage-independent growth and migration in vitro. Upon organic raft culturing, hsa-miR-9 hampered differentiation and induced proliferation in all strata of the epithelial layer. These findings support a potential oncogenic function of hsa-miR-9 in cervical cancer. In summary, differential expression of 106 miRNAs, partly associated with chromosomal alterations, was observed during cervical SCC development. Altered expression of hsa-miR-9 associated with a chromosomal gain of chromosome 1q was shown to be functionally relevant, underlining the importance of deregulated miRNA expression in cervical carcinogenesis.