HPV16 E6 gene variations in invasive cervical squamous cell carcinoma and cancer in situ from Russian patients

HPV16 is frequently seen in invasive cervical cancer (ICC) and cervical intraepithelial neoplasia (CIN). Its E6 gene has frequent sequence variations. Although some E6 variants have been reported to have different biochemical or biological properties, they do not show geographical identity. Moreover, the definition of ‘variant’ has been a source of confusion because it has been based on all departures from the ‘prototype’ once isolated randomly from an ICC case. We amplified the HPV16 E6 gene by PCR from fresh-frozen tissue of 104 cases of ICC and CIN from Russian patients and sequenced it in positive cases. We found that 32 of 55 (58.2%) ICC cases and 18 of 49 (36.7%) CIN cases were HPV 16-positive and we could identify 3 groups of E6 variants: group A was characterized by G at nt 350 where group B had T, and group M was a heterogeneous mixture of unique E6 variants; no significant difference existed in the distribution of the different groups between ICC and CIN; the clinically malignant (as defined by FIGO stage) order between the groups was M > A > B in ICC; in the cases with a single HPV16 E6 sequence, coexisting ICC, CIN and normal epithelium in the same patient shared the E6 variant; and 4 cases of ICC had double/multiple E6 variants. The results did not show any importance of E6 variants for ICC progression in Russian women. The results also indicated that the original HPV16 variant persisted during ICC progression, and that at a low frequency, double infections and/or mutation of variants might occur. © 2001 Cancer Research Campaign http://www.bjcancer.co

T35 Epigenetic changes in human cervical carcinomas associated with viral induced pathogenesis

Epigenetics investigates mechanisms that control inheritance of gene expression program during somatic cell divisions. These mechanisms include regulation by DNA methylation, histone post-translational modifications and nucleosome positioning, functioning of regulatory non-coding RNAs, control of alternative splicing of mRNA precursors and high-order chromatin organization. Genome-wide loss of epigenetic stability and increased epigenetic plasticity are common features of all tumor types. In normal tissues epigenetic plasticity allow cells to response on environment signals. Thus, in tumor cells its constitutive activation leads to epigenetic heterogeneity that are the additional hallmark of the most of the classical cancers. Cervical cancers are one of the most interesting models for the analysis of the role of epigenetic changes in tumor progression. These types of tumors are associated with infection of human papilloma viruses of so-called high-risk group (HR-HPV) and characterized by well-defined stages of malignant conversion from intraepithelial neoplasias to carcinomas. The viral DNA can persist in episomal form or integrates into the host-cell genome.Cellular genomes encode genetic information in their linear sequence, but appropriate gene expression requires chromosomes to fold into dynamic complex three-dimensional structures. Scaffold/matrix attachment regions (S/MARs) are specialized genomic DNA sequences that take part in organization of these structures. We demonstrated that methylation of S/MARs was required for their attachment to nuclear matrix and that methylation status of S/MARs was changed in cervical cancer cell compared to normal cells.DNA methylation plays an important role in the regulation of gene expression. We found that methylation of the regulatory sequences in the HPV16 genome specifically changes in transformed compared to the normal cervical epithelial cells. Next, we showed that methylation of the transcription factor binding sites modulates the viral oncogene expression. These data suggest that the HPV16 genome methylation may represent an important mechanism that initiates the development of HPV-associated tumors.Using next generation sequencing, we identified pattern of differentially expressed microRNAs in clinical samples of the cervical lesions. We confirmed expression of microRNAs that have been described previously as well as identified new microRNAs that can be potentially involved in the development and progression of cervical cancer. Spectrum of differentially expressed microRNAs includes microRNAs targeting tumor-suppressor genes as well as oncogenes.Telomerase is a key regulator of cell proliferation. This enzyme is silent in normal cells and activated in most of the tumors. Few forms of RNA (hTERT), encoded by telomerase gene were detected in different tumor cells and among them three forms (alfa, beta and gamma) are most well pronounced. We found that in cervical tumors expression of all three forms are significantly increased. In some cases, we also observe higher level of hTERT expression in neighboring “normal tissue”. The correlation between expression levels of these three forms varied on different stages of the disease (three stages on intraepithelial neoplasias and carcinomas). The function of these three hTERT forms is still not well understood

Эпигенетическая регуляция экспрессии генов в вирус-ассоциированных опухолях человека

Viruses are associated with nearly 20 % of human cancers worldwide. Until recently genetic abnormalities generated by oncogenic virusesin cells were the main object of studies. Understanding of the importance of epigenetics in the regulation of gene expression prompted the investigation of virus and host interactions at the epigenetic level. We review aspects such as common futures of oncogenic virus interactions with cell epigenetic system and virus-specific peculiarities of these interactions. Knowledge of the regulation of virus genomes by cell epigenetic system and disturbances of this system by viruses should provide us with markers for following cancer progression, as well as new tools for cancer therapyОпухоли, ассоциированные с вирусами, составляют около 20 % всех опухолей человека. До недавнего времени при исследовании молекулярных механизмов вирусного канцерогенеза основные усилия были направлены на генетические нарушения, вызываемые онкогенными вирусами в клетке. Успехи, достигнутые в понимании механизмов эпигенетической регуляции экспрессии генов, стимулировали исследования взаимодействия вирусов и клетки-хозяина на эпигенетическом уровне. В обзоре рассмотрены общие закономерности взаимодействия онкогенных вирусов с эпигенетической системой регуляции функционирования генома и специфические для каждого вируса особенности этого взаимодействия в процессе установления латентной инфекции и опухолевой трансформации. Исследования регуляции экспрессии вирусного генома эпигенетической системой клетки и, с другой стороны, нарушений этой системы вирусами вносят вклад в понимание механизмов вирусного канцерогенеза, выявление новых маркеров прогрессии опухолей и мишеней для таргетной терапии

Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences

BACKGROUND: In cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences. METHODS: Amplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used. RESULTS: Twelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression. CONCLUSIONS: Integration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA

Tissue restricted expression and chromosomal localization of the YB-1 gene encoding a 42 kD nuclear CCAAT binding protein.

YB-1 cDNA clones were isolated by binding site screening of a Hela expression library using a human papillomavirus type 18 enhancer oligonucleotide. YB-1 belongs to a family of transcription factors which bind to recognition sequences containing a core CCAAT element. YB-1 bound to its single stranded recognition sequence on the sense strand but not to the anti-sense strand. A synthetic peptide antiserum derived from the predicted YB-1 amino acid sequence identified a 42 kD nuclear protein in immunoblots. A protein with the same size was detected by binding site blotting experiments using the HPV18 enhancer oligonucleotide which bound YB-1. YB-1 gene expression was restricted in tissues from a human 24 week old fetus. High levels of YB-1 mRNA were present in heart, muscle, liver, lung, adrenal gland and the brain, in contrast, low amounts of YB-1 mRNA were found in thymus, kidney, bone marrow and spleen. In pancreas, bladder, stomach and testis YB-1 mRNA could not be detected by Northern hybridization. Finally, we have identified four YB-1 related loci in the mouse genome and have mapped these loci to four different mouse chromosomes by interspecific backcross analysis