Oleuropein prevents the progression of steatohepatitis to hepatic fibrosis induced by a high-fat diet in mice

Nonalcoholic steatohepatitis (NASH) is characterized by hepatocyte injury and inflammatory cell infiltration, which has been linked to peripheral insulin resistance and increased levels of triglycerides in the liver. The purposes of this study were to establish a mouse model of NASH by feeding mice a 60% high-fat diet (HFD) and to demonstrate the anti-fibrotic effects of oleuropein, which has been shown to have anti-oxidant and anti-inflammatory properties, in this HFD-induced mouse model of NASH. C57BL/6 mice were divided into three groups: a regular diet group (Chow), a HFD group and an oleuropein-supplemented HFD group (OSD), which was fed a 0.05% OSD for 6 months. The effects of oleuropein in this model were evaluated using biochemical, histological and molecular markers. The expression levels of alpha-smooth muscle actin (alpha-SMA)and collagen type I in the HFD and OSD groups were evaluated using real-time PCR and western blotting. The body weight, biochemical marker levels, nonalcoholic fatty liver disease activity score, homeostasis model of assessment-insulin resistance (HOMA-IR) and leptin levels observed in the HFD group at 9 and 12 months were higher than those observed in the Chow group. The HOMA-IR and leptin levels in the OSD group were decreased compared with the HFD group. In addition, alpha-SMA and collagen type I expression were decreased by oleuropein treatment. We established a NASH model induced by HFD and demonstrated that this model exhibits the histopathological features of NASH progressing to fibrosis. Our results suggest that oleuropein may be pharmacologically useful in preventing the progression of steatohepatitis and fibrosis and may be a promising agent for the treatment of NASH in humans.

Identification of TACC3 (Transforming Acidic Coiled-Coil 3) as a novel target of paclitaxel-mediated tumor therapy in cervical cancer cells / 부인종양

OBJECTIVE: Paclitaxel is currently used in the treatment of ovarian, breast, gastric, colorectal, lung and recurrent cervical cancer. Initial studies on the mechanism of action of paclitaxel have demonstrated that this drug alters microtubule assembly, by inhibiting microtubule depolymerization and changing microtubule dynamics. Although treatment of various tumor cells with paclitaxel induces apoptosis, but early paclitaxel-targeted proteins is not yet known. We tried to search paclitaxel-targeted proteins and to investigate its functions. METHODS: The effects of paclitaxel on HeLa cervical cancer cell growth were evaluated by cell proliferation assay, DAPI stain, and FACS analysis. We performed proteome analysis including 2-DE and MALDI-TOF-MS in nontreated-and paclitaxel-treated HeLa cells, as a result, we identified TACC3 protein that is down-regulated with paclitaxel treatment. We tried to characterize TACC3 functions through in vitro treatment of paclitaxel or RNAi technique. RESULTS: Paclitaxel- and TACC3 siRNA-treated cells are unable to proceed normally through the cell cycle and are arrested in G2/M phase and reveal apoptotic morphology. TACC3 levels after paclitaxel treatment decreased as a time- and dose- dependent manner both mRNA and protein levels. We confirmed that the role of TACC3 down-regulation for microtubule stabilization was similar to that of paclitaxel. Also, TACC3 is expressed at high levels in various cancer cells and tumor tissues. CONCLUSION: This study is proposed that the TACC3 protein may be participated in microtubule formation as an oncoprotein during mitosis and be regulated by paclitaxel as a novel target.

Antitumor activity of spinasterol isolated from Pueraria roots

We purified phytoestrogens from Pueraria root (Pueraria mirifica from Thailand and Pueraria lobata from Korea), which is used as a rejuvenating folk medicine in Thailand and China. Dried, powdered plant material was extracted with 100% ethanol and further separated by concentration, filtration, and thin layer silica gel chromatography. Using the fractions obtained during separation, we first investigated their cytotoxicity in several cancer cell lines from various tissues. The ethanol-extracted components (PE1, PE4) had significant antiproliferative effects on breast cancer cell lines, including MCF-7, ZR-75-1, MDA-MB-231, SK-BR-3, and Hs578T. Second, we compared these results with the cytotoxic effects of known flavonoids, sterols, and coumarins from Pueraria root. The known compounds were not as effective, and occurred in a different polarity region on HPLC. Third, further separation resulted in the isolation of eight different components (Sub PE-A to -H). One of these, PE-D, affected the growth of some breast cancer cell lines (MCF-7, MDA-MB-231) in a dose- and time-dependent manner, as well as the growth of ovarian (2774) and cervical cancer cells (HeLa). Finally, a transfection assay showed that this component had an estrogenic effect similar to 17beta-estradiol, which activates both estrogen receptor a (ER alpha) and ER beta. The NMR analysis determined that spinasterol (stigmasta-7, 22-dien-3beta-ol) is an active cytotoxic component of Pueraria root.

Differential protein expression of etoposide-treated CaSki cervical carcinoma cells / 부인종양

OBJECTIVE: This study was designed to examine the pharmaco-dynamic pattern of proteomic expression in cervical carcinoma cells (CaSki cell line; HPV-16 positive) after in vitro treatment by the etoposide. METHODS: We analyzed proteomic profiling in cervical carcinoma cells after etoposide treatment using two-dimensional gel electrophoresis (2-DE) with MALDI-TOF-MS used for protein identification. Then, we tested the several experimental methods for verification and functional identification, including MTT assay, PI staining, DNA fragmentation assay, FDA, FACS and Western blot analysis. RESULTS: Etoposide inhibited the CaSki cervical cancer cell growth in a dose-dependent manner and the optimal concentration of etoposide is 2micrometer(IC50) in the CaSki cervical cancer cells. The etoposide induced apoptosis, as determined by DNA fragmentation assay, FACS, and Western blot. The etoposide increased the protein expression of Fas (Apo-1/CD95), p53, pRb and caspase-3, but decreased the level of Bcl-2 and caspase-3 precursor and subsequently triggered the mitochondrial apoptotic pathway (release of cytochrome c and activation of caspase-9). To this end, we analyzed CaSki cancer cells using 2-DE. Eight proteins (XAP-5, HXC-36, serine/threonine protein phosphatase 2B catalytic subunit, G2/mitotic-specific cyclin B1, T-box transcription factor TBX20, diacylglycerol kinase, amiloride-sensitive amine oxidase, HEF-like protein, ras-related protein Rab-20) were down-regulated and nine proteins (RNA 3'-terminal phosphate cyclase-like protein, late endosomal/lysosomal Mp1 interacting protein, glia maturation factor, replication protein A 14 kDa subunit, mago sashi protein homolog, 14 kDa phosphohistidine phosphatase, protein C14 or f48, cyclin-dependent kinase 4 inhibitior A, retinoic acid-binding protein II) were up-regulated in etoposide-treated CaSki cells when compared with non-treated cells. CONCLUSION: Our results clearly indicate that etoposide induced cell death by apoptosis. These findings may provide insights into the mechanisms underlying the apparent anti-tumoral effects of etoposide.

Proteome Analysis of Differential Protein Expression in Cervical Cancer Cells after Paclitaxel Treatment / Journal of the Korean Cancer Association, 대한암학회지

@#PURPOSE: It is well known that infection with HPV (human papillomavirus) is the main cause of cervical cancer and certain types of HPV are recognized as carcinogens. At present, there is little information regarding the antineoplastic mechanism of paclitaxel against cervical carcinoma cells. We thus tried to analyze differential protein expression and antineoplastic mechanism-related proteins after paclitaxel treatment on cervical cancer cells by using a proteomic analysis and to investigate the mechanism of action. MATERIALS AND METHODS: Using proteomics analysis including 2-DE and MALDI-TOF-MS, we detected the antineoplastic mechanism-related proteins. Then, we performed western blot analysis for apoptosis- and transformation- related proteins to confirm expression patterns derived from proteome analysis after paclitaxel treatment. RESULTS: We identified several cellular proteins that are responsive to paclitaxel treatment in HeLa cells using proteomics methods. Paclitaxel treatment elevated main-ly apoptosis, immune response and cell cycle check point- related proteins. On the other hand, paclitaxel treatment diminished growth factor/oncogene-related proteins and transcription regulation-related proteins. Also, in the HPV-associated cervical carcinoma cells, paclitaxel demonstrated anti-proliferative activity through the membrane death receptor-mediated apoptotic pathway and the mitochondrial-mediated pathway. CONCLUSION: Identification and characterization of functionally modulated proteins involved in anti-cancer regulatory events should lead to a better nderstanding of the long-term actions of paclitaxel at the molecular level and will contribute to the future development of novel therapeutic drug treatments based upon current therapies.

GSTP1 Polymorphism, Cigarette Smoking and Cervical Cancer Risk in Korean Women

Previous studies have suggested that glutathione S-transferase (GST) genotypes may play a role in determining susceptibility to cervical cancer, though the data have often been conflicting. The objective of this study was to examine the effect of GSTP1 polymorphism on cervical carcinogenesis. The studied subjects, patients who were pathologically diagnosed with invasive cervical cancer yielding positive results for human papillomavirus (HPV) (n=342), were compared to healthy, normal, female controls (n=707). DNA from peripheral blood samples from studied subjects whose GSTP1 specific sequences had been determined by PCR with allele-specific primers were reviewed in comparison with the normal controls. The genetic susceptibility of GSTP1 (11q 13.1) in cervical carcinogenesis was determined by examining the effect of gene and environmental factors by the different histopathologic types of invasive cervical cancers. In assessing polymorphism GSTP1, the percentages of individuals homozygous for the A allele, homozygous for the G allele, and heterozygous for the two alleles were 66.8%, 3.9%, and 29.3%, respectively, in the control group, and 64.3%, 4.1%, and 31.6%, respectively, among in women with cervical cancer. Compared with GSTP1 G allele positive (GA or G/G), the odds ratio (OR) (95% confidence interval) for GSTP1 A/A was 1.0 (0.7 - 1.4) for invasive cervical cancer. However, the risk increased with GSTP1 A/A among ever smokers (3.9, 1.7 - 8.9, p-value=0.0012) compared with GSTP1 G allele positive among nonsmokers. In particular, this risk was higher among women with squamous cell carcinoma (4.7, 2.0 - 10.8, p=0.0003). Polymorphism of GSTP1 among smoking women was associated with a higher risk of developing cervical cancer.

Analysis of HPV-16 E6-related gene expression using cDNA microarray / 대한산부인과학회잡지

OBJECTIVE: To examine the effect of HPV-16 E6 expression on the transcription of cellular genes, we used cDNA microarray in HPV-16 E6 transfected stable cancer cell lines. METHODS: Using cDNA microarray consisting of 1,024 genes, we have performed a systematic characterization of gene expression in A549E6 human lung adenocarcinoma and RC10.1 human colon adenocarcinoma cell lines stably expressing HPV-16 E6 gene. The up-regulated and down-regulated genes were classified into the different functional categories; oncogenes, apoptosis, cell cycle, signal transduction, gene regulation, immune response, cell adhesion, protein transport, metabolism, redox control and angiogenesis. RESULTS: Among 1,024 known genes and ESTs (expressed sequence tags) tested, we found 27 up- regulated and 43 down-regulated genes in A549E6 (HPV-16 E6) compared to A549. The major up-regulated genes were as follows. GTPase-activating protein Rho 4, transcription factor D2, IKAROS, integrin-alpha 6, cadherin 11, ephrin-beta 2, RAN binding protein 2, branched-chain amino transferase 2. The major down-regulated genes were as follows. K-ras 2, CDC (cell division cycle) 37, CDC16, CDC7L1, IRF3, interferon-gamma-inducible protein 30, cadherin 6, desmoglein 1, desmocollin 2, endothelin 2. Also, we found 48 up-regulated and 34 down-regulated genes in RC10.1 (HPV-16 E6) compared to RKO. The major up-regulated genes were as follows. Colon cancer familial nonpolyposis type 1 (COCA 1), Bcl 2, jagged 1, MAP2K6, E2F1, ephrin receptor-beta 2, ephrin-beta 2, desmoglein 1, transforming growth factor-beta 3. The major down-regulated genes were as follows. KIT, Rad51C, Bcl 2 antagonist killer 1, STAT 4, epidermal growth factor receptor, high mobility group protein 2, cadherin 11, cadherin 12, cadherin 3, integrin-alpha 1, intergrin-alpha 8, chromosome segregation 1-like. CONCLUSION: Various expression patterns of cellular genes by HPV-16 E6 could be wholy grasped and classified into different functional groups using both cell line system stably expressed HPV-16 E6 and cDNA microarray analysis. These analysis methods must be helpful to understand multiple effects of a specific gene on cellular genes in a short period.

Growth Suppression of Ovarian Cancer Cells by Interferon-gama / Journal of the Korean Cancer Association, 대한암학회지

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.

Growth Regulation of Ovarian Cancer Cells through the Inactivation of AP-1 by Retinoid Derivatives / 대한암학회지

PURPOSE: The growth regulatory effect of retinoid derivatives could be mediated by the transcriptional inactivation of AP-1 oncogenic transcription factor. By using ovarian cancer cell lines we were to investigate the cross-regulation mechanism between retinoids and AP-1. MATERIALS AND METHODS: Cell proliferation assays were performed in 4 ovarian cancer cells (A2774, PA-1, OVCAR-3, SKOV-3) by increasing the concentrations of all-trans retinoic acid (ATRA), 9-cis retinoic acid (9RA), 13-cis RA (13RA), 4-hydroxyphenyl retinamide (4-HPR). Transient transfection and CAT ELISA were done to determine the selective activity of each retinoid on the RAR (alpha, beta, gamma), RXR (alpha, beta, gamma). and the negative activity on AP-1 (c-Jun). RESULTS: Antiproliferative effect of 4-HPR (IC50; 0.7~2.7 micrometer) was more potent than those of other retinoid derivatives (IC50; 2.7~9.0 micrometer). To assess the anticancer mechanism, we examined the effect of 4-HPR on the transriptional activity of retinoic acid receptors (RAR/RXR) and of c-jun. Contrary to other retinoid derivatives that are active for RAR and RXR with some different levels, 4-HPR showed weak activity only for RARgamma. However, 4-HPR exerted the strongest suppression on AP-1 (c-Jun) activity. CONCLUSION: Based on our results showing much 4-HPR's potent antiproliferative activity coupled with the most effectively inhibiting activity on AP-1 and minimum activity on RA receptor (selective for RARgamma) than other retinoid derivatives, we suggest that 4-HPR may be a novel, and very effective anticancer drugs for ovarian cancer.

The Effects of Interferon/Retinoic Acid on Cervical Cancer Cell Lines According to the Mutational Status of HPV-URR / 대한암학회지

PURPOSE: We investigated the effects of all-trans-retinoic acid (ATRA) and/or interferon-gamma (IFN-gamma) on the growth of various cervical cancer cell lines and HPV E6/E7 expression. The relationships between the functional activities of HPV-URR and the growth inhibition were identified. MATERIALS AND METHODS: Four groups of cell lines were included; i) with integrated form of HPV-16 DNA (SNU-17, CaSki), ii) episomal form of HPV-16 (SNU-523), iii) integrated form of HPV-18 (SNU-1160, HeLa) and iv) episomal form of HPV-18 (SNU-1245). The promoter activity of HPV-URR was confirmed by transient transfection assay in C33A using the HPV-18 URR-CAT reporter plasmid. RESULTS: Selective mutation was detected in TEF-1 (transcriptional enhancer factor) binding site in SNU-17, and the activity of URR in SNU-17 was higher than that of the prototype. The proliferation was more inhibited in SNU-17 by IFN-gamma (10 ng/ml) than in SNU-902, CaSki and HeLa. The increase of the HPV-URR activity might play a role in the inhibition of growth by interferon-g. The expression of HPV-16 E6/E7 were significantly decreased by ATRA or IFN-gamma. CONCLUSION: Point mutation at TEF-1 binding site of SNU-17 was related with the increased transcriptional activity of URR. Mutation in the HPV-URR and alteration of HPV-URR activity in SNU-17 might be related with significant growth suppression by IFN-gamma.

Novel Molecular Biologic Mechanism of Growth Suppressive Regulation by Retinoid / Interferon in Cervical Cancer Cells / 대한산부인과학회잡지

OBJECTIVE: Retinoic acids (RAs) and interferons (IFNs) have been implicated in the growth regulation of cervical cancer cells, which was suggested by clinical trials and in vitro experiments. However, the molecular mechanisms of growth regulation are not fully defined, The purpose of this study is to assess the effect of RA and/or IFN on human cervical carcinoma cells in vitro and to analyze their action mechanisms in HPV-positive cervical carcinoma cells by molecular biologic studies. METHODS: HPV-positive (CaSki, HeLa), HPV-negative (C33A, HT-3), and non-cervical cancer Cos-1 cell lines were treated with RA and/ar IFN. Their effects on cell growth were evaluated by the cell pmliferation assay and the following BrdU DNA incorporation assay. The molecular mechanism was further investigated by a series of immunoblottings and transient cotransfection assays, which were conducted in HeLa cells and C33A cells using the CAT reporter gene assay. To observe the down regulation of HPV E6/E7 gene expression by RA/IFN, reverse transcription-polymerase chain reaction (RT-PCR) was perforned. RESULTS: The powth of RA-treated cells was less suppressed than that of IFN-treated cells. Combined treatment of RA and IFN leads to additive effect on the growth suppression of HeLa and CaSki cells. The proliferation activity was most severely reduced in Hela cells by treatment of both all-trans-RA (AtRA) and IFN-r. Combined treatment of AtRA/IFN-r causes a great increase in the level of interferon regulatory factor-1 (IRF-1) protein in HeLa cells, whereas no induction of IRF-1 was observed in C33A cells. The CAT gene expression for IRF-1 was greatly induced by IFN-r in HeLa cells. Immunoblotting assays shows the concurrent induction of p21 CDK inhibitor and dephosphorylation of Rb protein in HeLa cells. In RT-PCR, an individual treatment of either RA or IFN reduced HPV E6/E7 mRNA levels and significantly cooperative when both RA and IFN were treated. By deaeasing E6 levels, the p53 level was increased in HeLs cells treated with RA and/or IFN. Transient cotransfection of IRF-1 and p53 as the transcription factors leads to the cooperative activation of a common p21 promoter to regulate the cell cycle. CONCLUSION: RA/IFN suppressed the growth of HPV-positive cervical cancer cells. When they were both treated, additive suppressive effects were observed in cellular proliferation as well as DNA synthesis. The growth suppressive effect is likely to be related to the increased expression of IRF-1 and p21 (antitumoral effect; p53-independent). The down regulation of HPV E6 gene suppression may account for the resultant increase of p53 levels (antiviral effect; p53-dependent). Both induced IRF-1 and p53 cooperatively augument tbe suppession of p21 CDK inhibitor, which results in dephosphorylation of pRb. Although clinical effects are likely complex and may include interactions of in vitro growth inhibitory effects with immunomodulatory and antiangiogeaetic effect, tbese results suggest the optimal clinical role for the combination of RA/IFN in the treatment of cervical canccers.

Inactivation of p73 Protein by Cervical Cancer-inducing HPV E6 Protein Novel p53-independent function of E6 / 대한산부인과학회잡지

OBJECTIVE: Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 and E7 oncoproteins play major roles by inactivation of cellular p53 and pRb tumor suppressor proteins, respectively. However, it has been recently suggested that p53 and/or pRb-independent functions of E6 and E7 are involved in cervical carcinogenesis. The purpose of this study is to identify novel a cellular target, p73, of E6 and to determine how E6 inactivates p73 function, METHODS: The interaction between E6 and p73 were identified by the yeast two-hybrid assay in vivo and the GST pull-down assay in vitro. The function of the interaction was determined by transient transfections using p21 promoter-CAT reporter plasmid. The molecular mechanism underlying the functional significance of the interaction was further assessed by in vivo and in vitro protein degradation assays, and gel mobility shift assays. RESULTS: 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. Transactivation domain (amino acid residues 1-49) is found to be absolutely required for this interaction. Transient co-expression of E6 significantly inhibits the p73-mediated activation of p21WAF1 promoter in a p53-defective C33A cell line. Using Ga14-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. The protein degradation assays in vivo and in vitro indicate that p73, unlike p53, is not susceptible to E6-dependent proteolysis. CONCLUSION: Throughout this study, we identified p73 as a novel cellular target of HPV-E6 protein and found that E6 binds p73 through the amino-terminal transactivation domain, and inhibits its transactivation function independent of the protein degradation and DNA binding. 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 cellular transformation.

Integration of HPV and the Antibody Respones to HPV Proteins in Patients with Cervical Cancer / 대한암학회지

PURPOSE: HPV (human papillomavirus) are known as the major causative agent for development of cervical cancer. High-risk HPVs, especially HPV-16 /18 DNA, are often found to be integrated into the human genome in high grade CINs as well as cervial cancer. Investigation of the relationship between the genomic states of HPV genes and their antibody response against the HPV-16 Ll/L2 virus-like particles (VLPs) and the in vitro translated E6 and E7 proteins may help to explain the mechanism of HPV-related cervical carcinogenesis and host immune responses. MATERIALS AND METHODS: Cervical cancer tissues obtained from 41 patients with cervical cancer were studied by PCR, Southern blot hybridization and the antibody response against HPV-16 Ll/L2 VLPs and HPV-16 E6, E7 proteins of serum were tested by ELISA and radioimmunoprecipitation assay (RIPA), respectively. RESULTS: Integrated forms of the HPV-16 DNA were found in 23 of the 38 patients (60.5%). The HPV-16 positive cervial cancer patients had a significantly higher prevalence (39.5%; 15/38) of antibodies to HPV-16 Ll/L2 VLPs than 8.7% (2/28) of the the control group (p0.05). CONCLUSION: Integrated forms of HPV-16 DNA were prevalent in most patients with cervical cancer. Antibodies to HPV-16 Ll/L2 VLPs, in vitro translated HPV-16 E6 and E7 proteins appeared in the significantly larger proportions of the HPV-associated cervical cancer patients than in the controls. Antibodies to HPV-16 Ll/L2 VLPs were more detectable in the cervical cancer patients with episomal form of HPV-16 DNA than those who having only integrated forms of HPV-16. Antibody responses to HPV-16 E6 and E7 proteins were not influenced by the different viral states. More numbers of studies would be necessary to determine the relationship between the genomic states of HPV and the immune responses to their proteins by the such genomic and serologic parameters.

Increased transcriptional activity by mutation of HPV-16URR in cervical cancers carrying episomal HPV-16 DNA / 대한부인종양콜포스코피학회잡지

HPV E2 protein is known to act as a negative regulator of transcription and the disruption of E2 open reading frame by HPV integration can release suppression of E6 and E7 mRNA expression, resulting in uncontrolled cellular growth and malignant transformation by inactivating tumor suppressor gene products (p53, pRb). YY1 mutation of HPV URR has been suggested as one of indicator that explains development of cervical neoplasia by episomal type of HPV. To extend this hypothesis, we examined whether mutation(s) in specific sites of HPV URR is functionally related to the invasiveness of cervical neoplasia and the physical status of HPV DNA. The URR sequences were obtained by PCR amplification of HPV-16 genome from CIN and invasive cancer patients, cloned into pUC18 for sequencing, and into pBLCAT8+ for functional CAT assay. Our previous data classified HPV-infected patients into three groups: 3 cancer cases carrying episomal HPV DNA; 12 cancer cases carrying integrated HPV DNA; 12 CIN cases carrying episomal HPV DNA. The specific variants in HPV-16 URR were found in Korean women: GA transition at nt 7520 (100%, 27/27), AC transition at nt 7729 (70%; 19/27), and GA transition at nt 7841 (78%; 21/27). Selective mutations were observed at the YY1-binding sites of HPV-16 URR in the 3 patients with invasive cervical cancer, who having the episomal forms of HPV-16 DNA: AC transition at nt 7484 and GA transition at nt 7488 (YY1-binding site 2; from 7481 to 7489). Additionally, CT transition at nt 7785 (YY1-binding site 3; from 7781 to 7790) was found from 2 of 3 patients. No YY1 site mutations were detected in the 12 CIN patients and in the HPV-integrated invasive cancer patients. To determine whether these mutations have effect on the expression of HPV E6/E7 genes driven by URR, the transient transfection assay was employed using URR-CAT reporter plasmid. The relative activities of three URR mutants from episomal HPV-16 DNA of cervical cancers were 2- to 4-fold higher than that of HPV-16 URR prototype. In contrast, the URRs from integrated HPV-16 DNA in cervical cancer and from episomal HPV-16 DNA in CIN, where no mutation of the YY1-binding site was detected, showed similar levels of promoter activity to that of URR prototype.Our results support the hypothesis that the mutation at YY1 binding site is functionally related to the development of cervical neoplasia caused by episomal HPV-16 DNA in Korean cervical cancer patient. Thus, mutation in YY1 site of episomal HPV-16 URR may play a role of HPV integration in the progression of cervical cancer.

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

BACKGROUNDS: Human papillomavirus (HPV) infection is known as the major causative phenomenon in the development of cervical cancer. E6 and E7 proteins of oncogenic HPV types can play critical roles in immortalization and malignant transformation of cervical epithelial cells. From the previous epidemiologic data, long term use of oral contraceptives may be one of the risk factor for cervical cancer. PURPOSE: Investigation of estrogenic and anti-estrogenic effects on the proliferation of cervical cancer cells and gene expression of HPV under the regulation of HPV upstream regulatory region (URR) would help to explain the role of estradiol in HPV-associated pathogenesis of cervical cancer. METHODS: Cervical cancer cells (HeLa, CaSki and C33A) were cultured in vitro in the presence of 17 beta-estradiol or tamoxifen and the numbers of cells were directly counted to observe the growth stimulatory or suppressive effect of the treatment. The correlation between the growth regulatory effect and HPV E6/E7 gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR). The estrogenic effect on the promoter activity of HPV URR was further confirmed by transient co-transfection assays, which were conducted in C33A cells using the HPV-18 URR-CAT reporter plasmid. Supplemental effect of estrogen receptor on the URR promoter activity was also evaluated. To analyze the growth suppressive function at the higher concentration of estradiol or tamoxifen in HeLa cells, DNA fragmentation assay was performed. RESULTS: The proliferation of HeLa and CaSki cells was stimulated by estradiol at the concentration of physiological level (< or =1 X 10-6M), reaching maximal growth at 0.5 X 10-6M. At concentration of 0.1 X 10-6M, tamoxifen also stimulated the proliferation of HeLa and CaSki cells. In contrast to HPV-positive cervical cells, C33A cells were not influenced to cell proliferation by addition of estradiol at the physiological level, indicating that HPV might play role in growth stimulatory effect of estrogen or tamoxifen. Interestingly, the proliferation of HeLa cells was totally suppressed by estradiol and tamoxifen at the higher concentration (5 and 10 X 10-6M), whereas those of CaSki and C33A cellswere not responded and little suppressed at the concentration, respectively. The levels of HPV-18 E6 and E7 mRNA were significantly increased after treatment of 0.5 X 10-6M estradiol as determined by RT-PCR. Furthermore, transient transfection experiments using the URR-CAT reporter plasmid indicated that the increased expression of HPV E6/E7 genes was related with the growth stimulatory effect of estradiol and tamoxifen. In addition, co-transfection of estrogen receptor (ER) leads to an over 4-fold increase in CAT activity after treatment of estradiol or tamoxifen with 0.5 X 10-6M. When estradiol or tamoxifen was treated at the concentration over 5 X 10-6M for 96 hr, a typical DNA ladder, a indicative of apoptosis, was observed in HeLa cells. However, DNA ladder was not detected in C33A cells of which growth was some suppressed under same concentration of estradiol. CONCLUSION: At the physiological levels, estradiol stimulated the growth of HPV-positive cervical cancer cells and tamoxifen also did at the concentration of 0.1 X 10-6M. The increased expression of HPV E6/E7 at the physiologic levels appeared to be related with the growth stimulation of HPV-positive cervical cancer cells. Growth suppression observed at the higher concentration (5 and 10 X 10-6M) might be a indicative of apoptosis shown by DNA fragmentation assay in HeLa cells. Taken together, these data suggested that the concentration of estradiol (< or =1 X 10-6M) could be a risk-factor in HPV-mediated cerivcal carcinogenesis.