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PubMed1999

Evaluation of the role of genital human papillomavirus in the pathogenesis of ungual squamous cell carcinoma.

Human papillomaviruses (HPV), and particularly HPV 16, have been associated with ungual squamous cell carcinoma (USCC). But their role in tumor development remains unclear. In genital carcinoma, where the oncogenic role of HPV is well established, integration of HPV DNA into the host cell genome seems to be important for malignant transformation. To clarify the issue, we have studied the physical state of HPV 16 in 3 cases of in situ USCC by the polymerase chain reaction and by in situ hybridization. HPV DNA was integrated into the human genome in 2 cases and episomal in 1 case. This particular physical state of HPV 16 in USCC, similar to those encountered in anogenital SCC, confirms the probable role of this kind of virus in the pathogenesis of USCC.

Aged↗

PubMed1994

Combined effects of human papillomavirus-18 and N-methyl-N'-nitro-N-nitrosoguanidine on the transformation of normal human oral keratinocytes.

We immortalized oral keratinocytes by transfecting them with recombinant human papillomavirus (HPV) type 18 DNA and established three cell lines. These lines were morphologically different from their normal counterpart, contained integrated entire HPV-18 DNA, and expressed the viral E6/E7 genes. The cells contained less p53 protein and more c-myc mRNA than normal cells. However, they proliferated only in keratinocyte growth medium (KGM) containing low calcium and were not tumorigenic in nude mice. To test the hypothesis that tumors result from the combined effect of a "high-risk" HPV and chemical carcinogens in the human oral cavity, we exposed the immortalized cells to the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Three chemically transformed cell colonies were isolated. These cells (a) proliferated well in both KGM and Dulbecco's modified minimum essential medium containing physiological levels of calcium; (b) were capable of proliferating in nude mice; (c) contained intact, integrated HPV-18 sequences; (d) transcribed substantially more HPV-18 E6/E7, transforming growth factor-alpha, and c-myc than the immortalized counterpart; and (e) contained, like the immortalized counterpart, less wild-type p53 protein and DCC message. These data indicate that human oral keratinocytes can be transformed by sequential exposure of normal keratinocytes to a "high-risk" HPV and chemical carcinogens.

Animals↗

PubMedDate not supplied

[The relation between human papilloma virus and cellular DNA content in epidermoid carcinoma of the head and neck].

The relation between the presence of gene sequences of human papilloma virus (HPV) and the development of abnormalities in cellular DNA content was analyzed in 31 squamous-cell carcinomas of the head and neck. The integration of HPV types 16 and 6b by PCR and DNA content was studied by flow cytometry in 31 specimens from patients with squamous-cell carcinoma of the head and neck. Eighteen (58%) cases were aneuploid. HPV DNA was present in seven tumors (22.5%), five of then HPV-6b and two of them HPV-16. Aneuploidy correlated with poorly differentiated tumors. No correlation was found between HPV integration and either cellular DNA content or the degree of histological tumor differentiation. Therefore, the presence of HPV gene sequences did not seem to be associated with a higher incidence of aneuploidy in squamous-cell carcinoma of the head and neck.

Adult↗

PubMed1990

Human cervical cells immortalized in vitro with oncogenic human papillomavirus DNA differentiate dysplastically in vivo.

Human papillomavirus (HPV) types 16 and 18 are associated with cervical dysplasia and carcinoma. In vitro integration of HPV-16 or HPV-18 DNA into cultured human cervical cells results in their immortalization. In this study, in vivo differentiation of human cervical cells immortalized with recombinant HPV-16 or HPV-18 DNA was studied by implanting confluent monolayers of HPV-16 and HPV-18 DNA immortalized cell lines under dorsal skin flaps of immunodeficient mice. Grafts of a human cervical cancer cell line (C4-1) and cultured normal cervical cells served as controls. Histologic analysis 2 to 3 weeks after grafting demonstrated dysplastic differentiation of the HPV-16 and HPV-18 cell lines as characterized by abnormal mitotic figures, nuclear pleomorphism, and loss of basal polarity. RNA in situ hybridization confirmed the presence of viral transcripts in the dysplastic grafts. G-banded chromosome analysis showed that both the HPV-16 and HPV-18 cell lines were aneuploid. Even after prolonged periods of implantation tumors did not form and invasion of underlying mouse stroma did not occur. Normal cervical cells differentiated normally and the C4-1 cell line formed tumors and demonstrated more pronounced nuclear abnormalities than the HPV immortalized grafts. Integration of HPV-16 and HPV-18 DNA into cervical cells leads to an indefinite growth capacity. These aneuploid cells differentiate abnormally in vivo, but do not form tumors or invade host tissue, consistent with a multistage theory of cervical carcinogenesis.

Animals↗

PubMed1996

Expression of HPV16 E6 or E7 increases integration of foreign DNA.

In most invasive cervical carcinomas, high-risk human papillomavirus (HPV) DNA is integrated into the host genome, while in pre-invasive cervical lesions the viral genome is typically maintained exclusively as an episome. In contrast, integration of low-risk HPV DNA is rare, as is the association of low-risk HPVs with carcinomas. High-risk HPV integration is associated with a selective growth advantage of affected cells, and hence, integration is likely to be an important genetic alteration contributing to cervical tumor progression. Expression of high-risk, but not low-risk, HPV E6 or E7 proteins disrupts the p53-dependent G1 arrest that cells normally display in response to DNA damage. Absence of this cell cycle checkpoint may predispose cells containing high-risk HPVs to genetic instability and to the accumulation of the genetic alterations that appear to be required for HPV-associated cervical tumor progression. We hypothesized that integration of high-risk HPV DNA into the host cell genome may be facilitated by E6- and/or E7-mediated disruption of the normal DNA damage response pathway. To test this hypothesis, we assessed the integration frequency of a reporter plasmid (pHyGal) in RKO cells expressing individual E6 or E7 genes of either high-risk (HPV16) or low-risk (HPV6, HPV11) type viruses. Cells expressing HPV16 E6 or HPV16 E7 exhibited a significantly increased frequency of pHyGal integration in comparison to RKO control cells or cells expressing low-risk HPV E6 or E7. Thus, expression of high-risk, but not low-risk, E6 and E7 proteins increases the frequency of foreign DNA integration into the host genome. These findings suggest that at least some of the difference in oncogenic potential observed between high-risk and low-risk HPV types may be determined by the increased ability of high-risk HPVs to integrate into host DNA.

Cinnamates↗

PubMed2003

Human papillomavirus type 16 status in cervical carcinoma cell DNA assayed by multiplex PCR.

Integration of human papillomavirus (HPV) DNA into host genome occurs early in cancer development and is probably an important event in malignant transformation of cervical cancer. The HPV genome integration usually disrupts E2 gene open reading frames. It results in the lack of E2 gene suppressor of the synthesis of E6 and E7 products which, in turn, leads to the overexpression of E6 and E7 genes. The oncogenic HPV types (HPV16, -18, -45, and -58) can be present as episomes or may integrate into human chromosomes. Sixty-six cervical cancer patients positive for HPV16 were tested for the presence of E6, E2, E1, and L1 genes. Multiplex PCR was carried out in all cases. Using cluster analysis, the calculated ratios of E1/E6, E2/E6, L1/E6, E1/E2, and E2/(E1*E6) gene amplification products were divided into two or three statistically different groups. These were used for statistical analysis of the prevalence of specific gene types in histological types of cancer, different levels of clinical staging, and histologically confirmed nodal metastases. The statistical analysis proved a significant correlation in the ratios of E2/E6 and E1/E2 only. The E2/E6 and E1/E2 were higher in carcinoma in situ than in advanced squamous cancers. The E2/E6 ratios were lower in higher clinical stages. The multiplex PCR estimation of the E2/E6 ratio could be a simple method for selecting patients with a high risk of a poor outcome in a standard stage-dependent treatment procedure.

DNA, Viral↗

PubMed2006

Detection of human papillomavirus type 16 integration in pre-neoplastic cervical lesions and confirmation by DIPS-PCR and sequencing.

BACKGROUND: Persistent infections with high-risk types of human papillomavirus (HR-HPV) favour integration of viral DNA into the host cells and are associated with cervical carcinoma. HPV16 is the prevalent HR-type worldwide associated to cervical cancer. Integration of viral DNA promotes a selective cell growth advantage, resulting a risk factor for cancer development. OBJECTIVES: To test physical status of HPV16 infection in pre-neoplastic cervical lesions using a quantitative real time-PCR (QRT-PCR) based method. To investigate reliability of this method in identification of HPV16 integrated sequences, by detection of integrated papillomavirus sequences (DIPS-PCR) assay and sequencing. STUDY DESIGN: One hundred and seventy HR-HPV positive archival cervical specimens were tested for presence of HPV16 DNA. In HPV16 positive samples, viral load and physical status were evaluated. RESULTS: HPV16 DNA was detected in 74/170 (43%) HR-HPV positive specimens. In 52/74 a QRT-PCR was performed, and 3 integrated, 13 mixed and 36 episomal forms were detected. Presence of integrated forms was confirmed by DIPS-PCR and sequencing. CONCLUSIONS: Presence of HPV integrated forms was detected and confirmed in pre-neoplastic cervical lesions. The QRT-PCR method we used is sensitive and specific for identification of HPV integration in cervical samples, and may be suitable for large scale investigations with prognostic and clinical implications in management of cervical cancer.

Base Sequence↗

PubMed2005

Tissue distribution of human papillomavirus 16 DNA integration in patients with tonsillar carcinoma.

PURPOSE: Human papillomavirus 16 (HPV-16) has been implicated as a causative agent in a subset of head and neck squamous cell carcinomas (HNSCC). This study was undertaken to discern the distribution and timing of HPV viral integration during tumorigenesis of the upper respiratory tract. EXPERIMENTAL DESIGN: A tissue array was assembled from a consecutive group of 176 patients with HNSCCs. The array was evaluated by HPV-16 in situ hybridization and p16 immunohistochemistry. Patients with HPV-positive tonsillar cancers who had undergone bilateral tonsillectomies were selected for more complete mapping of viral integration. RESULTS: HPV-16 was detected in 38 of the 176 (22%) cases by in situ hybridization. When stratified by site of origin, HPV-16 was detected in 37 of 45 cancers arising from the oropharynx but in only 1 of 131 tumors arising from nonoropharyngeal sites (82% versus 0.8%, P < 0.00001). P16 expression was associated with the presence of HPV-16: 31 of 38 HPV-positive tumors exhibited p16 expression, whereas only 9 of the 138 HPV-negative tumors were p16-positive (82% versus 6%, P < 0.00001). In the bilateral tonsil sections, hybridization signals were strictly limited to the invasive cancers and associated dysplasias. P16 staining was widely distributed throughout the nonneoplastic crypt epithelium of individuals with and without tonsillar cancer. CONCLUSIONS: HPV-16 is strongly associated with carcinomas arising from the oropharynx, and integration is tightly coupled to the neoplastic process. Viral integration does not occur as a field alteration throughout normal tonsillar epithelium. P16 expression localizes to HPV-positive cancers, and is intrinsic to the specialized epithelium of the tonsillar crypts. For risk assessment, early cancer detection and disease surveillance, evidence of HPV-16 integration may represent a meaningful finding, whereas high p16 expression, by itself, may not.

Carcinoma, Squamous Cell↗

PubMed2005

The role of viral integration in the development of cervical cancer.

The development of invasive cervical cancer is associated with human papillomavirus (HPV) infection and subsequent integration into the host epithelium. More than 99% of cervical cancers contain HPV sequences, and many of these contain a truncated HPV genome integrated into a single position within the host genome. Studies examining the role of viral integration in cervical cancer development have found that the sites of integration appear randomly distributed throughout the genome. This, and the observation that it frequently takes years after HPV infection for cervical cancer to develop, has led to the current paradigm that the site of HPV integrations is unimportant to the invasive cervical cancer that eventually develops. In our previous studies of HPV16 and HPV18 integration in cervical cancers, we also found integrations throughout the genome, but observed as well that more than half of the integrations occurred within common fragile site regions. To determine if HPV integration might play an important role in cervical cancer, we conducted two complementary studies. We first localized 40 new HPV16 integration sites from cervical tumors from women in Hong Kong; this, together with previous integration studies, provided a better picture of the distribution of integration sites throughout the genome. We then analyzed the sites of viral integration in an in vitro model of HPV integration. By comparing the sites of HPV integration in vivo (in multiple primary cervical tumors) to those obtained in vitro, the data can help to determine if HPV integrations observed in vivo are the result of random and nonselected integrations.

Base Sequence↗

PubMed1987

Papillomavirus sequences integrate near cellular oncogenes in some cervical carcinomas.

The chromosomal locations of cellular sequences flanking integrated papillomavirus DNA in four cervical carcinoma cell lines and a primary cervical carcinoma have been determined. The two human papillomavirus (HPV) 16 flanking sequences derived from the tumor were localized to chromosome regions 20pter----20q13 and 3p25----3qter, regions that also contain the protooncogenes c-src-1 and c-raf-1, respectively. The HPV 16 integration site in the SiHa cervical carcinoma-derived cell line is in chromosome region 13q14----13q32. The HPV 18 integration site in SW756 cervical carcinoma cells is in chromosome 12 but is not closely linked to the Ki-ras2 gene. Finally, in two cervical carcinoma cell lines, HeLa and C4-I, HPV 18 DNA is integrated in chromosome 8, 5' of the c-myc gene. The HeLa HPV 18 integration site is within 40 kilobases 5' of the c-myc gene, inside the HL60 amplification unit surrounding and including the c-myc gene. Additionally, steady-state levels of c-myc mRNA are elevated in HeLa and C4-I cells relative to other cervical carcinoma cell lines. Thus, in at least some genital tumors, cis-activation of cellular oncogenes by HPV may be involved in malignant transformation of cervical cells.

Attachment Sites, Microbiological↗

PubMed1995

Prognostic value of p53 protein accumulation in cancer cell nuclei in adenocarcinoma of the uterine cervix.

Adenocarcinoma of the uterine cervix (CxAd) is one of the most distressing malignancies of the female reproductive system because of its tendency to spread aggressively and to be resistant to radiation and systemic therapies. To clarify the prognostic significance of p53 alteration in CxAd, we immunohistochemically examined the incidence of p53 nuclear accumulation, which is considered to be mostly parallel with p53 gene mutation, and its association with clinicopathological parameters in 26 patients with CxAd. The overall incidence of p53 nuclear accumulation was 46% (12 of 26), being higher in groups with clinically advanced disease, higher degrees of cellular atypia, and deeper myometrial invasion, but significantly lower in patients with integration of human papillomavirus (HPV) type 16 or 18 DNA. Nuclear p53 immunoreactivity as well as lymph node status, depth of invasion and the absence of HPV-DNA integration were significant indicators of a poor prognosis. Examination of p53 nuclear accumulation could be applied to biopsy material, and would be of practical assistance in predicting the prognosis of CxAd both preoperatively and postoperatively.

Adenocarcinoma↗

PubMed1992

Sequential combined tumorigenic effect of HPV-16 and chemical carcinogens.

We immortalized oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines, human oral keratinocytes-16A (HOK-16A) and -16B (HOK-16B). These cell lines were morphologically different from the normal counterpart, contained HPV-16 DNA as integrated form and expressed numerous viral genes. However, these cells proliferated only in culture medium containing low calcium (0.15 mM) and are not tumorigenic in nude mice. To test the hypothesis that tumors can be developed by sequential combined effect of human papillomavirus and chemical carcinogens in the oral cavity, these immortalized cell lines were chemically transformed by exposure to either benzo[a]pyrene or methanesulfonic acid ethyl ester. Such transformants proliferated in medium containing physiological calcium levels (1.5 mM) and demonstrated enhanced growth potential in nude mice, whereas primary human oral keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. Chemically transformed cells contained integrated, intact HPV-16 sequences and transcribed significantly higher amount of HPV-16 E6/E7 messages and transforming growth factor-alpha (TGF-alpha) compared with the immortalized oral keratinocytes. Like the HPV-immortalized cell lines, the chemically transformed oral keratinocytes contained lower levels of newly synthesized, wild-type p53 proteins compared to normal cells, and expressed wild-type c-Ha-ras. These results indicate that this in vitro system is useful for investigating the mechanisms of multistep oral carcinogenesis.

Base Sequence↗

PubMed1995

Molecular biology of cervical cancer and its precursors.

Cervical cancer develops from well-defined precursor lesions referred to as either cervical intraepithelial neoplasia or squamous intraepithelial lesions. It is now known that specific types of human papillomaviruses (HPV) are the principal etiologic agents for both cervical cancer and its precursors. The high-oncogenic-risk HPV types associated with invasive cervical cancer produce two oncoproteins, designated E6 and E7, which interact with endogenous cell cycle regulatory proteins, including p53 and Rb. The interaction of virally derived and endogenous cellular proteins converges in deregulation of cell cycle progression and appears to be critical for the development of cervical cancers. However, the development of cervical cancer is a multistep process that cannot be explained simply by infection with specific types of HPV. One additional event that appears to play a role in tumor progression is integration of HPV DNA into the host genome. Integration of HPV DNA frequently disrupts the E2 open reading frames, resulting in overexpression of the E6 and E7 oncoproteins and possibly causing genomic instability. Additional cofactors and mutational events may be important in the pathogenesis of invasive cervical cancers and may include chromosomal rearrangements, loss of constitutional heterozygosity, and proto-oncogene activation.

Cell Transformation, Viral↗

PubMed2003

Preferential integration of human papillomavirus type 18 near the c-myc locus in cervical carcinoma.

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. Greater than 99% of all cervical tumors contain HPV DNA. Integration of high-risk HPV has been temporally associated with the acquisition of a malignant phenotype. Recent work from our lab has shown that HPV16, the most common high-risk HPV associated with cervical carcinoma, preferentially integrates at loci containing human common fragile sites (CFSs). CFSs are regions of genomic instability that have also been associated with deletions, translocations, and gene amplification during cancer development. The current work shows that HPV18, the second most prevalent high-risk HPV type found in cervical tumors, preferentially targets the CFSs. We identified 27 unique HPV18 integrations in cervical tumors, of which 63% (P<0.001) occur in CFSs. However, the distribution of HPV18 integrations found were profoundly different from those found for HPV16. Specifically, 30% of all HPV18 integrations occurred within the chromosomal band 8q24 near the c-myc proto-oncogene. None of the HPV16 integrations occurred in this region. Previous low-resolution mapping suggested that c-myc may be a target of HPV integration. Our data at nucleotide resolution confirm that in HPV18-positive cervical tumors, the region surrounding c-myc is indeed a hot spot of viral integration. These results demonstrate that CFSs are preferred sites of integration for HPV18 in cervical tumors. In addition, we have identified multiple cellular genes that have been disrupted by HPV18 integration in cervical tumors. Our results suggest that the sites of HPV18 integration are nonrandom and may play an important role in the development of cervical tumors.

Chromosome Mapping↗

PubMed1994

Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins.

Genomic integrity is maintained by a network of cellular activities that assess the status of the genome at a given point in time, provide signals to proceed with or halt cell cycle progression, and provide for repair of damaged DNA. Mutations in any part of these pathways can have the ultimate effect of disturbing chromosomal integrity. Recent work suggests that p53 performs this integrator function in mammalian cells. Our present study demonstrates that in mortal cells, the expression of E6 and E7 viral oncoproteins of type 16 human papillomavirus each disrupts the integration of these signals by diverged pathways. Cells expressing E6 protein, which binds and degrades the p53 protein, exhibited alterations in cell cycle control when placed in drug and displayed the ability to amplify the CAD gene. The expression of E7, which binds different cellular proteins important for transformation, including Rb, led to a p53-independent alteration in cell cycle control, a widespread cytocidal response, and polyploidy as a mechanism of drug resistance. These results demonstrate that diverse perturbations of molecular pathways can have different effects on chromosomal integrity.

Antineoplastic Agents↗

PubMed1992

Viral integration and fragile sites in human papillomavirus-immortalized human keratinocyte cell lines.

Human papillomavirus (HPV) types 16 and 18 integration sites were mapped in six HPV-immortalized human keratinocyte cell lines by fluorescence in situ hybridization (FISH). Mapping of HPV sequences in these cell lines revealed that HPV integration varied in copy number and location but that integration sites were stable over extended passages in culture. Integration occurred at different sites throughout the genome and did not correspond to the location of specific cellular genes. However, integration sites were consistent with integration near or within known fragile sites in five of the six cell lines. Induction of aphidicolin-sensitive fragile sites in one cell line prior to in situ hybridization revealed that integrated HPV DNA was disrupted by fragile-site expression, suggesting that integration occurred within a fragile site.

Cell Line, Transformed↗

PubMedDate not supplied

Role of human papilloma virus testing in cervical cancer prevention.

A clear causal relationship has been established between human papilloma virus (HPV) infection and the development of cervical cancer. Genital HPV infection is currently the most common sexually transmitted disease worldwide. The recent 2001 American Society for Colposcopy and Cervical Pathology Consensus Guidelines have included HPV testing for management of women with cervical cytological abnormalities. Clinicians now face the challenge of deciding when to use HPV testing in follow-up of abnormal Pap tests. This article includes updates on HPV, cervical cancer screening, and HPV testing technology. Recommendations for integration of HPV testing into clinical practice are provided.

Adult↗

PubMed1999

Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes.

Cervical cancer emerges from cervical intraepithelial neoplasia (CIN) induced by high-risk HPV (HR-HPV) infections. However, the vast majority of CIN lesions regresses spontaneously, and only a few lesions persist or progress to invasive carcinoma. On the basis of morphological criteria, it is not possible to differentiate high-grade lesions that will regress or persist from those that inevitably will progress to invasive cancers. In most cervical carcinomas, human papillomavirus (HPV) genomes are integrated into host cell chromosomes and transcribed into mRNAs encompassing viral and cellular sequences. In contrast, in early preneoplastic lesions, HPV genomes persist as episomes, and derived transcripts contain exclusively viral sequences. Thus, detection of HPV transcripts derived from integrated HPV genomes may specifically indicate both CIN lesions at high risk for progression as well as invasive cervical cancers. Here, we established a protocol for the amplification of papillomavirus oncogene transcripts (APOT) from cervical specimens that allows us to distinguish episome- from integrate-derived HPV mRNAs. Cervical swab and biopsy samples from 549 patients attending outpatient clinics for cervical dysplasia were screened for the presence of HPV DNA, and 155 samples that were positive for either HPV type 16 (n = 143) or 18 (n = 12) were subjected to the APOT assay. In samples derived from normal cervical epithelia (n = 19) or low-grade cervical lesions (CIN I, n = 10), no integrate-derived HPV transcripts were found. In contrast, in 1 (5%) of 22 samples derived from CIN II lesions, in 10 (16%) of 64 samples from patients with CIN III lesions, and in 35 (88%) of 40 samples from patients with cervical cancer, integrate-derived HPV transcripts were detected. Thus, detection of integrate-derived HPV transcripts in cervical swabs or biopsy specimens by the APOT assay points to advanced dysplasia or invasive cervical cancer.

Biopsy↗