HPV16 E6 Gene Research Articles

Genomic and Proteomic Expression Patterns in HPV-16 E6 Gene Transfected Stable Human Carcinoma Cell Lines

Genomic and Proteomic Expression Patterns in HPV-16 E6 Gene Transfected Stable Human Carcinoma Cell Lines

Genomic imbalances in 70 snap-frozen cervical squamous intraepithelial lesions: associations with lesion grade, state of the HPV16 E2 gene and clinical outcome

Host genomic abnormalities may determine the natural history of cervical squamous intraepithelial lesions (SILs). We undertook comparative genomic hybridisation analysis of epithelium carefully microdissected from 70 cervical SILs, the largest series to date. In contrast to previous studies, we used frozen sections for optimal DNA quality and examined whether patterns of DNA copy number imbalance (CNI) are characteristic of SIL grade, human papillomavirus (HPV) status and postoperative recurrence. We identified more CNIs in cervical SIL than previously described, with more CNIs per case in high-grade squamous intraepithelial lesion (HG-SIL) than in low-grade squamous intraepithelial lesion (LG-SIL) (P=0.04). While some CNIs were seen at similar frequencies in HG-SIL and LG-SIL, others, including gain on 1q, 3q and 16q, were found frequently in HG-SIL but not in LG-SIL. There were significantly more CNIs per case in HG-SILs showing loss of the HPV16 E2 gene (a repressor of viral oncogene transcription) (P=0.026) and in HG-SILs that subsequently recurred (P=0.04). Our data are consistent with sequential acquisition of CNIs in cervical SIL progression. Higher frequency of CNI in association with E2 gene loss supports in vitro evidence that high-risk HPV integration is associated with genomic instability. Further investigation of the clinical value of specific host genomic abnormalities in cervical SIL is warranted.

The prevalence of HPV-18 and variants of E6 gene isolated from cervical cancer patients in Taiwan

Cervical cancer is the second most common cancer in women worldwide. It has been considered that human papillomavirus (HPV) is associated with cervical cancer. Currently, more than 80 different serotypes of HPV have been characterized and they are divided into low- and high-risk groups. The most common types that lead to cervical cancer are HPV-16 and -18. The viral oncogenes E6 and E7 are associated with the development of cervical cancer. In previous study, the variants of HPV-16 E6 gene have been reported. It suggests that variants may influence the morbidity of carcinogenesis, but the variant study on HPV-18 remains unknown. To identify the variants of integrated HPV-18 E6 gene in the prevalent infection of HPV-18 of cervical cancer patients. 25 cervical cancer patients were clinically identified and the biopsies were obtained. The infectious HPV types were identified by PCR and Southern blotting analysis. The DNA fragments of the integrated HPV-18 E6 were amplified by PCR and cloned. The nucleotide sequences were obtained by sequencing. The prevalence of HPV infection in our 25 cases was HPV-18 (100%) and 7 out of these 25 cases (28%) were co-infected with HPV-16. The most dominant mutation among 25 tested patients was a silence mutation C183G of the E6 coding region. The prevalent HPV infectious serotype is HPV-18, which differs from the worldwide prevalent type. The identified HPV-18 E6 variants had a unique silence mutation located on C183G in E6 coding region.

Detection of HPV16 E6 gene in cervical tissues by quantitative polymerase chain reaction

To establish a method for detection of Human Papillomavirus (HPV) type16 E6 gene in Cervical carcinomas Specimens. To study the relationship between the quantities of HPV16 E6 (Human papillomavirus type16 E6 gene) in cervical tissues and the course of cervical disease in Xinjiang. HPV16E6 gene and beta-actin was detected in parallel by FQ-PCR (fluorescence quantitative PCR). The number of copies of HPV16 E6 gene and beta-actin was detected in parallel by FQ-PCR (fluorescence quantitative PCR) in tissues of 69 cervical cancer, 65 cervical intraepithelial neoplasia (CIN), 33chronic cervicitis and samples of 96 cervical smear samples of vaginitis and cervicitis. The variation in HPV copies per genomic DNA equivalent can be estimated by dividing the HPV copy number by the beta-actin copy number. The positive rate of HPV16 E6 gene was 83.0%, 75.7%, 93.3% and 3.3% in tissues of cervical cancer, cervical intraepithelial neoplasia (CIN), chronic cervicitis and samples of cervical smear respectively. The amount of HPV16 E6 gene was gradually higher by the developing of the course of cervical disease. They have positive rank correlation, r = 0.83, P < 0.01. The study underscores the importance of the relationship between the HPV16 E6 gene and the course of cervical disease in Xinjiang. It also suggests that the quantification of HPV16 E6 gene may be useful as a prognostic tool to identify women who are at increased risk of developing cervical cancer. This method may be applied to studies of a number of issues related to the natural history of cervical cancer, such as the amounts of HPV in high- and low-grade lesions.

Evidence for an association of human papillomavirus and breast carcinomas.

Human papillomavirus (HPV) DNA has been detected in breast carcinoma by different laboratorial techniques, suggesting the virus could play a role in the pathogenesis of this tumor. The aim of the present study is to investigate the presence of HPV in patients with breast carcinoma and the correlation of the viral infection with prognostic factors for the disease outcome. Between June 2001 and July 2002, 101 paraffin embedded breast carcinoma specimens were analyzed through polymerase chain reaction (PCR) and sequencing of HPV-E6 gene. Twenty specimens of reduction mammoplasty and 21 specimens of fibroadenomas were also studied as a non-malignant control group. Two different specific primer sets targeting E6 region of the HPVs 16 and 18 were used for the analysis. The HPV DNA was detected in 25 breast carcinomas (24.75%), but in none of the benign breast specimens ( p < 0.001). Out of the 25 positive cases, 14 were HPV-16 positive (56%) and 10 were HPV-18 positive (40%). An original finding was the detection of both HPV-16 and -18 in a single tumor (4%). The amplified viral sequences confirmed the presence of HPV-16 and -18. No correlation between the presence of HPV DNA and specific prognostic predictors for the disease outcome was observed. Our results suggest that the presence in the breast of either HPV-16 or -18 might be related to development of the malignant phenotype. Further studies are warranted.

Antitumor efficacy of Venezuelan equine encephalitis virus replicon particles encoding mutated HPV16 E6 and E7 genes

An effective vaccine for treating human papillomavirus (HPV)-associated malignancies such as cervical cancer should elicit strong T cell-mediated immunity (CMI) against the E6 and/or E7 proteins necessary for the malignant state. We have developed Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) vaccines encoding the HPV16 E6 and E7 genes and tested their immunogenicity and antitumor efficacy. The E6 and E7 genes were fused to create one open reading frame and mutated at four or at five amino acid positions to inactivate their oncogenic potential. VRP encoding mutant or wild type E6 and E7 proteins elicited comparable cytotoxic T lymphocyte (CTL) responses to an immunodominant E7 49–57 epitope and generated comparable antitumor responses in several HPV16 E6 +E7 + tumor challenge models: protection from either C3 or TC-1 tumor challenge was observed in 100% of VRP-vaccinated mice. Eradication of C3 tumors was observed in approximately 90% of mice following therapeutic VRP vaccination. Eradication of HLF16 tumors lacking the E7 49–57 epitope was observed in 90% of human leukocyte antigen (HLA)-A ∗0201 transgenic mice following therapeutic VRP vaccination. Finally, the predicted inactivation of E6 and E7 oncogenic potential was confirmed by demonstrating normal levels of both p53 and retinoblastoma proteins in human mammary epithelial cells (MEC) infected with VRP expressing mutant E6 and E7 genes. These promising results support the continued development of mutant E6 and E7 VRP as safe and effective candidates for clinical evaluation against HPV-associated disease.

Loss of p53 induces M-phase retardation following G2 DNA damage checkpoint abrogation

Most cell lines that lack functional p53 protein are arrested in the G2 phase of the cell cycle due to DNA damage. When the G2 checkpoint is abrogated, these cells are forced into mitotic catastrophe. A549 lung adenocarcinoma cells, in which p53 was eliminated with the HPV16 E6 gene, exhibited efficient arrest in the G2 phase when treated with adriamycin. Administration of caffeine to G2-arrested cells induced a drastic change in cell phenotype, the nature of which depended on the status of p53. Flow cytometric and microscopic observations revealed that cells that either contained or lacked p53 resumed their cell cycles and entered mitosis upon caffeine treatment. However, transit to the M phase was slower in p53-negative cells than in p53-positive cells. Consistent with these observations, CDK1 activity was maintained at high levels, along with stable cyclin B1, in p53-negative cells. The addition of butyrolactone I, which is an inhibitor of CDK1 and CDK2, to the p53-negative cells reduced the floating round cell population and induced the disappearance of cyclin B1. These results suggest a relationship between the p53 pathway and the ubiquitin-mediated degradation of mitotic cyclins and possible cross-talk between the G2-DNA damage checkpoint and the mitotic checkpoint.

P53 and p14 increase sensitivity of gastric cells to H. pylori-induced apoptosis.

H. pylori infection of the gastric mucosa is associated with increased epithelial cell apoptosis. In vitro, interferon-gamma and TNF-alpha have been shown to increase the sensitivity of cells to apoptosis induced by H. pylori. The p53 tumor suppressor gene is frequently mutated in many cancers, including gastric cancer. Since p53 protein can induce apoptosis, we sought to determine whether or not p53 increases the ability of gastric epithelial cells to undergo apoptosis in response to H. pylori-induced cell injury. Human gastric epithelial cell lines, AGS (p53 wild-type) cells and AGS cells infected with HPV E6 gene (AGS-E6) to inactivate p53 were exposed to H. pylori. The p53, p21, and p14ARF proteins were measured in gastric epithelial cells by immunoelectrophoresis. Gastric epithelial cell apoptosis was measured by DNA end-labeling assay (TUNEL) and subG0 cell fractions using flow cytometry, and by agarose gel electrophoresis of DNA. Exposure to H. pylori increased the levels of p53, p21, and p14ARF proteins two fold in AGS cells. Gastric AGS cells with fragmented DNA increased from 1.1% to 68% in after exposure to H. pylori for 24 hr. However, AGS-E6 cells were relatively resistant to apoptosis induced by H. pylori (only 15% of cells underwent apoptosis). In additional experiments, mouse embryonic fibroblasts (MEFs) were used to further investigate the role of ARF in stabilizing p53 after exposure to H. pylori. Wild-type and p19ARF-/- MEFs were exposed to H. pylori and evaluated for activation of p53, p19ARF, and apoptosis. As with AGS cells, H. pylori stimulated a 2-fold increase in p53 and p19ARF in wild-type MEFs; however, there was no increase in p53 in ARF-null MEFs. H. pylori easily stimulated apoptosis in wild-type MEFs, although, the absence of p19ARF significantly reduced the ability of H. pylori to induce apoptosis in these cells. Activation of ARF by H. pylori is important in stabilizing p53 resulting in increased apoptosis. Thus, inactivation of either ARF or p53 in gastric cells may reduce their ability to undergo apoptosis in response to injury induced by H. pylori.

Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference.

Selective silencing of mammalian gene expression has recently been achieved using short interfering RNA (siRNA). Synthetic siRNA targets homologous mRNA for degradation and the process is highly efficient. Here we demonstrate siRNA silencing of pathogenic viral gene expression. As a well characterized model we chose cervical carcinoma cells positive for human papillomavirus type 16. Over 90% of human cervical cancers are positive for papillomavirus and abnormal cell proliferation is driven by co-operative effects of viral E6 and E7 genes. We sought to silence HPV E6 and E7 gene expression using siRNAs to target the respective viral mRNAs. Our results indicate selective degradation of E6 and E7 mRNAs. Silencing was sustained for at least 4 days following a single dose of siRNA. E6 silencing induced accumulation of cellular p53 protein, transactivation of the cell cycle control p21 gene and reduced cell growth. In contrast, E7 silencing induced apoptotic cell death. HPV-negative cells appeared unaffected by the anti-viral siRNAs. Thus we demonstrate for the first time (i) that siRNA can induce selective silencing of exogenous viral genes in mammalian cells, and (ii) that the process of siRNA interference does not interfere with the recovery of cellular regulatory systems previously inhibited by viral gene expression.

Protection against human papillomavirus type 16‐E7 oncogene‐induced tumorigenesis by in vivo expression of dominant‐negative c‐jun†

Expression of the human papillomavirus (HPV) type 16 E6 and E7 gene products is a risk factor for human cervical carcinogenesis as well as skin and oral carcinogenesis. Expression of the HPV-16 E7 gene in mouse skin induces hyperplasia and enhances tumor promotion. Expression of dominant-negative c-jun (TAM67) in the mouse skin protects mice from 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced papillomagenesis without blocking mitogen-induced hyperproliferation. To determine the role of activator protein-1 (AP-1) in HPV-induced cancer, we crossed HPV-16 E7 mice with TAM67 mice and analyzed the effects of DMBA/TPA on tumor promotion. We showed that expression of TAM67 protected mice from HPV-16 E7-enhanced tumorigenesis, suggesting AP-1 as a target for prevention of HPV-induced cancer.

Sodium arsenite suppresses human papillomavirus-16 E6 gene and enhances apoptosis in E6-transfected human lymphoblastoid cells

The p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV) in most cervical cancer cells. The E6 proteins, which could mediate p53 degradation, are related to cellular immortalization, transformation, and tumor formation. In order to study the E6 abrogated p53 function in stress, we transfected HPV-16 E6 gene to TK6 cells in this study. Here we showed that HPV-16 E6 mRNA levels decreased in a dose dependent manner after sodium arsenite (SA) treatment, but not after X-irradiation. P53, p21, and MDM2 were induced in E6-transfected TK6 cells, as well as in parental TK6 cells after arsenite treatment. But the above proteins were only induced in TK6 cells after X-irradiation. It indicated that arsenite, but not X-ray, could suppress the transcription of E6 gene and therefore activate the p53 tumor suppressor pathway in TK6-E6 cells. After arsenite treatment, TK6-E6 cells showed more sub-G1 apoptosis, activated caspase-3/CPP32 fragment, DNA ladder, and less viability than parental TK6 cells, indicating that arsenite enhanced apoptosis in E6-transfected TK6 cells. In contrast, after X-irradiation, TK6-E6 cells showed less sub-G1 apoptosis and higher viability than parental TK6 cells. Thus, it would be another possible strategy to promote arsenite as another potential candidate for the therapeutic purpose in HPV-positive cancer cells. J. Cell. Biochem. 84: 615–624, 2002. © 2001 Wiley-Liss, Inc.

Sodium arsenite suppresses human papillomavirus‐16 E6 gene and enhances apoptosis in E6‐transfected human lymphoblastoid cells

The p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV) in most cervical cancer cells. The E6 proteins, which could mediate p53 degradation, are related to cellular immortalization, transformation, and tumor formation. In order to study the E6 abrogated p53 function in stress, we transfected HPV-16 E6 gene to TK6 cells in this study. Here we showed that HPV-16 E6 mRNA levels decreased in a dose dependent manner after sodium arsenite (SA) treatment, but not after X-irradiation. P53, p21, and MDM2 were induced in E6-transfected TK6 cells, as well as in parental TK6 cells after arsenite treatment. But the above proteins were only induced in TK6 cells after X-irradiation. It indicated that arsenite, but not X-ray, could suppress the transcription of E6 gene and therefore activate the p53 tumor suppressor pathway in TK6-E6 cells. After arsenite treatment, TK6-E6 cells showed more sub-G1 apoptosis, activated caspase-3/CPP32 fragment, DNA ladder, and less viability than parental TK6 cells, indicating that arsenite enhanced apoptosis in E6-transfected TK6 cells. In contrast, after X-irradiation, TK6-E6 cells showed less sub-G1 apoptosis and higher viability than parental TK6 cells. Thus, it would be another possible strategy to promote arsenite as another potential candidate for the therapeutic purpose in HPV-positive cancer cells.

HPV16 E6 gene variations in invasive cervical squamous cell carcinoma and cancer in situ from Russian patients.

HPV16 is frequently seen in invasive cervical cancer (ICC) and cervical intraepithelial neoplasia (CIN). Its E6 gene has frequent sequence variations. Although some E6 variants have been reported to have different biochemical or biological properties, they do not show geographical identity. Moreover, the definition of ‘variant’ has been a source of confusion because it has been based on all departures from the ‘prototype’ once isolated randomly from an ICC case. We amplified the HPV16 E6 gene by PCR from fresh-frozen tissue of 104 cases of ICC and CIN from Russian patients and sequenced it in positive cases. We found that 32 of 55 (58.2%) ICC cases and 18 of 49 (36.7%) CIN cases were HPV 16-positive and we could identify 3 groups of E6 variants: group A was characterized by G at nt 350 where group B had T, and group M was a heterogeneous mixture of unique E6 variants; no significant difference existed in the distribution of the different groups between ICC and CIN; the clinically malignant (as defined by FIGO stage) order between the groups was M > A > B in ICC; in the cases with a single HPV16 E6 sequence, coexisting ICC, CIN and normal epithelium in the same patient shared the E6 variant; and 4 cases of ICC had double/multiple E6 variants. The results did not show any importance of E6 variants for ICC progression in Russian women. The results also indicated that the original HPV16 variant persisted during ICC progression, and that at a low frequency, double infections and/or mutation of variants might occur. © 2001 Cancer Research Campaign http://www.bjcancer.com

Modified HPV16 E7 Genes as DNA Vaccine against E7-Containing Oncogenic Cells

Therapeutic vaccines against tumors associated with human papillomaviruses (HPV) should elicit cellular immune responses against early HPV antigens, primarily the oncoproteins E7 and E6. Because of safety concerns, the direct use of an unmodified oncogene is impossible in human DNA vaccination. Therefore, we introduced three point mutations into the pRb-binding site of HPV16 E7 oncogene to eliminate its transformation potential. The resultant gene was denoted E7GGG. The rates of expression and the cellular localization of E7 and E7GGG proteins were comparable. In immunization-challenge experiments, the efficacy of plasmids containing the E7, E7GGG, or fusion genes of HPV16 E7, viz. L1ΔCE71–60 (M. Muller et al., 1997, Virology 234, 93–111), and Sig/E7/LAMP-1 (T. C. Wu et al., 1995, Proc. Natl. Acad. Sci. USA 92, 11671–11675), was compared. While tumors developed in all animals immunized with the wild-type E7 gene, a significant proportion of mice remained tumor-free after vaccination with the E7GGG gene. The fusion gene L1ΔCE71–60 induced negligible protection, but Sig/E7/LAMP-1 conferred the highest protection. Intradermal immunization by gene gun proved superior to i.m. inoculation. In “therapeutic” experiments, a 1-day delay between inoculation of oncogenic cells and the start of DNA immunization resulted in partial therapeutic effect, but a 3-day delay produced a substantially lower immunization effect. A combination of Sig/E7/LAMP-1 and E7GGG genes did not enhance the immune response. These results demonstrate a significant enhancement of HPV16 E7 immunogenicity after mutagenesis of the pRb-binding site, but the mutated E7 gene did not excel the Sig/E7/LAMP-1 fusion gene.

Mammary gland tumor in transgenic mice expressing targeted beta-casein/HPV16E6 fusion gene.

The human papillomaviruses (HPV)-16 and HPV-18 referred to as high-risk HPVs are strongly associated with anogenital malignancies as well as benign epithelial cysts. It has been demonstrated that transgenic mice carrying HPV-16 E6-E7 under the control of the MMTV LTR developed malignant tumors including salivary gland carcinoma, lymphoma, skin histiocytomas and testicular tumors in a non-mammary gland specific manner. Another regulatory unit of rat beta-casein gene can confer the expression of fusion gene preferentially in the mammary glands of transgenic mice in a developmentally regulated manner. In order to generate mammary tumor formation in transgenic mice directing HPV16E6 gene alone into the mammary gland, this regulatory unit was fused to the E6 gene of HPV-16 type to constructing fusion gene. By screening 51 newborn founder transgenic mice, three mice carrying transgenes were identified. One line termed TG32 developed in a mammary gland tumor with large subcutaneous mass in the left rib region at 17 months of age. The levels of E6 transcript in the mass-tumor of TG32 line were lower than those in non-tumor mammary gland of identical TG32 and of TG250. In each tissue of TG32 line, high expression of E6 transcript was detected both in the mammary gland and brain. Histological analysis showed that cells from mammary gland tumor of the TG32 line had also hyperplasia appearance, with irregular or increased total number of mitotic rate. These observations suggest that developing phenotype and the level of E6 transcripts in the process of malignant transformation may have different mechanisms involving the capacity to bind and destabilize p53, although for confirmation it is necessary to investigate many more transgenic mice.

Retrovirus-Mediated Delivery of HPV16 E7 Antisense RNA Inhibited Tumorigenicity of CaSki Cells

Objective. In cervical cancer, high-risk human papillomavirus (HPV) genes are expressed solely in cancerous cells and have been proposed to be the most important etiological factors for cervical cancer, thus making them suitable targets for gene therapy. In this study, we aim to inactivate the HPV16 E7 in CaSki cells and test the possibility of reducing the tumorigenicity of these cells.Methods. The full-length HPV16 E7 cDNA was cloned in the pBabe-puro or pWZL-Hygro retrovirus vector in reverse orientation and was stably transfected into CaSki cells by replication-defective retrovirus infection giving rise to CaSki-E7AS and CaSki-E7AS2X cells. Immunoprecipitation/Western analysis and real-time RT-PCR were performed to document the levels of HPV16 E7 gene product. Flow cytometry was performed to study changes in the cell cycle in response to reduced E7 protein. The expression of bcl-2, RB, and E2F-1 was studied using Western blot analysis. Tumorigenicity of CaSki, CaSki-E7AS, and CaSki-E7AS2X cells was assayed with subepidermal tumor growth in nude mice.Results. We have documented that the delivery of the antisense gene construct resulted in the reduction of HPV16 E7 protein expression and cell proliferation in CaSki cells. Furthermore, we demonstrated that these changes were accompanied by cell cycle arrest, up-regulation of RB, and down-regulation of E2F-1 and bcl-2 proteins. More importantly, dose-dependent transduction of the antisense HPV16E7 construct was able to inhibit and/or retard the tumorigenicity of CaSki cells in vivo.Conclusions. Down-regulation of HPV16 E7 with antisense RNA is beneficial in reducing the tumorigenicity of CaSki cells and can potentially be useful for HPV-associated malignancy gene therapy.

HPV-16 E2 gene disruption and sequence variation in CIN 3 lesions and invasive squamous cell carcinomas of the cervix: relation to numerical chromosome abnormalities

Aim—To test the hypothesis that, because the human papillomavirus (HPV) E2 protein represses viral early gene transcription, E2 gene sequence variation or disruption could play a part in the induction...

Uneven distribution of HPV 16 E6 prototype and variant (L83V) oncoprotein in cervical neoplastic lesions.

A previous Swedish study revealed that both prototype and variant HPV16 E6 oncoprotein, occur in about equal numbers in high-grade cervical intraepithelial neoplasia (HCIN), whereas variant HPV16 predominates in invasive cervical squamous carcinoma. Most of the malignant HPV16 variants contain a common mutation, L83V, in the E6 oncoprotein. In the present investigation, 28 HPV16 positive, invasive cervical adenocarcinomas were collected from a total number of 131 adenocarcinomas. These HPV16-positive cases were evaluated with analysis of the E6 gene, using a recently described PCR-SSCP method for identification of the specific mutation (L83V) in the E6 gene. The results obtained were correlated to findings in 103 preinvasive, HCIN, and 31 invasive cervical squamous carcinomas also infected with HPV16. The HPV16 E6 variant L83V was present in 40% of the HCIN lesions, in 54% of the invasive adenocarcinomas, in comparison to 81% of the invasive squamous carcinomas. The difference between HCIN and squamous carcinomas was statistically significant, P< 0.001, whereas the difference between HCIN and invasive adenocarcinomas was not statistically significant, P = 0.604. Prototype HPV16 and its E6 variant L83V are both prevalent in preinvasive and invasive cervical lesions in Swedish women. However, the obvious predominance of HPV16 variant in squamous carcinomas was not seen in adenocarcinomas. A single amino-acid shift in the HPV16 E6 gene appears to result in a different transforming potential in squamous and glandular cervical lesions. © 2000 Cancer Research Campaign

Human papillomavirus type 16 E7 impairs the activation of the interferon regulatory factor-1.

We had previously observed that HPV-16 E7 disturbs the Guanylate Binding Protein (GBP)-ISRE reporter activation by IFN-gamma thus suggesting an alteration of the IRF-1 function. In this study we examined the mechanism by which E7 affects the IFN-gamma signals driving the activation of gene transcription. Using 14/2 BRK cells containing dexamethasone-inducible HPV-16 E7 gene, we observed a large inhibition of the IRF-1 DNA binding activity upon E7 induction. Concomitantly, there was no significant change in the levels of IRF-1, indicating that this was not due to reduced levels of IRF-1 expression. Likewise, in vitro translated E7 did not affect the IRF-1 DNA binding activity in nuclear extracts derived from IFN-induced cells, thus indicating that the effects of E7 are upstream of IRF-1's binding to its DNA recognition site. Finally, NFkappaB DNA binding activity was also inhibited under conditional expression of E7. These data indicate that HPV-16 E7 inhibits the IRF-1 and NFkappaB function and this could lead to the impairment of the IFN response in HPV-infected cells. Furthermore, the findings suggest that different events of the IFN inducible signal cascade seem to be target for HPV-16 E7.

Human Papillomavirus Types 16 E6 and E7 Contribute Differently to Carcinogenesis

High-risk human papillomaviruses (HPVs) are etiologically implicated in human cervical cancer. Two viral genes, E6 and E7, are commonly found expressed in these cancer cells. We have previously shown that mice transgenic for the HPV-16 E6 gene or E7 gene, in which the E6 or E7 was expressed in the basal layer of epithelia, developed skin tumors. The spectrum of tumors derived from E6 and E7 mice differed, however; although most tumors derived from the E7-transgenic mice were benign, the majority of the tumors from the E6-transgenic mice were malignant. These findings led us to hypothesize that E6 and E7 play different roles in carcinogenesis. To assess at what stages in carcinogenesis E6 and E7 act, we treated the skin of K14E6- and K14E7-transgenic mice with chemical carcinogens known to contribute to distinct stages in carcinogenesis. Both E6 and E7 were found to synergize with chemical carcinogens in causing tumor formation. E6 was found to act weakly at the promotion stage of carcinogenesis in the formation of benign tumors but strongly at the progression stage which involves the malignant conversion of benign tumors. In contrast, E7 primarily affected the promotion stage of carcinogenesis. These results provide direct evidence that E6 and E7 contribute differently to carcinogenesis; E7 promotes the formation of benign tumors, and E6 acts primarily to accelerate progression of these benign tumors to the malignant stage. Consistent with this model, we found E6 and E7 to cooperate in inducing tumor formation in mice expressing both oncogenes.