HPV16 DNA Integration Research Articles

Quantitative measurement of human papillomavirus type 16 e5 oncoprotein levels in epithelial cell lines by mass spectrometry.

The high-risk human papillomavirus type 16 (HPV-16) E5 protein (16E5) induces tumors in a transgenic mouse model and may contribute to early stages of cervical carcinogenesis. Although high-risk E5 expression is generally thought to be lost during the progression to cervical carcinoma following integration of HPV DNA into the host genome, episomal viral DNA has been documented in a subset of HPV-16-positive malignant lesions. Numerous studies have shown that transcripts that could potentially encode 16E5 are present in cervical biopsy specimens and cervical cancer cell lines, but the presence of E5 protein has been demonstrated in only two reports that have not been corroborated. In the present study, we show that trypsin cleavage of 16E5 generates a unique four-amino-acid C-terminal peptide (FLIT) that serves as a marker for E5 expression in transfected cells and epithelial cell lines containing integrated and episomal HPV-16 DNA. Following trypsin cleavage, reversed-phase chromatography and mass spectrometry (MS) were used to detect FLIT. Immunoprecipitation assays using a newly generated anti-16E5 antibody confirmed that 16E5 was solely responsible for the FLIT signal, and deuterated FLIT peptide provided an internal standard that enabled us to quantify the number of 16E5 molecules per cell. We show that 16E5 is expressed in the Caski but not in the SiHa cervical cancer cell line, suggesting that 16E5 may contribute to the malignant phenotype of some cervical cancers, even in cells exclusively containing an integrated HPV genome.

Non-Random Integration of the HPV Genome in Cervical Cancer

HPV DNA integration into the host genome is a characteristic but not an exclusive step during cervical carcinogenesis. It is still a matter of debate whether viral integration contributes to the transformation process beyond ensuring the constitutive expression of the viral oncogenes. There is mounting evidence for a non-random distribution of integration loci and the direct involvement of cellular cancer-related genes. In this study we addressed this topic by extending the existing data set by an additional 47 HPV16 and HPV18 positive cervical carcinoma. We provide supportive evidence for previously defined integration hotspots and have revealed another cluster of integration sites within the cytogenetic band 3q28. Moreover, in the vicinity of these hotspots numerous microRNAs (miRNAs) are located and may be influenced by the integrated HPV DNA. By compiling our data and published reports 9 genes could be identified which were affected by HPV integration at least twice in independent tumors. In some tumors the viral-cellular fusion transcripts were even identical with respect to the viral donor and cellular acceptor sites used. However, the exact integration sites are likely to differ since none of the integration sites analysed thus far have shown more than a few nucleotides of homology between viral and host sequences. Therefore, DNA recombination involving large stretches of homology at the integration site can be ruled out. It is however intriguing that by sequence alignment several regions of the HPV16 genome were found to have highly homologous stretches of up to 50 nucleotides to the aforementioned genes and the integration hotspots. One common region of homologies with cellular sequences is between the viral gene E5 and L2 (nucleotides positions 4100 to 4240). We speculate that this and other regions of homology are involved in the integration process. Our observations suggest that targeted disruption, possibly also of critical cellular genes, by HPV integration remains an issue to be fully resolved.

Correlation between ebv co-infection and HPV16 genome integrity in Tunisian cervical cancer patients

Infection with high risk Human papillomavirus (HR-HPV) is necessary but not sufficient to cause cervical carcinoma. This study explored whether multiple HR-HPV or coinfection with Epstein-Barr virus (EBV) influence the integration status of HPV16 genome. The presence and typing of HPV in a series of 125 cervical specimens were assessed by polymerase chain reaction (PCR) using the specific primers for the HPV L1 region. As for EBV infection, the viral EBNA1 gene was used for its detection through PCR amplification. Disruption of the HPV E2 gene was assessed by amplification of the entire E2 gene with single set of primers, while E2 transcripts were evaluated by a reverse transcription PCR method (RT-PCR). The overall prevalence of HPVDNA was of 81.8% in cervical cancers versus 26.9% in benign lesions. In HPV positive cases, HPV16 and HPV18 were the most prevalent types, followed by HPV types 33, 31. EBV EBNA1 prevalence was statistically more frequent in cervical carcinomas than in benign lesions (29.5%, vs 9.6%; P=0.01). No viral infection was detected in healthy control women. The uninterrupted E2 gene was correlated with the presence of E2 transcripts originating from the HPV episomal forms. It was observed that integration was more common in HPV18 and EBV coinfection. The presence of EBV caused a five-fold [OR= 5; CI= 1.15-21.8; P = 0.04] increase in the risk of HPV16 genome integration in the host genome. This study indicates that EBV infection is acting as a cofactor for induction of cervical cancer by favoring HPVDNA integration.

HPV-DNA Integration and Carcinogenesis: Putative Roles For Inflammation And Oxidative Stress

HPV-DNA integration into cellular chromatin is usually a necessary event in the pathogenesis of HPV-related cancer; however, the mechanism of integration has not been clearly defined. Breaks must be created in both the host DNA and in the circular viral episome for integration to occur, and studies have shown that viral integration is indeed increased by the induction of DNA double strand breaks. Inflammation generates reactive oxygen species, which in turn have the potential to create such DNA strand breaks. It is plausible that these breaks enable a greater frequency of HPV-DNA integration, and in this way contribute to carcinogenesis. Consistent with this idea, co-infections with certain sexually transmitted diseases cause cervical inflammation, and have also been identified as cofactors in the progression to cervical cancer. This article examines the idea that inflammation facilitates HPV-DNA integration into cellular chromatin through the generation of reactive oxygen species, thereby contributing to carcinogenesis.

Human papillomavirus infection induces NF-κB activation in cervical cancer: A comparison with penile cancer.

This study aimed to determine the relationship between human papillomavirus (HPV) and nuclear factor-κB (NF-κB) in cervical cancer using 62 tissues of cervical cancer, and to compare the findings to penile cancer. HPV-DNA integration is a crucial factor for malignant transformation in cervical cancer and can be identified using in situ hybridization. Of the 62 cases, HPV infection was detected in 28 (45.2%). This frequency was lower than in penile cancer (68.2%) as shown by our previous study. The earliest age of onset of cervical and penile cancer was 18 and 35, respectively, whereas the mean age of the initial diagnosis of cervical and penile cancer was 50.1 and 59.6, respectively. The discrepancies of HPV prevalence, earliest ages of onset and mean ages between cervical and penile cancer patients may result from the gender-based synergistic action of HPV associated with multiple epidemiological co-factors. Of the 28 HPV-infected cases, NF-κB expression was observed in the nucleus in 18 (64.3%), in the cytoplasm in 19 (67.9%) and in the nucleus and/or cytoplasm in 27 cases (96.4%). The overexpression of NF-κB in cervical cancer cases suggests that NF-κB activation is a key modulator in driving chronic inflammation to cancer.

Distribution of Human Papillomavirus Genotypes in Sardinian Patients with Oral Squamous Cell Carcinoma~!2010-03-01~!2010-05-10~!2010-07-13~!

Human papillomaviruses (HPVs) seem to play an important role in the pathogenesis of gynecological carcinomas and in head and neck carcinomas. The aim of this study was to detect and genotype HPVs in fresh oral squamous cell carcinoma (OSCC) from a Sardinian population, and to determine whether HPV presence was significantly associated with the development of OSCC.The oral mucosa tissues were obtained from 120 samples (68 OSCC and 52 control samples) taken from a Sardinian population seen at the Dental Clinic of the Department of Surgery and Odontostomatological Sciences, University of Cagliari (Italy) and the “ Ospedale SS Trinità”, Cagliari (A.S.L. 8) between 2007 and 2008. PCR was used for the detection of HPV DNA and the genotype was determined by DNA sequencing. The frequency of HPV infection was evaluated in relation to age, sex, smoking and alcohol use. Statistical analysis was performed using the SPSS 11.5 software.The results showed the presence of HPV-DNA in 60.3% of OSCC with HPV-16 (51.2%) being the most frequent genotype. In these Sardinian OSCC patients, HPV-DNA was detected more in males (65.8%) than in females (34.1%) while controls show a 0% of HPV presence. HPV positive was highly associated with OSCC among subjects with a history of heavy tobacco and alcohol use and among those with no such history.A greater frequency of high risk HPV presence was observed in patients with OSCC compared to health control patients. In addition these results suggested that oral HPV presence could be associated in OSCC subjects. Our results need more analyses to detect the HPV-DNA integration into tumoral cells.

New molecular method for the detection of human papillomavirus type 16 integration

Human papillomavirus (HPV) infection is the cause of cervical cancer. Integration of HPV-16 DNA in cervical cells is considered to be a key event in the progression towards invasive cancer, but little is known about this event in anal carcinogenesis. The integration could be a useful biomarker for cancer progression. Optimized assays are needed to determine the value of real-time detection of HPV integration in longitudinal studies, and this approach is only possible with a high-throughput assay. The aim of this study was to develop a new multiplex real-time PCR assay based on simultaneous amplification of the E2 and E6 HPV open reading frames (ORFs) in order to assess the physical status (episomal and/or integrated) of HPV-16 in anal cells of HIV-positive men. The comparative threshold (Ct) cycle values for E2 and E6 obtained for SiHA cells and artificial mixtures of episomal and integrated DNA were as expected: similar Ct for episomal forms and absence of E2 amplification for integrated forms. The multiplex real-time PCR was tested in 77 consecutive samples from individual HIV-infected patients with HPV-16 anal infection. The integration of HPV-16 was detected in 25 (32%) patients: 23 as mixed (episomal and integrated) and two as completed integrated forms. The integration occurs in the early stage of anal lesions and was associated with the severity of the lesions (p 0.004). The multiplex real-time PCR assay developed in the course of this study was shown to be a simple, sensitive, specific and inexpensive technique which may be applied routinely to detect HPV-16 integration.

Frequent genomic structural alterations at HPV insertion sites in cervical carcinoma

To investigate whether integration of HPV DNA in cervical carcinoma is responsible for structural alterations of the host genome at the insertion site, a series of 34 primary cervical carcinomas and eight cervical cancer-derived cell lines were analysed. DNA copy number profiles were assessed using the Affymetrix GeneChip Human Mapping 250K Sty array. HPV 16, 18 or 45 integration sites were determined using the DIPS-PCR technique. The genome status at integration sites was classified as follows: no change, amplification, transition normal/gain, normal/loss or gain/LOH. A single HPV integration site was found in 34 cases; two sites were found in seven cases; and three sites in one case (51 sites). Comparison between integration sites and DNA copy number profiles showed that the genome status was altered at 17/51 (33%) integration sites, corresponding to 16/42 cases (38%). Alterations detected were amplification in nine cases, transition normal/loss in four cases, normal/gain in three cases, and gain/LOH in one case. A highly significant association was found between genomic rearrangement and integration of HPV DNA (p < 10(-10)). Activation of the replication origin located in viral integrated sequences in a cell line derived from one of the primary cervical carcinomas induced an increase of the amplification level of both viral and cellular DNA sequences flanking the integration locus. This mechanism may be implicated in the triggering of genome amplification at the HPV integration site in cervical carcinoma. Structural alterations of the host genome are frequently observed at the integration site of HPV DNA in cervical cancer and may act in oncogenesis.

Human Papillomavirus 16 Integration and Risk Factors Associated in Anal Samples of HIV-1 Infected Men

The integration of HPV-16 DNA into the host genome is considered an important event in the progression of premalignant cervical lesions to cervical cancer. The aim of our study was to assess the prevalence of HPV-16 integration in anal cytologic specimens of HIV-1 infected men and its association with risk factors. PATIENTS METHODS: This cross-sectional study included 269 HIV-infected males. Detection and typing of HPV-infection was done by multiplex PCR, and integration of HPV-16 by real-time PCR. The overall anal HPV-infection prevalence was 78% (209/269), 29% (77/269) for HPV-16 infection, and 9% (25/269) for HPV-16 integration. In HPV-16 infected group, the integration prevalence represented 32% (25/77). The only risk factor associated with HPV-16 integration was the time since HIV diagnosis (OR = 1.2, 95% CI: 1.0-1.3; P = 0.010). The risk factors associated with abnormal cytology results were: HPV infection (OR = 17.8, 95% CI: 6.8-46.6), HPV-16 infection (OR = 4.6, 95% CI: 2.5-8.4), and presence of HPV-16 integrated forms (OR = 11.7, 95% CI: 1.5-93.5). Moreover, in the multivariate analysis, the HPV-16 integration continued representing the most important risk factor (OR = 20, 95% CI: 1.6-226) for anal cytologic abnormalities. HPV-16 infection and its integration in anal cells were highly prevalent in HIV-infected men. The assessment of HPV-16 integration rather than HPV-infection could be a good biomarker for predicting anal precancerous lesions in HIV-positive men.

Human Papillomavirus DNA and Oncogene Alterations in Colorectal Tumors

The aim of the present study is to determine the presence and molecular integrity of high-risk HPV types in colorectal adenocarcinomas and to assess whether viral DNA is related to common proto-oncogene alterations, such as k-ras mutations and c-myc gene amplification, in colorectal cancer. Seventy-five colorectal adenocarcinomas were screened for HPV infection using nested-PCR (MY09/11-GP5+/6+). HPV typing was performed by type-specific PCR for HPV 16 and HPV 18 DNA. Unidentified samples were subsequently sequenced to determine the viral genotype. The physical status of HPV was determined by a nested PCR approach for type-specific E2 sequences. C-myc amplification was assessed by co-amplification with β-globin as control locus, and mutation in k-ras codons 12 and 13 by ARMS-PCR. Overall, HPV was detected in thirty-three colorectal specimens (44%). HPV 16 was the prevalent type (16/75), followed by HPV 18 (15/75), HPV 31 (1/75) and HPV 66 (1/75). E2 disruption was detected in 56.3% of HPV 16 and in 40% of HPV 18 positive tumors. C-myc amplification was detected in 29.4% of cases, while k-ras mutations in 30.7%. There was no significant trend for HPV infection in tumors harboring either k-ras or c-myc alterations. This study demonstrates HPV DNA and viral integration in colorectal tumors, suggesting a potential role of this virus in colorectal carcinogenesis. There was no concurrence, however, of k-ras and c-myc activation with viral infection.

Recombination of human papillomavirus‐16 and host DNA in exfoliated cervical cells: A pilot study of L1 gene methylation and chromosomal integration as biomarkers of carcinogenic progression

Human papillomavirus-16 DNA replicates in productive infections in circular form, but is found in most carcinomas integrated into the host cell DNA. Because this transition is essential for carcinogenesis, detailed research is desirable and may help to triage patients with abnormal Pap smears. Previous studies addressed the chromosomal integration of HPV-16 DNA in biopsies of tumors by an indirect biomarker, methylation of the viral L1 gene and by reverse ligation polymerase chain reaction (rliPCR). The pilot study reported here asked whether these techniques can be targeted successfully at DNA prepared from exfoliated cervical cells. Abnormal Pap smears of 21 patients that were positive for HPV-16 were analyzed for (i and ii) methylation of the L1 gene after bisulfite modification and PCR amplification by direct sequencing and indirectly in cloned DNA and (iii) recombination with chromosomal DNA by rliPCR. Four of these 21 patients contained highly methylated L1 DNA, which was integrated in three of the samples with sufficient DNA for rliPCR analysis. Seven patients contained sporadically methylated L1 DNA, which was integrated in two and episomal in three samples with sufficient DNA. Ten patients contained only unmethylated DNA, which was episomal in six but possibly integrated in two samples. It is concluded that HPV-16 is found integrated chromosomally in a fraction of precancerous infections, and with higher frequency in methylated than in low or unmethylated samples. Since L1 gene methylation indicates integration, it has the potential to be used as a clinical marker of cancer progression.

Human papillomavirus reduces the prognostic value of nodal involvement in tonsillar squamous cell carcinomas

Assessment of the prognostic value of nodal status in relation to human papillomavirus (HPV) status and the various treatment modalities in tonsillar squamous cell carcinomas (TSCC). Retrospective 5-year survival analysis. A 5-year follow-up of disease-free, disease-specific, and overall survival in a group of 81 patients with TSCC was conducted. The nodal status and integration of HPV-DNA in the genome (detected with fluorescence in situ hybridization) as prognostic indicators were examined while correcting for other clinical parameters (smoking habits, alcohol consumption, treatment modality, differentiation, TNM classification). Of TSCCs, 41% were positive for HPV type 16. In these TSCCs, the primary tumor was significantly smaller when compared to HVP-negative TSCCs (P = .04), whereas the percentage of cases with cervical metastases was identical. In the total population, it was not nodal involvement, but rather HPV manifestation, which was related to patient prognosis. Within the treatment modalities (surgery combined with radiotherapy and radiotherapy alone), neither nodal status nor HPV were prognostic indicators. Since a substantial percentage of TSCCs are HPV-positive and metastasizes to cervical lymph nodes in less advanced primary tumors, the N status is an unreliable prognostic indicator in TSCCs. HPV is only prognostically relevant in the total tumor population, but loses its value within patient groups receiving a single treatment modality. The value of HPV for prognosis of patients with TSCC requires further study.

HPV in situ hybridization signal patterns as a marker for cervical intraepithelial neoplasia progression

Objective HPV-DNA integration is one factor for malignant transformation and can be identified using in situ hybridization (ISH), where a diffuse signal represents episomal HPV and punctate, integrated. The aim is to verify if a punctate pattern could be a marker of CIN1 that progresses. Methods 74 CIN1 biopsies were studied. In the follow up, a second biopsy was performed and 65% showed CIN1 or no lesion (group without progression) and 35% CIN2/3 (with progression). ISH was carried out with HR-HPV GenPoint® in the first biopsy looking for the positive distribution in epithelium regions (basal, intermediate, superficial) and reaction pattern (diffuse and punctate). The Mann-Whitney and Fisher tests were used to compare the groups ( p ≤ 0.05). Results The mean age of patients without progression was 26 and, with progression 31 ( p = 0.02). ISH was positive in 22 cases, 8 with CIN2/3 in the second biopsy. The punctate signal was observed in all epithelial layers, and the mean coefficient between the number of cells with punctate and diffuse signals was 3.5 times more common in the progression group ( p = 0.08). The average percentage of punctate nuclei patterns in the basal region in cases without progression was 0.5% and 11% in those with progression ( p= 0 .05). However, in superficial layer this was not correlated with progression. Conclusion Progression was observed in 35% of CIN1 and associated with age; 30% of the cases were positive by ISH, but must be carefully interpreted. Punctate signals were related to progression only in basal cells, identifying CIN1 with potentially aggressive behavior.

Integration of human papillomavirus type-16 and type-18 is a very early event in cervical carcinogenesis

Aim:Human papillomavirus (HPV) integration is a critical event in cervical carcinogenesis. The aim of this study was to explore the physical status of HPV-16 and HPV-18 during the progression of...

Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells

Human papillomaviruses (HPVs) are involved in the pathogenesis of cancer of the cervix (CaCx). MicroRNA (miRNA) expression analysis using Ambion (Austin, TX, USA) arrays showed that three miRNAs were overexpressed and 24 underexpressed in cervical cell lines containing integrated HPV-16 DNA compared to the normal cervix. Furthermore, nine miRNAs were overexpressed and one underexpressed in integrated HPV-16 cell lines compared to the HPV-negative CaCx cell line C-33A. Based on microarray and/or quantitative real-time PCR and northern blot analyses, microRNA-218 (miR-218) was specifically underexpressed in HPV-positive cell lines, cervical lesions and cancer tissues containing HPV-16 DNA compared to both C-33A and the normal cervix. Expression of the E6 oncogene of high-risk HPV-16, but not that of low-risk HPV-6, reduced miR-218 expression, and conversely, RNA interference of E6/E7 oncogenes in an HPV-16-positive cell line increased miR-218 expression. We also demonstrate that the epithelial cell-specific marker LAMB3 is a target of miR-218. We also show that LAMB3 expression is increased in the presence of the HPV-16 E6 oncogene and this effect is mediated through miR-218. These findings may contribute to a better understanding of the molecular mechanisms involved in cervical carcinogenesis.

Retrovirus-mediated delivery of short hairpin RNA targeting human papillomavirus (HPV) 16 E6 and E7 oncogenes and induction of apoptosis in oropharyngeal squamous cell cancer (OSCC) cell line

6002 Background: Human papillomavirus type 16 is identified in almost 50% of the cases of OSCC. The E6 and E7 genes of HPVs encode oncoproteins that bind and degrade the tumor suppressor proteins p53 and Rb, respectively. We are exploring the potential use of short hairpin RNA (sh RNA) for gene therapy of HPV-positive OSCC. Methods: Small hairpin RNAs targeting E6 or E7 genes were delivered by a retrovirus vector to 93VU147T (bearing integrated HPV16 DNA) and 92VU040T (HPV negative) oropharyngeal cancer cell lines. Flow cytometry analysis was used to assess apoptosis after the retrovirus infection. The E6 and E7 mRNA downregulation was assessed by reverse transcription polymerase chain reaction (RT-PCR). At protein level p53 and Rb expression were evaluated with Western blotting analysis. Results: Apoptosis was seen in over 90% of 93VU147T cells 48 hours after infection whereas 92VU040T cells were not affected. RT-PCR demonstrated that HPV16 E6/E7 mRNA levels decreased significantly in infected 93VU147T cells. 93VU147T infected cells also showed a marked increase in p53 and Rb protein levels. Conclusions: Downregulation of E6/E7 gene expression in HPV16+ OSCC cells results in apoptosis and reactivation of p53 and Rb tumor suppression pathways. These results have significant implications in treating HPV-associated OSCC with HPV-targeted gene therapy. No significant financial relationships to disclose.

Integration of HPV-16 and HPV-18 DNA in vulvar intraepithelial neoplasia

Integration of HPV-16 and HPV-18 DNA in vulvar intraepithelial neoplasia

Integration of HPV-16 and HPV-18 DNA in vulvar intraepithelial neoplasia

Integration of HPV-16 and HPV-18 DNA in vulvar intraepithelial neoplasia

HPV prevalence, E6 sequence variation and physical state of HPV16 isolates from patients with cervical cancer in Sichuan, China

Infection with high-risk human papillomavirus (hr-HPV) is an important factor associated with cervical cancer. The genetic mutation of HPV16 E6 and integration of HPV16 DNA in the cervical carcinoma tissues are considered important genetic changes in cervical lesion progression. But the studies of hr-HPV epidemiology are relatively less in the area of Sichuan, China. Therefore, we investigated the prevalence of 9 high-risk subtypes and analyzed the genetic mutation characteristic of HPV16 E6 and physical state of HPV16 DNA. The fragments of L1 and E6 genes were amplified by PCR or nested PCR and then directly sequenced. Further, the multiplex PCR for HPV16 E2 and E6 genes was performed for detection of integration. HPV16, 58 and 18 were prominent, accounting for 78.6%, 20.0% and 9.7%, respectively in 145 isolates. E6 variants revealed that the European (EP) prototype and East Asia (EA) strain were 26 (23.0%) and 34 (30.1%), respectively. Furthermore, there were 14 base substitutions in E6 regions of the study group, of which 12 resulted in amino acid changes and the rest was silent mutation. Significantly, the 240G substitution exactly located the P53 degradation site. Overall, 8 of 114 (7.0%) isolates only contained integrated HPV16 DNA, 43 (37.7%) only contained episomal DNA and 63 (55.3%) contained both integrated and episomal DNA. The proportion of disruption of an intact E2 gene in the patients with cervical cancer is much lower than that in the previous studies. HPV16, 58 and 18 were mainly prevailing subtypes in patients with cervical cancer from Sichuan areas, China and EP/EA strains were predominant in these areas. Some mutations of E6 gene, which lead to the amino acid changes, may be more potentially carcinogenic and the proportion of disruption of an intact E2 gene is much lower.

Integration of HPV-16 and HPV-18 DNA in Vulvar Intraepithelial Neoplasia

Integration of HPV-16 and HPV-18 DNA in Vulvar Intraepithelial Neoplasia