Quantification of HPV‐16 E6‐E7 transcription in cervical intraepithelial neoplasia by reverse transcriptase polymerase chain reaction

Hsu, E. M.; McNicol, Patricia J.; Guijon, Fernando B.; Paraskevas, M

doi:10.1002/ijc.2910550311

Cited by 47 publications

(23 citation statements)

References 25 publications

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“…Total RNA (0.1-1 Ag) was reverse transcribed using an oligo(dT) 17 -primer coupled to a linker sequence p3 (32) and 50 units of Moloney murine leukemia virus reverse transcriptase (SuperScript; Life Technologies) for 1 h at 42jC in a final volume of 20 AL. To control RNA integrity and first-strand cDNA quality, PCR reactions using glyceraldehyde-3-phosphate dehydrogenase-specific primers were performed as described previously (33). First-strand cDNAs encompassing viral oncogene sequences were subsequently amplified by PCR using HPV E7-specific primer [p1] as forward primers (Table 1) and linker p3 as the reverse primer; and 1.5 units of Taq DNA Polymerase (Life Technologies) in a total volume of 50 AL.…”

Section: Methodsmentioning

confidence: 99%

“…It is now widely accepted that almost all cervical carcinomas and the majority of vaginal, vulval, penile, and anal cancers arise as consequence of persistent infections with about 13 different HR-HPVs (1). Most of these human papillomavirus (HPV) types show phylogenetic similarities to either HPV16 (31,33,35,52, and 58) or HPV 18 (39,45,59, and 68; ref. 2).…”

Section: Introductionmentioning

confidence: 99%

“…Even in the presence of a large excess of episomal nonintegrated viral genomes, single or few integrated viral genome copies can be detected. We compared the integration frequencies of the five most frequent HPV types 16,18,31,33, and 45 and analyzed the median age of the patients at diagnosis of precancer and cancer with respect to HPV type and HPV integration status. The reported data suggest that integration of HR-HPV genomes rather reflects the level of chromosomal instability conferred by distinct HR-HPV types.…”

Section: Introductionmentioning

confidence: 99%

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Type-Dependent Integration Frequency of Human Papillomavirus Genomes in Cervical Lesions

Vinokurova¹,

Wentzensen²,

et al. 2008

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Chromosomal integration of high-risk human papillomavirus (HR-HPV) genomes is believed to represent a significant event in the pathogenesis of cervical cancer associated with progression from preneoplastic lesions to invasive carcinomas. This hypothesis is based on experimental data suggesting that integration-dependent disruption of HR-HPV E2 gene functions is important to achieve neoplastic transformation and on clinical data gathered by analyzing lesions induced by human papillomavirus (HPV) 16 and 18 that revealed integrated viral genome copies in the vast majority of cervical cancer cells. However, a substantial fraction of cervical cancers is associated with other HR-HPV types for which virtually no data concerning their integration status have been reported so far. Here, we compared integration frequencies of the five most common oncogenic HPV types (HPV16, 18, 31, 33, and 45) in a series of 835 cervical samples using a specific mRNA-based PCR assay (Amplification of Papillomavirus Oncogene Transcripts). Most precancerous lesions displayed exclusively episomal viral genomes, whereas 62% of the carcinomas had integrated viral genomes. However, the frequency of integrated HR-HPV genomes showed marked differences for individual HR-HPV types. HPV16, 18, and 45 were found substantially more often in the integrated state compared with HPV types 31 and 33. The analysis of the median age of patients with high-grade precancerous lesions and invasive cancers suggests that precancers induced by HPV types 18, 16, and 45 progress to invasive cervical cancer in substantially less time compared with precancers induced by HPV types 31 and 33. These findings suggest that integration of oncogenic HPV genomes in cervical lesions is a consequence rather than the cause of chromosomal instability induced by deregulated HR-HPV E6-E7 oncogene expression. Distinct HR-HPV types apparently provoke chromosomal instability in their host cells to a different extent than is reflected by their integration frequencies in advanced lesions and the time required for CIN 3 lesions to progress to invasive cancer.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Type-Dependent Integration Frequency of Human Papillomavirus Genomes in Cervical Lesions

Vinokurova¹,

Wentzensen²,

et al. 2008

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“…Primers used to amplify the cyclin D1 gene were the following: dogD1S, 5Ј-CTGGCCATGAACTACCTGGA-3Ј and dogD1AS, 5Ј-GGAAGTGCTCGATGAAGTCG-3Ј. As control, we used the following specific primers to the GAPDH gene (Hsu et al, 1993): primer 1, 5Ј-CATCTCT-GCCCCCTCTGCTGA-3Ј and primer 2, 5Ј-GGATGACCTTGCCCACAGCCT-3Ј.…”

Section: Rt-pcrmentioning

confidence: 99%

Cyclin D1 Is Transcriptionally Down-Regulated by ZO-2 via an E Box and the Transcription Factor c-Myc

Huerta¹,

Muñoz²,

Tapia

et al. 2007

MBoC

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Recent reports have indicated the participation of tight junction (TJ) proteins in the regulation of gene expression and cell proliferation. Here, we have studied the role of zona occludens (ZO)-2, a TJ peripheral protein, in the regulation of cyclin D1 transcription. We found that ZO-2 down-regulates cyclin D1 transcription in a dose-dependent manner. To understand how ZO-2 represses cyclin D1 promoter activity, we used deletion analyses and found that ZO-2 negatively regulates cyclin D1 transcription via an E box and that it diminishes cell proliferation. Because ZO-2 does not associate directly with DNA, electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay were used to identify the transcription factors mediating the ZO-2-repressive effect. c-Myc was found to bind the E box present in the cyclin D1 promoter, and the overexpression of c-Myc augmented the inhibition generated by ZO-2 transfection. The presence of ZO-2 and c-Myc in the same complex was further demonstrated by immunoprecipitation. ChIP and reporter gene assays using histone deacetylases (HDACs) inhibitors demonstrated that HDACs are necessary for ZO-2 repression and that HDAC1 is recruited to the E box. We conclude that ZO-2 down-regulates cyclin D1 transcription by interacting with the c-Myc/E box element and by recruiting HDAC1.

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“…Commercial nucleic acid sequence-based amplification in a real-time format allows the reliable type-specific detection of E6 and E7 mRNA from HPV types 16, 18, 31, 33, and 45. Several authors have thus suggested that RNA-based assays could be more effective than DNA testing in risk assessment (16,21,22,35,36,38,48,55). In the current study, we test this hypothesis using cytological and histological findings to compare the sensitivities, specificities, positive predictive values (PPVs), and negative predictive values (NPVs) of RNA and DNA testing.…”

mentioning

confidence: 99%

Clinical Performance of Human Papillomavirus E6 and E7 mRNA Testing for High-Grade Lesions of the Cervix

et al. 2009

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Infection with high-risk (HR) human papillomavirus (HPV) is the major cause of cervical cancer. However, relatively few infections progress to malignant disease. Progression to malignancy requires the overexpression of the E6 and E7 genes in the integrated HPV genome. It follows that the E6 and E7 transcripts could be useful markers of disease progression. The study presented here tests this possibility, using data from colposcopy and from cytological and histological tests to compare RNA assays for the E6 and E7 genes with DNA testing. A total of 180 women underwent colposcopy, cytology, and biopsy of suspected lesions (143 cases). Cervical brush specimens were analyzed for HPV DNA and for E6 and E7 mRNA. DNA from HR HPV was found in 57.8% of the specimens; E6 and E7 transcripts were found in 45%. The rates of detection of HPV DNA and of E6 and E7 transcripts were 33.3% and 25%, respectively, for specimens with normal findings; 51.4% and 31.9%, respectively, for specimens with cervical intraepithelial neoplasia grade 1 (CIN1); and 61.1% and 44.2% for specimens with CIN2, respectively. All specimens with CIN3 and 95.5% of specimens from patients with squamous cell carcinoma were positive by both assays. Thirty-seven patients with normal colposcopy findings did not undergo biopsy. HPV DNA and mRNA transcripts were found in 32.4% and 18.9% of these cases, respectively. Comparisons with cytological tests produced similar results. Overall, the mRNA tests showed a higher specificity than the DNA tests for high-grade lesions (72.7% and 56.2%, respectively) and a higher positive predictive value (59.3% and 49.0%, respectively). These findings suggest that mRNA assays could be more powerful than DNA testing for predicting the risk of progression and offer a strong potential as a tool for triage and patient follow-up.Carcinomas of the anogenital tract, particularly cancer of the cervix, represent the second most frequent type of neoplasm worldwide (43, 57). The major cause of these cancers is infection with high-risk (HR) human papillomavirus (HPV). DNA from HPV has been detected in more than 99% of cervical squamous cell carcinomas (SCCs) and a smaller proportion of adenocarcinomas (3,4,31,33). However, most HPV infections regress spontaneously or progress only after a long period of latency. As a result, the number of infections is far higher than the number of women who develop cancer.The most common types of HPV found in cancer patients are types 16, 18, 31, 33, and 45 (5, 10, 40, 53). Persistent infection with these types is regarded as a significant risk factor (40). The role of HPVs in the etiology of cervical cancer is tightly correlated with the overexpression of two oncogenes (E6 and E7) due to a specific opening in the E2 open reading frame in the integrated viral genome (23, 28). Studies of cervical cancer cell lines and cancer biopsy specimens have shown that the continuous expression of the genes is a necessary condition for the transformation and maintenance of neoplastic and dysplastic cells (46,56,57).Ce...

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Quantification of HPV‐16 E6‐E7 transcription in cervical intraepithelial neoplasia by reverse transcriptase polymerase chain reaction

Cited by 47 publications

References 25 publications

Type-Dependent Integration Frequency of Human Papillomavirus Genomes in Cervical Lesions

Type-Dependent Integration Frequency of Human Papillomavirus Genomes in Cervical Lesions

Cyclin D1 Is Transcriptionally Down-Regulated by ZO-2 via an E Box and the Transcription Factor c-Myc

Clinical Performance of Human Papillomavirus E6 and E7 mRNA Testing for High-Grade Lesions of the Cervix

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