Persistence of human papillomavirus as a predictor for treatment failure after loop electrosurgical excision procedure.

We aimed to investigate whether postconization human papillomavirus (HPV) DNA testing can predict treatment failure and improve the accuracy of conventional follow-up in women with high-grade cervical intraepithelial neoplasia (CIN). Between March 2001 and October 2005, 120 patients with confirmed CIN 2 or 3 were treated with loop electrosurgical excision procedure (LEEP) and were enrolled. Six patients were lost to the follow-up. Postconization follow-up was performed at every 3-6 months during the first year and then annually. Specimens were tested for the presence of HPV, using the Hybrid Capture 2 (Digene Co, Gaithersburg, MD) and HPV DNA chip (Mygene Co, Seoul, Korea) test. Persistent HPV infection was defined as persistently (two times or more) positive HPV tests with the same HPV subtype(s) at initial diagnosis. Twenty-two (19.3%) patients showed treatment failure after conization. The only significant risk factor for redevelopment of CIN after conization was persistence of the same HPV subtype (P < 0.0001). And women with recurrent or residual CIN had higher HPV load during the 6-month follow-up postconization. In conclusion, the persistence of the same HPV subtype after LEEP conization was an important predictor of treatment failure. The follow-up protocol after conization of CIN should include both cervical cytology and HPV test, and HPV DNA chip test is needed to detect a persistent HPV infection.

Human papillomavirus genotype as a prognostic factor in carcinoma of the uterine cervix.

The clinical implications of specific human papillomavirus (HPV) types in invasive cervical carcinomas are only now beginning to be appreciated. The objective of this study was to determine the clinical implications and prognostic value of the HPV genotype in cervical carcinomas. In this study, we employed an HPV DNA chip to detect the type-specific sequence of HPV from cervical swabs taken from women with biopsy-proven neoplastic lesions of the cervix. We divided the patients into four groups: HPV-negative, HPV-16-related, HPV-18-related, and intermediate risk type-related. Associations with clinicopathologic data (stage, histologic type, lymph node status, parametrial invasion, lymphvascular space invasion, tumor size, vaginal involvement) and overall survival were assessed. HPV DNA was detected in 81.4% of the patients, and 19.0% harbored multiple HPV variants. HPV-16-related was the predominant type and was detected in 47.4% (46/97) of the patients. The HPV-16-related types were detected more frequently in patients with squamous cell carcinomas, whereas the HPV-18-related types were more prevalent in cases of adenocarcinomas and adenosquamous carcinomas (P < 0.05). Otherwise, no significant correlations were detected between the HPV genotype and any other clinicopathologic parameters. After a median follow-up of 30 months, the 5-year survival rate was lower in the HPV-18-related patients, but this difference was not found to be statistically significant, according to the results of the log-rank test. We conclude that neither the presence nor type of HPV DNA bears any prognostic significance in cases of cervical carcinoma.

Distinctive cell cycle regulatory protein profiles by adenovirus delivery of p53 in human papillomavirus-associated cancer cells.

In this study, microarray analyses were performed to determine the time course of gene expression profiles in SiHa cells after infection with an adenovirus-expressing p53 (Adp53). We then investigated the consequences of Adp53 gene transfer on the expression level of six genes associated with cell cycle control and on apoptosis and cell cycle arrest in SiHa cells and compared these results with those from CaSki and HeLa cells. Gene expression profiling of the p53-targeted genes in SiHa cells revealed that p21, p53, and mdm2 protein expression was significantly upregulated at 24 and 48 h. Western blot results revealed that p21 and p53 expression levels had significantly increased after Adp53 infection. Cyclin-dependent kinase 4 levels were decreased 48 h after treatment in SiHa and CaSki cells. Proliferating cell nuclear antigen levels were unchanged after Adp53 infection. Only SiHa cells exhibited significant cell death. Cell cycle arrest at the G1 phase was induced in the SiHa and HeLa cells but was not induced at the G2/M and S phases in the CaSki cells. These data support the notion that the understanding of p53-dependent apoptosis and cell growth arrest could be applicable to advanced strategies in the development of preferential tumor cell-specific delivery.

Development of anticancer gene vaccine interact with human papillomavirus oncoprotein inhibition.

Adeno-associated virus (AAV) Rep 78 protein is known to inhibit the promoter site of several oncogenes and viral genes, including the human papillomavirus (HPV) type 16 E6 transforming genes. The biochemical studies of Rep 78 have been reported, but the effects of Rep 78 gene-mediated inhibition of HPV 16 E6 promoter activity on the various human cervical carcinoma cells have not been characterized. pEGFP-N1 vector, cloned by AAV-mediated Rep 78, is transfected into cervical carcinoma cells. Transfection efficiency of Rep 78 was approximately 30-60% different. Messenger RNA (mRNA) and protein expression of Rep 78 gene was significantly higher on day 1 of the transfection of Rep 78 DNA in CaSki cells, and DNA level of HPV 16 E6 was decreased on day 1 of the transfection. The growth of CaSki cervical cancer cells was only 10-15% inhibited by Rep 78, and the other cervical cells, HeLa, HeLaS3, HT3, and QGU, were unaffected by Rep 78 transfection. In spite of the high efficiency of Rep 78 gene transformation and expression rate, we could not show the significant growth inhibition in various cervical cancer cell lines. Taken together, long-term expression of Rep 78 strategy might be needed for cervical carcinoma gene therapy using AAV vector.

Cellular process classification of human papillomavirus-16-positive SiHa cervical carcinoma cell using Gene Ontology.

This study utilized mRNA differential display and the Gene Ontology (GO) analysis to characterize the multiple interactions of a number of genes involved in human papillomavirus (HPV)-16-induced cervical carcinogenesis. We used HPV-16-positive cervical cancer cell line (SiHa) and normal human keratinocyte cell line (HaCaT) as a control. Each gene has several biological functions in the GO, and hence, we chosen the several functions for each gene. and then, the specific functions were correlated with gene expression patterns. The results showed that 157 genes were up- or down-regulated above two-fold and organized into mutually dependent subfunction sets depending on the cervical cancer pathway, suggesting the potentially significant genes of unknown function. The GO analysis suggested that cervical cancer cells underwent repression of cancer-specific cell-adhesive properties. Also, genes belonging to DNA metabolism such as DNA repair and replication were strongly down-regulated, whereas significant increases were shown in protein degradation and in protein synthesis. The GO analysis can overcome the complexity of the gene expression profile of the HPV-16-associated pathway and identify several cancer-specific cellular processes as well as genes of unknown function. Also, it can become a major competing platform for the genome-wide characterization of carcinogenesis.

Natural killer cell activity and quality of life were improved by consumption of a mushroom extract, Agaricus blazei Murill Kyowa, in gynecological cancer patients undergoing chemotherapy.

A mushroom extract, Agaricus blazei Murill Kyowa (ABMK), has been reported to possess antimutagenic and antitumor effects. Here, we investigate the beneficial effects of ABMK consumption on immunological status and qualities of life in cancer patients undergoing chemotherapy. One hundred cervical, ovarian, and endometrial cancer patients were treated either with carboplatin (300 mg / m(2)) plus VP16 (etoposide, 100 mg / m(2)) or with carboplatin (300 mg / m(2)) plus taxol (175 mg / m(2)) every 3 weeks for at least three cycles with or without oral consumption of ABMK. We observed that natural killer cell activity was significantly higher in ABMK-treated group (ANOVA, n = 39, P < 0.002) as compared with nontreated placebo group (n = 61). However, no significant difference in lymphokine-activated killer and monocyte activities was observed in a manner similar to the count of specific immune cell populations between ABMK-treated and nontreated groups. However, chemotherapy-associated side effects such as appetite, alopecia, emotional stability, and general weakness were all improved by ABMK treatment. Taken together, this suggests that ABMK treatment might be beneficial for gynecological cancer patients undergoing chemotherapy.

Necrosis-like death with plasma membrane damage against cervical cancer cells by photodynamic therapy.

In order to elucidate the antitumor effect of photodynamic therapy (PDT) using the photosensitizing agent hematoporphyrin derivative (Photogem) and a diode laser, we evaluated the cell death of uterine cancer cell lines (CaSki, HT3, HeLa, and SKOV-3) and mice transplanted with TC-1 lung cancer cells. Morphological changes, MTT assay, flow cytometry, cytotoxicity, and tumor growth-inhibition study were evaluated at various time intervals after PDT. The results showed that the survival rates of each cell line decreased with time and dose-response after performing PDT. Also, PDT-induced damage of cancer cells was almost entirely confined to necrosis of the tumor cells in the early time courses. The irradiation of CaSki cells in the presence of Photogem induced plasma membrane disruption and cell shrinkage, indicating the plasma membrane as the main target for Photogem. In the experiment in vivo, the time courses of Photogem with irradiation showed significantly longer survival and a significantly smaller tumor size compared to those in the untreated control groups, and resorption of the tumor after PDT treatment was observed. Collectively, our results indicated that Photogem possesses tumor-specific affinity, and necrosis-like death with plasma membrane damage was postulated to be the principal mechanism of the antitumor effect of PDT using Photogem.

Anti-cancer effect of adenovirus p53 on human cervical cancer cell growth in vitro and in vivo.

To evaluate anti-tumor effects of recombinant adenovirus p53, time-course p53, E6 expression, and cell growth inhibition were investigated in vitro and in vivo using cervical cancer cell lines such as CaSki, SiHa, HeLa, HeLaS3, C33A, and HT3. The cell growth inhibition was studied via cell count assay, MTT assay and neutral red assay. After transfecting AdCMVp53 into SiHa cells-xenografted nude mice, the transduction efficiency and anti-tumor effect were investigated for a month. The results showed that adenoviral p53 expression induced significant growth suppression on the cancer cells, in which E6 transcript was strongly repressed, and that the expression of p53 and E6 were remarkably dependent on each cell type. The transduction efficiency was highly maintained in vivo as well as in vitro, and the size of tumor was remarkably decreased in comparison with AdCMVLacZ control. The results suggest that the adenovirus-mediated p53 gene transfection was done very effectively in vitro and in vivo experiment, and the cell growth was suppressed via p53-dependent apoptotic cell death, and that the anti-tumor effect could be related to E6 and p53 expression pattern.

Gene-gene and gene-environmental interactions of p53, p21, and IRF-1 polymorphisms in Korean women with cervix cancer.

BACKGROUND: The aim of this study was to identify gene-gene and gene-environmental factors affecting cervix carcinogenesis in Korean women. METHODS: We evaluated 530 subjects composed of 185 female cervix cancer patients and 345 normal healthy women. The single nucleotide polymorphisms (SNPs) of p53 codon 72, p21 codon 31, and interferon regulatory factor-1 (IRF-1) intron 6 were evaluated from extracted DNA of peripheral blood with an automatic DNA sequencer. The differences of each SNP, gene-gene and gene-environmental interactions between normal controls and patients were evaluated in the adjusted environmental background. RESULTS: In the environmental aspect, the rate of cervix cancer increased in the women with a lower level of education, a younger age at first sexual intercourse and more childbearing. The women who had p53 (Arg/Arg), IRF-1 (T/T), and <6 years of education showed a 14.7-fold increased risk of cervix cancer compared to the women who had p53 ( approximately Pro), IRF-1 ( approximately C), and >15 years of education. The women who had p53 (Arg/Arg), p21 (Ser/Ser), and >3 children showed a 6.4-fold increased risk of cervix cancer compared to the women who had p53 ( approximately Pro), p21 ( approximately Arg), and no children. The women who had p53 (Arg/Arg), IRF-1 (T/T), and first sexual intercourse before 22 years old showed a 5.5-fold increased risk of cervix cancer compared to the women who had p53 ( approximately Pro), IRF-1 ( approximately C), and first sexual intercourse after 26 years old. CONCLUSIONS: We found that the level of education, the age at first intercourse, and the number of children were independent risk factors in cervix carcinogenesis. The specific combinations of p53, p21, and IRF-1 gene-gene and gene-environmental interactions were significantly noted in the cervix carcinogenesis of Korean women.

The effect of codon 98 of the FHIT gene on cervical cancer in Korean women.

The Fragile Histadine Triad (FHIT) is a putative tumor suppressor gene involved in different tumors. The objective of this study was to examine the effect of codon 98 of FHIT on cervical carcinogenesis. The study subjects were patients who were pathologically diagnosed with cervical neoplasia and who had a positive result for human papillomavirus (n = 567) compared to normal healthy women as normal controls (n = 506). The FHIT-specific sequences of DNA from peripheral blood samples from study subjects were determined by PCR using allele-specific primers and were compared with those of the controls. The genetic susceptibility of codon 98 of the FHIT gene (3p14.2) in cervical carcinogenesis was determined by examining the effect of the gene and environmental factors vs. the different stages of cervical intraepithelial lesions and the different histopathologic types of invasive cervical cancers. On assessing FHIT polymorphisms, the percentages of individuals homozygous for the T allele, homozygous for the C allele, and heterozygous for these two alleles were 42.1%, 11.3, and 46.6% in the control group. The corresponding figures were 39.5%, 14.8%, and 45.7% among in women with cervical cancer. Compared with FHIT T/ T, odds ratio (95% confidence interval) for FHIT C/C was 1.4 (0.8-2.5) for invasive cervical cancer and 1.7 (0.9-3.1) for cervical intraepithelial neoplasia (CIN) II or III. The risks for invasive cervical cancer were higher with early onset cervical carcinogenesis (2.3, 1.0-5.5, P = 0.0438), than with late onset (1.0, 0.5-2.1, P = 0.9306). The risks of FHIT C/C or C/ T also increased for ever smokers or women with two or more children compared with FHIT T/ T. Polymorphisms of FHIT are associated with a higher risk of developing cervical cancer, in particular early onset cervical carcinogenesis.

Protective effects of green tea extracts (polyphenon E and EGCG) on human cervical lesions.

We investigated clinical efficacy of green tea extracts (polyphenon E; poly E and (-)-epigallocatechin-3-gallate [EGCG]) delivered in a form of ointment or capsule in patients with human papilloma virus (HPV) infected cervical lesions. Fifty-one patients with cervical lesions (chronic cervicitis, mild dysplasia, moderate dysplasia and severe dysplasia) were divided into four groups, as compared with 39 untreated patients as a control. Poly E ointment was applied locally to 27 patients twice a week. For oral delivery, a 200 mg of poly E or EGCG capsule was taken orally every day for eight to 12 weeks. In the study, 20 out of 27 patients (74%) under poly E ointment therapy showed a response. Six out of eight patients under poly E ointment plus poly E capsule therapy (75%) showed a response, and three out of six patients (50%) under poly E capsule therapy showed a response. Six out of 10 patients (60%) under EGCG capsule therapy showed a response. Overall, a 69% response rate (35/51) was noted for treatment with green tea extracts, as compared with a 10% response rate (4/39) in untreated controls (P<0.05). Thus, the data collected here demonstrated that green tea extracts in a form of ointment and capsule are effective for treating cervical lesions, suggesting that green tea extracts can be a potential therapy regimen for patients with HPV infected cervical lesions.

Protein kinase C modulates telomerase activity in human cervical cancer cells.

Telomerase, a ribonucleoprotein reverse transcriptase that extends telomeres of eukaryotic chromosomes is repressed in normal somatic cells but is activated during development and neoplasia. The regulation mechanism of telomerase activity in cancer cells is not clearly known. In this report, a possible affect of PKC on telomerase activity was examined using HeLa and CUMC-6 cervical cancer cell lines. Exposure of cells to PKC inhibitor, bisindolylmaleimide I and Gö6976, and high levels of PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA) resulted in the inhibition of PKC activity in both cells. Telomerase activities were also inhibited by bisindolyl-maleimide I and Gö6976, respectively, in a time-dependent manner. As PKC activity changes in TPA-treated cervical cancer cells, telomerase activities were increased at low dose of TPA and decreased at high dose. The expression levels of human telomerase subunits, human telomerase RNA (hTR) were not influenced by PKC modulating drugs. In contrast, the expression of full-length human telomerase reverse transcriptase (hTERT) was decreased after exposure to bisindolylmaleimide I and Gö6976 in a time-dependent manner. hTERT expression was not affected by low dose of TPA. In contrast, high dose of TPA inhibited hTERT expression level. But the expression patterns of beta-deletion transcript of hTERT after 72 h of treatment with PKC inhibitors or high dose of TPA exposure were not discernable as compared with those of full-length hTERT transcripts to PKC modulating drugs. These results suggest that PKC-modulating drugs altered telomerase activities by affecting full-length hTERT expression profile in human cervical cancers.

Functional inactivation of p73, a homolog of p53 tumor suppressor protein, by human papillomavirus E6 proteins.

Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 binds to and inactivates p53 tumor suppressor protein by ubiquitin/proteasome-dependent degradation. Recently, p73, a novel family of p53, has been identified and demonstrated, like p53, to activate p21(WAF1). Here we show that p73 is also inactivated by HPV-E6, but ubiquitin-mediated proteolysis is not responsive. Yeast two-hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro, respectively. The transactivation domain (amino acid residues 1 to 49) is found to be absolutely required for the interaction. Transient co-expression of E6 significantly inhibits the p73-mdiated activation of p21(WAF1) promoter in a p53-defective C33A cell line. Using Gal4-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by a direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. Co-transfection of E6 mutants reveals that the same portion of E6 appears to be responsible for the inactivation of p53 and p73 function. However, the inactivation mechanism of p73 is clearly different from that of p53, because p73, unlike p53, is inactivated by both high- and low-risk E6s and is not susceptible to E6-dependent proteolysis. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV-E6 may, at least in part, contribute to E6-mediated transformation and hyperproliferation of cervical cells.

Growth Suppression of Ovarian Cancer Cells by Interferon-gama.

PURPOSE: Growth regulation of cancer cells very frequently involves tumor suppressor gene p53, Rb and cell cycle regulator, however the molecular biologic mechanisms of growth regulation in ovarian carcinoma cells are not fully defined. To assess the mechanism of growth suppression, we treated IFN-gama in ovarian carcinoma cells. MATERIALS AND METHODS: Growth suppression by treatment of IFN-gama was determined by cell proliferation assay in ovarian carcinoma cell lines. Apoptosis was determined by DNA fragmentation assay and electron microscopy. Molecular mechanism of the apoptosis in ovarian carcinoma cell by IFN-gama was further analyzed by the western blot. RESULTS: We found that IFN-gama had remarkable growth- suppressive effects in PA-1 and A2774 ovarian carcinoma cells in a time-dependent manner. Apoptosis was observed in PA-1 and A2774 cell following treatment of IFN- gama by DNA fragmentation assay and EM. The expression of IRF-1 protein from A2774 and PA-1 cell extracts was elevated by increasing the concentration of IFN-gama. IFN-gama caused an increased expression of the important apoptosis-related gene, ICE (interleukin-1beta-converting enzyme) protein in A2774 and PA-1. CONCLUSION: The coordinate induction of IRF-1 and ICE by IFN-gama in ovarian carcinoma cells suggests a functional relationship between these proteins in programmed cell death. The significance of this study is the molecular biologic background of IFN-gama considered as an alternative treatment trial of ovarian cancers.

Combined analysis of germline polymorphisms of p53, GSTM1, GSTT1, CYP1A1, and CYP2E1: relation to the incidence rate of cervical carcinoma.

BACKGROUND: The authors established the genotype frequencies of cytochrome P450 (CYP1A1/MspI, CYP2E1/PstI, and CYP2E1/DraI), glutathione-S-transferase (GSTM1 and GSTT1), and p53 (exon 4/AcclI and intron 3/16-base pair duplication) gene polymorphisms in cervical carcinoma patients and controls and evaluated the association between the specific genotype or genotype combinations of these polymorphisms and the risk of cervical carcinoma. METHODS: In this case-control study, the genotypes of 181 human papillomavirus (HPV)-16 or HPV-18 positive cervical carcinoma patients and 1-to-1 age-matched controls were determined using a polymerase chain reaction-based technique. RESULTS: Among these polymorphisms, the individuals carrying arginine/proline genotypes of p53 showed a 9.5-fold increase of cervical carcinoma risk (95% confidence interval [CI], 4.9-18.6) compared with those individuals carrying arginine/arginine genotypes. The frequency of overall GSTT1 null genotypes also was significantly higher in cervical carcinoma patients compared with that of GSTT1 positive genotypes (P = 0.003; odds ratio [OR] = 1.9; 95% CI, 1.2-2.9). The genotype combination of p53 and GST played a more important role in describing the relative risk of cervical carcinoma. The individuals carrying both the arginine/proline genotype of p53 and the null genotype of GSTT1 showed a 3.5-fold increase of cervical carcinoma risk (95% CI, 1.8-7.1) compared with those individuals carrying both the arginine/arginine genotype of p53 and the GSTT1 positive genotype. In the patients who were stratified into the two age groups, the null genotypes of GSTT1 (69.1% vs. 45.5%; P = 0.016) and GSTM1 (61.8% vs. 40.0%; P = 0.028) in cervical carcinoma were significantly overrepresented in the younger age subgroup (age 40 years or younger) compared with those of controls. Especially in this age group, the individuals carrying both null genotypes of GSTT1 and GSTM1 showed a 17.8-fold increase of cervical carcinoma risk (95% CI, 2.2-141.0) compared with the individuals carrying both positive genotypes of GSTT1 and GSTM1. CONCLUSIONS: The results of the current study suggested that the arginine/proline genotype of p53, independently or in conjunction with the GSTT1 null genotype, could affect the genetic susceptibility for cervical carcinoma, and HPV positive women carrying both null genotypes of GSTT1 and GSTM1 have an increased risk of cervical carcinoma developing before age 40 years.

AAC-11 overexpression induces invasion and protects cervical cancer cells from apoptosis.

To identify the genes involved in cervical carcinogenesis, we applied the mRNA differential display (DD) method to analyze normal cervical tissue, cervical cancer, metastatic lymph node, and cervical cancer cell line. We cloned a 491-bp cDNA fragment, CC231, which was present in metastatic tissue and cervical cancer cell line, but absent in normal cervical and cervical cancer tissues. The 491 bp cDNA fragment has 98% homology to the previously published sequence, AAC-11 (antiapoptosis clone 11). The levels of AAC-11 mRNA expressions in nine normal cervical and nine primary cervical cancer tissues were low. Its expression was higher in three metastatic tissues and five cervical cancer cell lines (HeLa, CaSki, SiHa, CUMC-3, and CUMC-6). Invasion of matrigel and adhesion to laminin by AAC-11 transfected CUMC-6 cells were increased by approximately 2-fold and 4-fold, respectively. Northern blot analysis showed that matrix metalloproteinase (MMP)-2 and membrane type 1 MMP (MT1-MMP) genes were found to be expressed in high levels in AAC-11-transfected cancer cells. But MMP-2 and MT1-MMP were not expressed in cells transfected with vector alone or wild-type cells. AAC-11-transfected cells expressed an elevated level of MMP-2 protein as assessed by immunoblotting. On the contrary, tissue inhibitor of MMP (TIMP-2) expression was detectable in cells transfected with vector alone or wild-type cells, respectively. Its expression was undetectable in AAC-11 transfected cells. In cervical cancer cells transfected with AAC-11, the expression of beta-catenin was up-regulated. These suggest that overexpressions of MMP-2 and MT1-MMP, loss of TIMP-2 expression, and up-regulation of beta-catenin by AAC-11 transfection may contribute to the development of cervical cancer invasion. AAC-11 gene transfection increased cervical cancer cell colonization. The effect of AAC-11 on cultured cervical cancer cells was associated with antiapoptotic process. Approximately 50% of the AAC-11 transfected cells in serum-free medium died after 2 weeks, compared to 1 week for vector alone or wild-type cells. These results suggest that AAC-11 may serve as a candidate metastasis-related and apoptosis-inhibiting gene in human cervical cancer.

Inactivation of interferon regulatory factor-1 tumor suppressor protein by HPV E7 oncoprotein. Implication for the E7-mediated immune evasion mechanism in cervical carcinogenesis.

In studying biological roles of interferon regulatory factor (IRF)-1 tumor suppressor in cervical carcinogenesis, we found that HPV E7 is functionally associated with IRF-1. Binding assays indicate a physical interaction between IRF-1 and HPV E7 in vivo and in vitro. The carboxyl-terminal transactivation domain of IRF-1 was required for the interaction. Transient co-expression of E7 significantly inhibits the IRF-1-mediated activation of IFN-beta promoter in NIH-3T3 cells. Co-transfection of E7 mutants reveals that the pRb-binding portion of E7 is necessary for the E7-mediated inactivation of IRF-1. It was next determined whether histone deacetylase (HDAC) is involved in the inactivation mechanism as recently suggested, where the carboxyl-terminal zinc finger domain of E7 associates with NURD complex containing HDAC. When trichostatin A, an inhibitor of HDAC, was treated, the repressing activity of E7 was released in a dose-dependent manner. Furthermore, the mutation of zinc finger abrogates such activity without effect on the interaction with IRF-1. These results suggest that HPV E7 interferes with the transactivation function of IRF-1 by recruiting HDAC to the promoter. The immune-promoting role of IRF-1 evokes the idea that our novel finding might be important for the elucidation of the E7-mediated immune evading mechanism that is frequently found in cervical cancer.

Antiproliferative effects of retinoic acid/interferon in cervical carcinoma cell lines: cooperative growth suppression of IRF-1 and p53.

Retinoids and interferons have been implicated in the growth regulation of cervical cancer cells. However, the molecular mechanisms are not fully defined. To analyze detailed mechanisms, HPV-positive (HeLa, CaSki), HPV-negative (C33A, HT-3) and non-cervical Cos-1 cell lines were treated with I microM all-trans-retinoic acid (RA) and/or 10 ng/ml interferon-gamma (IFN-gamma). The growth of RA-treated HeLa cells was less effectively suppressed than that of IFN-gamma-treated ones. A combination of RA and IFN-gamma leads to an additive antiproliferative effect on the cell growth. CaSki cell growth was also inhibited by IFN-gamma but was little stimulated by RA treatment, and the IFN effect was attenuated when IFN-gamma was combined with RA. HPV-negative C33A and HT-3 cells, which are defective in p53 and Rb, and Cos- 1 cells were weakly or not responsive to all combined treatments. The molecular mechanism underlying the differential effects of RA/IFN on HeLa and C33A cells was investigated. Combined RA/IFN-gamma treatment caused a marked increase in the level of IFN regulatory factor-1 (IRF-1) in HeLa, whereas no induction of IRF-1 was observed in C33A, consistent with the findings that IFN signaling is functional in HeLa but is defective in C33A cells. The increase of p53 in HeLa cells might account for the down-regulation of HPV-18 E6 gene expression by RA/IFN-gamma. Furthermore, RA/IFN-gamma treatment resulted in the concurrent induction of p21WAF1 CDK inhibitor and dephosphorylation of Rb protein. Transient co-expression of IRF-1 and p53 led to the cooperative activation of the p21WAF1 promoter. Our results indicate that 2 transcription factors, increased in response to RA/IFN-gamma, cooperate functionally to regulate the cell cycle through the activation of a common p21WAF1 gene in HeLa cells.

Regulation of cell growth and HPV genes by exogenous estrogen in cervical cancer cells.

Human papillomavirus (HPV) infection is known as the major cause of the development of cervical cancer. The E6 and E7 proteins of oncogenic HPV can play critical roles in immortalization and malignant transformation of cervical epithelial cells. From the previous epidemiologic data, it has been determined that long-term use of oral contraceptives may be a risk factor for cervical cancer. Investigation of the estrogenic and antiestrogenic effects on the proliferation of cervical cancer cells and the gene expression of HPV would help to explain the role of estrogen in the HPV-associated pathogenesis of cervical cancer. In this study, cervical cancer cells (HeLa, CaSki, and C33A) were cultured in vitro in the presence of 17beta-estradiol or tamoxifen to observe their regulatory growth effect and HPV E6/E7 gene expression. The estrogenic effect on the promoter activity of HPV URR was further confirmed by transient transfection assay, which was conducted in C33A cells using the HPV-18 URR-CAT reporter plasmid. The supplemental effect of estrogen receptors on URR promoter activity was also evaluated. The proliferation of HeLa and CaSki cells was stimulated by estradiol at physiologic concentration levels (

Antitumor effect of GnRH agonist in epithelial ovarian cancer.

OBJECTIVE: The effects of the gonadotropin releasing hormone (GnRH) agonist (D-Trp(6)) were examined in two human ovarian cancer cell lines and in severe combined immune deficiency (SCID) mice to evaluate its potential as a cytocidal, cytostatic, or differentiating antitumor agent. METHODS: We treated the human ovarian cancer cell lines OVCAR-3 and SKOV-3 for 5 or 7 days and sex-matched SCID mice with GnRH agonist for 29 days. The antitumor effect of GnRH agonist were studied in various aspects. To confirm the antiproliferative effect, we used 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide colorimetric assay, in vitro, and a serial measurement of tumor growth in vivo. The disturbances of progression in the cell cycle and the changes of cyclin-dependent kinase 1 following treatment with GnRH agonist were evaluated with flow cytometric analysis in vitro. The induction of apoptosis following treatment with GnRH agonist was studied using in situ terminal deoxyribonucleotidyl transferase (Tdt) and further quantitated with ELISA in vitro. The presence of telomerase activity following treatment with GnRH agonist was measured by PCR-based telomeric repeat amplification protocol and ELISA detection in cell lines and xenografts in vitro and in vivo. RESULTS: Continuous exposure of cell lines and xenografts to GnRH agonist resulted in growth inhibition of cancer cells in a dose- and time-dependent manner. In cultured cells, the GnRH agonist blocked cell cycle progression in G0/G1 phase and thus reduced the number of cells in S and G2/M phases. The phenomenon of apoptosis was documented in cultured cells treated with GnRH agonist by in situ Tdt assay. The frequency of apoptotic cells in the in situ Tdt assay was 5-6% compared with control, 4-5%. Apoptosis quantified by ELISA revealed a high incidence in cultured cells treated with GnRH agonist. The activities of telomerase in cell lines and xenografts were not decreased by GnRH agonist. There were not any significant changes of expression of CA-125 by flow cytometry and of the cellular morphology observed with light microscopy. CONCLUSIONS: Our results indicate that the antiproliferative effect of GnRH agonist in epithelial ovarian cancer cells may be mainly attributed to cytostatic activities resulting in blocking of cell cycle progression in the G0/G1 phase and minimally related to the induction of apoptosis.