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PubMed1991

Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm.

The integration of human papillomavirus (HPV) DNA into the human genome has been generally accepted as a characteristic of malignant lesions. To gain a better understanding of this phenomenon, genomic DNA from 181 cervical biopsy specimens was isolated and analyzed for HPV type and physical state of the HPV genome. These specimens represented the full spectrum of cervical disease, from condyloma to invasive carcinoma. Discrimination between integrated and episomal HPV DNA was accomplished by the detection of HPV-human DNA junction fragments on Southern blots. In most cases in which ambiguous Southern blot results were obtained, the specimens were reanalyzed by two-dimensional gel electrophoresis. Of the 100 biopsy specimens of cervical intraepithelial neoplasia analyzed, only 3 showed integrated HPV DNA, in contrast to 56 (81%) of 69 cervical carcinomas (P less than 0.001) showing integrated HPV DNA. Of the 40 carcinomas containing HPV 16 DNA, 29 (72%) had integrated HPV DNA, of which 8 (20%) also had episomal HPV DNA. In 11 (27%) cancers, only episomal HPV 16 DNA was detected. All 23 HPV 18-containing carcinomas had integrated HPV DNA, and 1 also had episomal HPV 18 DNA. The difference between HPV types 16 and 18 with respect to frequency of integration was statistically significant (P less than 0.01). The results of this study indicate that detectable integration of HPV DNA, regardless of type, occurs infrequently in cervical intraepithelial neoplasia. The absence of HPV 16 DNA integration in some carcinomas implies that integration is not always required for malignant progression. In contrast, the consistent integration of HPV 18 DNA in all cervical cancers examined may be related to its greater transforming efficiency in vitro and its reported clinical association with more aggressive cervical cancers.

Blotting, Southern↗

PubMed2006

Interferon-beta treatment of cervical keratinocytes naturally infected with human papillomavirus 16 episomes promotes rapid reduction in episome numbers and emergence of latent integrants.

Following integration of human papillomavirus (HPV) into the host genome, overexpression of the viral oncogenes E6 and E7 requires loss of the transcriptional repressor functions of E2. A key step in HPV-related carcinogenesis is therefore clearance of residual viral episomes, which encode E2. As spontaneous loss of HPV-16 episomes in vitro is associated with increased expression of antiviral genes inducible by type I interferon (IFN), we used the W12 model to examine the effects of exogenous IFN-beta on cervical keratinocytes containing HPV-16 episomes as a result of 'natural' infection in vivo. In contrast to studies of cells transfected with HPV-31 or bovine papillomavirus, IFN-beta caused rapid reduction in numbers of HPV-16 episomes. This was associated with the emergence of cells bearing previously latent integrants, in which there was increased expression of E6 and E7. Our data indicate that integrated HPV-16 can exist in a minority of cells in a mixed population without exerting a selective advantage until episome numbers are reduced. The kinetics of cell death and changes in viral transcription and translation that we observed support a model where integrants are initially present in cells also containing episomes, with generalized episome clearance by IFN-beta resulting in integrant de-repression. We conclude that IFN-beta can hasten the transition from episomal to integrated HPV-16 in naturally infected cervical keratinocytes. Greater emphasis should be placed on episome loss in models of HPV-related carcinogenesis. We provide the strongest evidence to date that treating HPV-16 lesions by inducing an IFN response may cause clinical progression.

Animals↗

PubMed2005

High levels of HPV-16 DNA are associated with high-grade cervical lesions in women at risk or infected with HIV.

OBJECTIVE: To examine associations between levels of episomal and integrated human papillomavirus (HPV) 16 DNA and the grade of cervical disease. DESIGN: Cross-sectional data were obtained from a cohort of women with and without HIV infection and with high-risk sexual behaviour. METHODS: Episomal and integrated HPV-16 DNA loads were measured in cervicovaginal lavages collected from 75 women (58 HIV seropositive, 17 HIV seronegative) using real-time polymerase chain reaction assays, controlling for cell content and the presence of inhibitors. RESULTS: HPV-16 viral loads were significantly higher in women with high-grade squamous intraepithelial lesions (n = 6) than in women with normal cytology (n = 44), whether total (10(8.28) versus 10(5.10) HPV-16 DNA copies/microg DNA), episomal (10(7.99) versus 10(4.61)) or integrated (10(7.95) versus 10(4.77)) HPV-16 viral loads were measured (P < 0.02 for each comparison). Thirty-nine women had colposcopy [11 normal cervix, 16 cervical intraepithelial neoplasia (CIN) 1, six CIN 2, six CIN 3] and 24 additional women had three consecutive normal cytology smears. Controlling for age, race, CD4 cell count and HIV status, total (OR 3.5, 95% CI 1.2-10.4; P = 0.02), episomal (OR 2.9, 95% CI 1.2-7.4; P = 0.02,) and integrated (OR 1.6, 95% CI 1-2.6; P = 0.05) HPV-16 DNA loads were significantly associated with CIN 2,3, but the differences between CIN 1 and CIN 2,3 were not significant (P > 0.06). A greater amount of cellular DNA was collected from women with CIN 2,3 (P = 0.007). CONCLUSION: Higher HPV-16 DNA loads are associated with cervical lesions detected by either histology or cytology. No additional information is gained by measuring integrated or episomal over total HPV-16 DNA loads.

Adult↗

PubMed1987

Molecular analysis of integrated human papillomavirus 16 sequences in the cervical cancer cell line SiHa.

Human papillomavirus (HPV) 16 is frequently found integrated into cervical cancer cell genomes and these integrations are thought to play a role in tumorigenesis. To investigate the mechanisms of HPV integration and its effect on transcription and chromosomal sequence organization, we have cloned and analyzed the HPV16 integration from the cervical cancer cell line SiHa. Restriction analyses and Southern blotting indicated that approximately 95% of an HPV16 genome was integrated without gross rearrangement. Sequence analysis of the cellular-viral DNA junctions revealed that integration had occurred within the E2 and E4 ORFs where 251 bp of viral sequence was deleted. One viral terminus occurred within sequences of an Alu repeat and a 4-bp homology was present at the site of recombination. Using unique cellular flanking DNA probes, a 4.8-kb deletion of cellular sequences was detected at the site of viral integration. The chromosomal location of the viral integration and cellular deletion were mapped to chromosome 13 using a rodent X human somatic cell hybrid panel. Northern blot analysis using viral subgenomic and 3' cellular probes revealed transcription from the 3' portion of integrated HPV16 (E6, E7, E1) and flanking cellular sequences. The observation of viral-cell transcripts and chromosomal deletions associated with HPV integration may indicate that such events are part of a multistep mechanism leading to the development of cervical cancer.

Base Sequence↗

PubMed1991

The physical state of human papillomavirus type 16 DNA in cervical carcinomas of Hong Kong Chinese.

The presence of human papillomavirus (HPV) in 15 cervical carcinoma specimens obtained from Hong Kong Chinese patients was analyzed by Southern blot hybridization studies. In nine (60%) of them, HPV 16 genomes were detected, while two others (13.3%) were found to harbor HPV DNA of unknown type closely related to HPV 16. All of them were classified as squamous cell carcinomas according to WHO guidelines. In addition, the presence of HPV 18 was shown in another two (13.3%) squamous cell carcinoma samples. Among the nine tumors harboring HPV 16, four specimens (44.4%) have HPV in integrated forms, while four others (44.4%) have HPV in episomal forms. The simultaneous presence of both episomal and integrated forms was demonstrated in the remaining tissue sample (11.2%). The result obtained here indicates a strong association between HPV infection and cervical carcinogenesis in Hong Kong Chinese, with HPV 16 prevalent in squamous cell carcinoma. Moreover, the persistence of HPV 16 episomes in some of the tumor specimens suggests that extrachromosomal HPV DNA, possibly acting synergistically with other oncogenic factors, is also capable of inducing cervical cancer.

Blotting, Southern↗

PubMed2003

Biology and evolution of cervical squamous intraepithelial lesions: a hypothesis with diagnostic prognostic implications.

Recent advances in the understanding of HPV-associated cervical squamous intraepithelial lesions, specifically with respect to HPV DNA integration into basal cervical epithelial cells, need to be incorporated into strategies for diagnosing and classifying these lesions. The biology and evolution of HPV-associated cervical squamous intraepithelial lesions is reviewed, along with recent developments using tyramide-based in situ hybridization and MIB-1 immunoreactivity. It is proposed that HPV DNA integration into the basal cells of cervical squamous epithelium precedes the transformation of low-into high-grade lesions. The HPV DNA tyramide-based in situ hybridization system may prove to be a powerful diagnostic/prognostic tool in this regard. It is also proposed that the presence of mitoses (especially atypical forms) in the upper layers may be a discriminatory hallmark in the morphologic distinction between low- and high-grade lesions. Further, since the biologic changes manifest between these two lesions are reflected in their respective phenotype, it appears plausible to adopt the Bethesda System two-tiered/binary classification of LGSIL and HGSIL for histopathologic diagnoses.

DNA, Neoplasm↗

PubMed1998

APM-1, a novel human gene, identified by aberrant co-transcription with papillomavirus oncogenes in a cervical carcinoma cell line, encodes a BTB/POZ-zinc finger protein with growth inhibitory activity.

Integration of human papillomavirus (HPV) DNA into the host cell genome is an important step in cervical carcinogenesis. In tumour cells with integrated HPV DNA, transcription of viral oncogenes E6 and E7 continues into the flanking cellular sequences thereby producing viral-cellular fusion transcripts. Analysis of cellular sequences flanking the integrated HPV68 DNA in the cervical carcinoma cell line ME180 revealed homozygosity of the mutant allele in ME180 cells. We speculated that this could indicate the existence of a cellular tumour suppressor gene in the integration region. We report here the identification of a novel human gene, named APM-1, which is co-transcribed with the HPV68 E6 and E7 genes and is present in the 3'-cellular part of the ME180 viral-cellular fusion transcripts. The APM-1 gene encodes a protein with a BTB/POZ domain and four zinc fingers, and is located at chromosome 18q21. APM-1 transcripts are detected in normal cervical keratinocytes, but not in the majority of cervical carcinoma cell lines analysed. The APM-1 gene caused a reduction of clonal cell growth in vitro of HeLa and CaSki tumour cells. These characteristics make APM-1, the first novel human gene identified in a HPV integration region, a likely candidate for the postulated tumour suppressor gene.

Alleles↗

PubMed1997

Human papillomavirus type 18 DNA sequences in adenocarcinoma and adenosquamous carcinoma of the uterine cervix.

Human papillomavirus (HPV) DNA sequences were detected by Southern blot hybridization and polymerase chain reaction (PCR) in 10 out of 19 patients (52.7%) with adenocarcinoma [15] and adenosquamous [4] carcinoma of the uterine cervix. HPV 18 DNA was detected in 8 of these 19 patients (42.1%), HPV 16 DNA in 1 patient (5.3%) and HPV type X (unknown) in another (5.3%). Of the 10 HPV positive samples HPV 18 was found in 6 out of 6 pure adenocarcinomas (100%), and in 2 of 4 (50%) adenosquamous carcinomas. HPV 16 and HPV X were each detected in 1 out of 4 (25%) adenosquamous carcinomas. The physical state of the viral DNA was investigated in 5 of the 10 HPV-positive cases. All the specimens from these 5 cases showed HPV to be integrated into the host genome, except for one adenosquamous specimen, which showed both episomal and integrated forms of HPV 16. Six of 8 HPV 18 DNA positive specimens were from cases of pure adenocarcinoma and it was found by PCR that five of these 6 specimens retained fragments of E6/E7, LCR/E7 and early sequence of E1 fragment (sequence: 1188-1373) but deleted most part of E1.

Adenocarcinoma↗

PubMed2003

The fidelity of HPV16 E1/E2-mediated DNA replication.

Human papillomaviruses (HPV) are causative agents in a variety of human diseases; for example over 99% of cervical carcinomas contain HPV DNA sequences. Often in cervical carcinoma the HPV genome is integrated into the host genome resulting in unregulated expression of the viral transforming proteins E6 and E7. Therefore viral integration is a step toward HPV-induced carcinogenesis. Integration of the HPV genome could occur following double-strand DNA breaks that could arise during viral DNA replication. We investigated the fidelity of HPV 16 E1- and E2-mediated DNA replication of non-damaged and UVC-damaged templates in a variety of cell lines with different genetic backgrounds; C33a (derived from an HPV-negative cervical carcinoma), XP30RO (deficient in the by-pass polymerase eta (poleta)), XP30eta (expressing a restored wild-type poleta), XP12RO (nucleotide excision repair defective), and MRC5 (derived from a 14-week-old human fetus). The results demonstrate that the fidelity of E1- and E2-mediated DNA replication is reflective of the genetic background in which the assays are carried out. For example, restoring poleta to the XP30 cell line results in a 3-fold drop in the number of mutants obtained following replication of a UVC-damaged template. A relatively high percentage of the mutant-replicated molecules arise as a result of genetic rearrangement. This is the first time such studies have been carried out with an HPV replication system, and the results are discussed in the context of the HPV life cycle and what is known about HPV genomes in human cancers.

Blotting, Southern↗

PubMed1991

Episomal and integrated human papillomavirus in cervical neoplasia shown by non-isotopic in situ hybridisation.

It was postulated that non-isotopic in situ hybridisation (NISH) signal types 1-3 for human papillomavirus in cervical biopsy specimens represent episomal or integrated virus. The aim of this study was to validate this hypothesis by independent molecular techniques. Fresh cervical intraepithelial neoplasia (CIN) and squamous cell cancer (SCC) tissue were examined for NISH signal pattern by hybridising with digoxigenin labelled HPV 16. DNA was extracted from the same samples and analysed by restriction endonuclease digestion and Southern blotting to determine the physical state of the viral genome. Six CIN biopsy specimens showed a type 1 NISH signal for HPV 16. On Southern analysis these biopsy specimens contained only episomal HPV 16. Three SCC with a type 2 NISH signal contained integrated HPV 16 by Southern analysis. Two specimens, a CIN 3 and an SCC with a type 3 NISH signal for HPV 16, showed the presence of both episomal and integrated HPV 16 with conventional Southern analysis and two dimensional gel electrophoresis. These results show that episomal HPV can be reliably determined by NISH type 1 signal, integrated HPV by type 2, and a combination of both episomal and integrated HPV, by a type 3 signal in archival paraffin wax embedded cervical biopsy specimens. This will add another variable to the epidemiological studies of HPV infection. In particular, it will now allow retrospective studies to be done to define the role of episomal and integrated HPV in the evolution of cervical intraepithelial neoplasia and other cervical disease associated with this virus.

Blotting, Southern↗

PubMed2003

Integrations of the hepatitis B virus (HBV) and human papillomavirus (HPV) into the human telomerase reverse transcriptase (hTERT) gene in liver and cervical cancers.

Chronic infections with the hepatitis B virus (HBV) and high-risk human papillomaviruses (HPVs) are important risk factors for hepatocellular carcinoma (HCC) and cervical cancer (CC), respectively. HBV and HPV are DNA viruses that almost invariably integrate into the host genome in invasive tumors. The viral integration sites occur throughout the genome, leading to the presumption that there are no preferred sites of integration. A number of viral integrations have been shown to occur within the vicinity of important cancer-related genes. In studies of HBV-induced HCC and HPV-induced CC, we have identified two HBV and three HPV integrations into the human telomerase reverse transcriptase (hTERT) gene. Detailed characterization of the integrations revealed that four integrations occurred within the hTERT promoter and upstream region and the fifth integration occurred in intron 3 of the hTERT gene. None of the integrations altered the hTERT coding sequence and all resulted in juxtaposition of viral enhancers near hTERT, with potential activation of hTERT expression. Our work supports the hypothesis that the sites of oncogenic viral integration are nonrandom and that genes at the sites of viral integration may play important roles in carcinogenesis.

Base Sequence↗

PubMed1998

Precancer of the human cervix.

The tumour biology of cervical precancer is unusual. A large variety of individually distinct forms crudely divided into slight, moderate, severe dysplasia and carcinoma in situ exist. Virtually all contain genital human papillomavirus (HPV) either as infectious virions or as episomal or integrated DNA. HPV, which occurs as hundreds of types, subtypes and variants, has a high prevalence in all human populations. Most males are symptomless reservoirs, whereas a proportion of infected women develop condyloma, precancer and subsequently, in a minority, invasive cancer. HPV has unequivocal features of a sexually transmitted infectious agent. Risk of precancer is statistically related to infection with genital HPV, but differences in risk between populations with high and low prevalence of HPV are larger than expected from a direct correlation. Findings fit with HPV as a major risk factor, but other factors must also be operative. These may include shifts in number of target cells, depending on regeneration and infection by various micro-organisms, hormones, smoking and immunity. Final proof of necessity of HPV infection for precancer can probably be delivered only after its elimination by successful vaccination. Genital condyloma, which is not precancerous, is caused by HPV low risk types, typically 6 or 11, in analogy with papilloma formation in skin and mucosa in a large variety of species. This benign lesion is the hallmark of mammalian HPV pathology and a source of interindividual spread of virus. Slight dysplasia is heterogeneous. Many lesions seem to be polyclonal, self limited cell proliferative responses to infection with low grade HPV. A small proportion are associated with either simultaneous presence or subsequent development of higher grades of dysplasia, in situ or invasive cancer. Evidence exists for two mechanisms: clonal selection of cells with increasingly undifferentiated phenotypes, and independent development of different morphological types of precancer. The relative importance of the two is unknown. High risk HPV, typically 16 or 18, is preferentially associated with high grade dysplasia and in situ cancer, either because it increases risk of clonal progression to these forms or induces them de novo. Severe dysplasia, in situ and invasive cancer always present as monoclonal lesions. Genetic links indicate that these pathologies arise by clonal selection from less advanced precursors. The number of potential target cells for precancer confined to a narrow transformation zone is small. Risk of precancer and malignant transformation per target cell is therefore probably far higher than in any other human tissue subject to cancer. Spontaneous mutation rate and physicochemical carcinogens seem insufficient for the creation of a malignant phenotype in cells of the transformation zone. Currently HPV is the only strong candidate for such a feat. Any or all of the following mechanisms may play a role: overexpression of viral E6 and E7 genes, often triggered by disruption of control elements upon integration of viral DNA into the cellular genome, activity of specific (E6?) configurations in certain HPV variants, inactivation of TP53 with decreased capacity for DNA repair and enhanced likelihood of accumulation of "transforming" mutations and viral integration at sites controlling function of cellular oncogenes and/or suppressor genes. Target cells within the transformation zone have the capacity for bidirectional (squamous and/or glandular) differentiation. HPV types seem to drive cells preferentially in different directions after infection/transformation. Low risk types are almost always associated with squamous differentiation, HPV 16 usually also with squamous differentiation and HPV 18 with adenosquamous or adenomatous differentiation. (ABSTRACT TRUNCATED)

Carcinoma in Situ↗

PubMed1994

Improvements in visualisation and localisation of human papillomavirus DNA in CaSki cells by fluorescence in situ hybridization, laser scanning confocal microscopy and three-dimensional image reconstruction.

The visual interpretation and localisation of specific DNA sequences in three dimensions in cell nuclei was investigated by fluorescence in situ hybridization (FISH) and laser scanning confocal microscopy (LSCM) using CaSki cells containing 600 copies per cell of human papillomavirus (HPV) DNA type 16 integrated in cellular DNA. Biotinylated DNA probes were used and DNA-DNA hybrids were revealed by a three-step reaction involving a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and a streptavidin-fluorescein isothiocyanate complex. The DNA from cell nuclei was counterstained with propidium iodide. With standard fluorescence microscopy, some dense fluorescent spots were seen in the cell nuclei. Similarly, with LSCM, some hybridization spots were observed in the cell nuclei but they were at different levels of the nuclei as shown by successive nuclear sections taken along the z axis. The visualisation of multiple hybridization spots confirmed the presence of multiple integration sites of HPV 16 DNA in CaSki cells. Association of LSCM with three-dimensional reconstructions lead to spatial images of hybridization spots obtained by stacking (x,y) images from consecutive confocal planes. Rotation of the reconstructed cell nuclei around the y axis makes it possible to distinguish closely adjacent spots. The combination of these techniques improves the detection of hybridization spots and may be of interest to further determine whether the HPV DNA is episomal or integrated in infected cells.

DNA, Viral↗

PubMed1987

Integration of human papillomavirus type 16 DNA sequences: a possible early event in the progression of genital tumors.

The keratinocyte line SK-v harbors only integrated human papillomavirus type 16 (HPV 16) DNA sequences, although it originated from vulvar Bowenoid papules predominantly containing multiple copies of free HPV 16 genomes. We have cloned a fragment of cell DNA that contains the integrated HPV 16 DNA sequences and have shown that integration interrupts the HPV 16 genome in open reading frames E2 and L2 and creates a deletion of 813 base pairs. This allows the expression of open reading frames E6 and E7, as actually substantiated by Northern (RNA) blot analysis of SK-v RNAs with subgenomic HPV 16 RNA probes. Using a unique flanking cellular DNA sequence as the probe, we have shown that the integration of HPV 16 sequences had already occurred in the premalignant lesions from which the SK-v cell line was derived.

Base Sequence↗

PubMed1994

Possible role for human papillomavirus 16 in squamous cell carcinoma of the finger.

There are about 20 types of human papillomaviruses (HPVs) which are generally believed to be confined to the anogenital tract. HPV 16 is the most common of these. Four squamous cell carcinomas of the finger from three individuals were analyzed for the presence of HPV DNA by Pst 1 restriction enzyme digest and Southern blotting. Under high stringency conditions, all were positive for HPV 16. The DNA from each patient was digested with a panel of restriction enzymes so that integration of HPV into the genome could be evaluated. Three of three samples contained high molecular weight oligomeric circular DNA arranged as catanates. None showed integration. To determine if the HPV was transcriptionally active, RNA was isolated, reverse transcribed, and amplified using primers that amplified the unspliced E6 transcript and the E6I and E6II spliced transcripts. All of the patient biopsies assayed expressed the unspliced E6 transcript and the spliced E6I transcript with the E6I transcript being the most abundant. The E6II transcript was not detected in any of the samples. These findings indicate that HPV plays a role in the development of squamous cell carcinoma of the finger and the role of the malignant genital HPV needs to be carefully looked at in areas outside the genital region. It also suggests that the natural history of HPV in areas outside the cervix may not be identical to that of HPV in the anogenital region.

Base Sequence↗

PubMed1992

Frequent occurrence of p53 gene mutations in uterine cancers at advanced clinical stage and with aggressive histological phenotypes.

The clinical and pathological significance of mutation of the p53 tumor-suppressor gene was examined in 108 cases of primary uterine cancers using single-strand conformation polymorphism and direct DNA sequencing analyses. Mutation of the p53 gene was detected in 19 (31%) of 62 cases of cancer of the uterine corpus and was more frequent in groups at an advanced clinical stage and/or with aggressive histology. Among four adenocarcinomas arising in the lowest portion of the uterine corpus, three showed integration of human papillomavirus (HPV) types 16 and/or 18 DNA, and two of them also showed p53 mutation. In cancer of the uterine cervix, p53 mutations were rare; 7% (3/46) in total, 3% (1/30) of cases with integration of HPV types 16 and/or 18 DNA and 13% (2/16) of cases without HPV DNA integration. Three mutations were detected among two cases at clinical stage IV and two cases of undifferentiated cervical carcinoma. Immunohistochemically, all five cases of uterine cancer which showed diffuse (> 50% of cancer cells) nuclear staining of p53 protein also carried the p53 mutation. Therefore, p53 alterations were suggested to be involved in the development of uterine cancers showing aggressive biological behavior. Although a high incidence of HPV DNA integration and a low incidence of p53 mutation were confirmed in cancer of the uterine cervix, there was no inverse association between integration of HPV types 16 and/or 18 DNA and p53 mutation.

Adenocarcinoma↗

PubMed1997

Physical status and expression of HPV genes in cervical cancers.

It is known that E2 protein of oncogenic human papillomavirus (HPV) inhibits the expression of E6 and E7 genes from their major promoters in vitro and suppresses the proliferation of cervical cancer cells. This suggests that the loss of functional E2 gene may provide selective advantages in the development of cervical cancer. Investigation of the relationship between the disruption of HPV-16/18 E2 genes by DNA integration and clinical outcome of cervical cancer may not only help to understand the mechanism of HPV-related cervical carcinogenesis, but may also provide novel management of cervical cancer. It was noted that integrated HPV-16/18 DNA was predominant in most patients with cervical cancers, marking 51 of a total of 68 cases (75%); episomal HPV DNAs were found in 5 cases (7.4%), and finally mixed forms of HPV DNAs with episome and integration were found in 12 cases (17.6%). Whole portions of E2 DNA of HPV-16 could be amplified by PCR in 19 (36.5%) of 52 cases of cervical intraepithelial neoplasia. It was shown that there was not statistically significant association with the different stages, but integrated HPV DNAs were detectable only in the patients with far-advanced stage of cervical cancers, which also means no episomal forms were detected. Episomal forms of HPV DNA were detectable in 14 (25.9%) of 54 squamous cell carcinomas (4 pure episomal forms and 10 mixed forms), whereas only 1 (8.3%) of 12 adenocarcinomas and adenosquamous cell carcinomas contained episomal viral DNA. When HPV DNA forms were compared with initial tumor size, lymphovascular space involvement, and frequency of nodal metastasis, statistically significant relationships were not found. The association of DNA integration with invasive cervical cancers was seen regardless of HPV type; however, there were differences between the integration profiles of HPV-16 and HPV-18 DNA. Of the 51 HPV-16-containing cancers, 36 (70.6%) revealed purely integrated HPV DNA, and another 10 cases (19.6%) displayed both integrated and episomal HPV DNAs. However, 5 (9.8%) cases showed only episomal copies of the HPV-16 genome. In contrast, all 17 HPV-18-containing cancers (5 cases positive for HPV-18 and 12 cases positive for both HPV-16 and -18) revealed only the integrated form of HPV-18 DNA. The expression of E6 and E7 transcripts of HPV-16/18 is uniformly correlated with the physical status of HPV DNAs. HPV E2 mRNAs were constantly expressed in the presence of the intact virus in cases with episome and mixed forms of HPV DNA. In general, amplified signals from HPV E2 RT-PCR are more intensive than those from DNA-PCR in the same patients. It is suggested that RT-PCR is a valuable method to evaluate dynamic expression of the specific gene and seems to be more sensitive than the DNA-PCR method in detecting intact E2 gene because of the gene copy numbers.

Base Sequence↗

PubMed1996

Molecular analysis of cellular loci disrupted by papillomavirus 16 integration in cervical cancer: frequent viral integration in topologically destabilized and transcriptionally active chromosomal regions.

To discern the structural features of cellular loci that are disrupted by type 16 human papillomavirus (HPV-16) integration in cervical cancer, a polymerase chain reaction (PCR)-based strategy was employed for direct amplification and sequence analysis of four such cellular loci in cancer biopsy samples. One of the HPV-16-disrupted loci was found to be the microtubule-associated protein (MAP-2) gene and the other three loci were uncharacterized and were designated PID-1 to -3 (for papillomavirus integration-disrupted). The junctional sequences of the viral integration sites in the four loci analyzed are bracketed by long tracts of homogeneous purine or pyrimidine or alternating purine-pyrimidine which are known to destabilize the B-form conformation of the DNA structure. Using a panel of human/hamster hybrid cell DNAs and PCR analysis, the four loci were assigned to chromosomes 2 (MAP-2), 9 (PID-1), 1 (PID-2) and 8 (PID-3), respectively. These chromosomes carry numerous other previously determined viral integration and chromosomal fragile sites and the myc oncogenes. The PID-1 locus was further found in Southern analysis to be rearranged and amplified in another cervical cancer biopsy and a cervical carcinoma cell line (CaSki). On Northern analysis, the PID-1 and -3 probes detected a 3.0- and a 3.6-kb transcript, respectively, in normal cervical cells and in cervical cancer cell lines. The findings suggest that HPV-16 genome integrates frequently into topologically destabilized and transcriptionally active chromosomal sites. It remains to be elucidated whether the MAP-2 and the PID loci contribute to the pathogenesis of cervical cancer.

Base Sequence↗