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Integration of human papillomavirus 18 DNA in esophageal carcinoma 109 cells.

ABSTRACT: To detect human papillomavirus (HPV) DNA in esophageal carcinoma (EC) 109 cells and investigate the relationship between HPV and EC.Genomic DNA and total RNA from EC109 cells were isolated. HPV DNA was detected by polymerase chain reaction (PCR) with the general primer sets of My09/11 and GP5 +/6 + for the HPV L1 gene and type-specific primer sets for HPV18 E6 and HPV18 E6-E7. Reverse transcription (RT) of mRNA isolated from EC109 cells was performed to produce a cDNA. And then a PCR-based protocol for the amplification of papillomavirus oncogene transcripts was used to analyze HPV18 DNA and integrated transcripts of HPV18 in the chromosomes of EC109 cells. The final nested PCR products were cloned into a pMD-18T vector and sequenced to analyze the chromosomal location of HPV integration.HPV18 DNA was detected in EC109 cells by PCR using the general primer sets of My09/11 and GP5 +/6 + for HPV L1 and the type-specific primer sets for HPV18 E6 and E6-E7 to generate products of 450 bp, 150 bp, 335 bp and 944 bp, respectively. Approximately 600 bp of integrated HPV18-specific transcript was identified. The final nested PCR product of integrated HPV18 DNA was cloned into a pMD-18T vector and sequenced to analyze the chromosomal location of HPV integration. Sequence alignment showed that the HPV18 sequence from EC109 cells was identical to that of the encoded early protein E7-E1 of the standard HPV18 strain X05015, and another partial gene sequence was identical to a partial sequence of human chromosome 8.Integration of the HPV genome into the host cell chromosome suggests that persistent HPV infection is vital for malignant cell transformation and carcinogenesis.

SUBMITTER: Zhang K

PROVIDER: S-EPMC3208371 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Integration of human papillomavirus 18 DNA in esophageal carcinoma 109 cells.

Zhang Ke K Li Jin-Tao JT Li Shu-Ying SY Zhu Li-Hua LH Zhou Ling L Zeng Yi Y

World journal of gastroenterology 20111001 37

<h4>Aim</h4>To detect human papillomavirus (HPV) DNA in esophageal carcinoma (EC) 109 cells and investigate the relationship between HPV and EC.<h4>Methods</h4>Genomic DNA and total RNA from EC109 cells were isolated. HPV DNA was detected by polymerase chain reaction (PCR) with the general primer sets of My09/11 and GP5 +/6 + for the HPV L1 gene and type-specific primer sets for HPV18 E6 and HPV18 E6-E7. Reverse transcription (RT) of mRNA isolated from EC109 cells was performed to produce a cDNA ...[more]

PMID: 22072858

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Project description:BackgroundThe role of human papillomavirus (HPV) in the causation of esophageal squamous cell carcinoma is unclear. We examined the associations between esophageal squamous cell carcinoma and 28 centrally measured HPV serological markers in serum from six existing case-control studies conducted in regions with differing background risks of esophageal cancer.MethodsWe used centralized multiplex serology to test serum samples from 1561 case subjects and 2502 control subjects from six case-control studies for antibodies to the major HPV capsid protein (L1) and/or the early proteins E6 and/or E7 of eight high-risk, two low-risk, and four cutaneous HPV types. Study-specific odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were estimated using conditional logistic regression with adjustment for smoking, alcohol consumption, and other potential confounders. Pooled odds ratios and 95% confidence intervals were calculated using either a linear mixed-effects approach or a joint fixed-effects approach. All statistical tests were two-sided.ResultsWe found statistically significant associations between esophageal squamous cell carcinoma and antibodies to E6 for HPV16 (OR = 1.89, 95% CI = 1.09 to 3.29, P = .023) and HPV6 (OR = 2.53, 95% CI = 1.51 to 4.25, P < .001) but not for other tested HPV types. There were no statistically significant associations between esophageal squamous cell carcinoma and antibodies to E7 for any of the tested HPV types. Simultaneous seropositivity for HPV16 E6 and E7 was rare (four case subjects, two control subjects; OR = 5.57, 95% CI = 0.90 to 34.35; P = .064). We also found statistically significant associations between esophageal squamous cell carcinoma and capsid antibodies for the high-risk mucosal type HPV33 L1 (OR = 1.30, 95% CI = 1.00 to 1.69; P = .047) and the low-risk mucosal types HPV6 (OR = 1.22, 95% CI = 1.05 to 1.42; P = .010) and HPV11 (OR = 1.30, 95% CI = 1.09 to 1.56, P = .0036).ConclusionsWe found limited serological evidence of an association between esophageal squamous cell carcinoma and HPV in the populations studied. Although HPV does not appear to be an important risk factor for esophageal squamous cell carcinoma, we cannot exclude the possibility that certain HPV types may be involved in a small subset of cancers.

Project description:Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3'-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3'-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.

Project description:BackgroundPersistent infection with oncogenic Human Papillomavirus (HPV) is associated with the development of cervical cancer with each genotype differing in their relative contribution to the prevalence of cervical disease. HPV DNA testing offers improved sensitivity over cytology testing alone but is accompanied by a generally low specificity. Potential molecular markers of cervical disease include type-specific viral load (VL), integration of HPV DNA into the host genome and methylation of the HPV genome. The aim of this study was to evaluate the relationship between HPV type-specific viral load, integration and methylation status and cervical disease stage in samples harboring HPV16, HPV18, HPV31 or HPV45.MethodsSamples singly infected with HPV16 (n=226), HPV18 (n=32), HPV31 (n=75) or HPV45 (n=29) were selected from a cohort of 4,719 women attending cervical screening in England. Viral load and integration status were determined by real-time PCR while 3'L1-URR methylation status was determined by pyrosequencing or sequencing of multiple clones derived from each sample.ResultsViral load could differentiate between normal and abnormal cytology with a sensitivity of 75% and a specificity of 80% (odds ratio [OR] 12.4, 95% CI 6.2-26.1; p<0.001) with some variation between genotypes. Viral integration was poorly associated with cervical disease. Few samples had fully integrated genomes and these could be found throughout the course of disease. Overall, integration status could distinguish between normal and abnormal cytology with a sensitivity of 72% and a specificity of 50% (OR 2.6, 95% CI 1.0-6.8; p=0.054). Methylation levels were able to differentiate normal and low grade cytology from high grade cytology with a sensitivity of 64% and a specificity of 82% (OR 8.2, 95% CI 3.8-18.0; p<0.001). However, methylation varied widely between genotypes with HPV18 and HPV45 exhibiting a broader degree and higher magnitude of methylated CpG sites than HPV16 and HPV31.ConclusionsThis study lends support for HPV viral load and CpG methylation status, but not integration status, to be considered as potential biomarkers of cervical disease.

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Integration of human papillomavirus 18 DNA in esophageal carcinoma 109 cells.

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Integration of human papillomavirus 18 DNA in esophageal carcinoma 109 cells.

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