In an attempt to understand the molecular mechanisms for the different clinical features between adenocarcinoma/adenosquamous carcinoma (AC/ASC) and squamous carcinoma (SC) of the uterine cervix, we analyzed gene expression profiles of different histological subtypes of cervical cancer. Cancer specimens and the surrounding normal tissue counterparts were separately collected from cervical cancer patients undergoing type III radical hysterectomy. Paired total RNA (cancer and normal tissues) was isolated and analyzed with cDNA microarrays containing duplicate spots of 7 334 sequence-verified human cDNA clones. Selected differentially expressed genes specific for AC or SC were further verified using real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemistry. Genes, including CEACAM5, TACSTD1, S100P and MSLN were upregulated in AC. Contrarily, genes involved in epidermal differentiation complex such as S100A9 and ANXA8 were upregulated in SC. Cross-validation of the results using an independent but comparable group of patients with known long-term outcomes (n 5 63, median follow-up 70.3 months; range, 4-208 months) showed that the correlation between the selected 6 differentially expressed genes and histology was highly significant. CEACAM5 (p < 0.0001) and TACSTD1 (p 5 0.009) were significant prognostic factors by multivariate Cox proportional hazards regression analysis. The combination of cDNA microarray, RTQ-PCR and immunohistochemical results of this study showed that it is possible to define different gene profiles for AC and SC. Moreover, TACSTD1 expression may be a novel poor prognostic factor. ' 2006 Wiley-Liss, Inc.

Taxol is a mitotoxin widely used to treat human cancers, including of the breast and ovary. However, taxol resistance (txr) limits treatment efficacy in human patients. To study chemoresistance in ovarian cancer, we established txr ovarian carcinoma cells derived from the SKOV3 cell lineage. The cells obtained were cross-resistant to other mitotoxins such as vincristine while they showed no resistance to the genotoxin cisplatin. Transcriptomic analysis identified 112 highly up-regulated genes in txr cells. Surprisingly, FK506-binding protein 5 (FKBP5) was transiently up-regulated 100-fold in txr cells but showed decreased expression in prolonged culture. Silencing of FKBP5 sensitized txr cells to taxol, whereas ectopic expression of FKBP5 increased resistance to the drug. Modulation of FKBP5 expression produced similar effects in response to vincristine but not to cisplatin. We observed that a panel of newly identified txr genes was trancriptionally regulated by FKBP5 and silencing of these genes sensitized cells to taxol. Notably, immunoprecipitation experiments revealed that FKBP5 forms a protein complex with the androgen receptor (AR), and this complex regulates the transcriptional activity of both proteins. Furthermore, we found that the Akt kinase pathway is regulated by FKBP5. These results indicate that the FKBP5/AR complex may affect cancer cell sensitivity to taxol by regulating expression of txr genes. Our findings suggest that mitotoxin-based treatment against ovarian cancer should be avoided when the Akt/FKBP5/AR axis is activated.

A human cell line selected for cisplatin resistance (CPR) was irradiated with UV light and showed cross-resistance to UV light. Applying a modified chloramphenicol acetyltransferase assay, we observed that CPR cells acquired enhanced host cell reactivation of a transfected plasmid carrying UV damage. Gel mobility shift analysis indicated that two nuclear factors that recognize UV-modified DNA were overexpressed in CPR cells. In addition, factors that bind UV-modified DNA were independent from the factors that bind cisplatinmodified DNA. The significance of the identified binding factors, possibly DNA repair enzymes, is discussed.

We conducted a population-based cohort study to evaluate the complementary value of HPV testing to Papanicolaou (Pap) smear and the prevalence and genotype distribution of HPV in Taiwan. In this report, we described the design of the whole study and analyzed the cross-sectional results. Female residents (age 30 years) of Taoyuan, Taiwan were invited. After signing informed consent, every participant had a Pap smear and a HPV testing. Patients with Pap atypical squamous cell of undetermined significance (Group I) or those with HPV-positive but normal cytology (Group II) were referred for a colposcopic examination. A total of 10,014 women were eligible. The overall HPV prevalence was 10.8% (95% confidence interval 10.5%-11.4%) in the study population. A total of 37 types of HPV were identified and the leading three were HPV-52, -18 and -58. There was a significant positive correlation of HPV prevalence with older age, postmenopausal status, current-user of oral contraceptives and never-user of hormone replacement therapy. Past users of oral contraceptives and never users of Pap were associated with higher risk of abnormal Pap, while age 40-49 strata had lower risk. Fifty-nine cases of cervical intraepithelial neoplasia (CIN) 2 from Group I and additional 11 from Group II were identified. The improvement of sensitivity with additional HPV testing was 15.3%. Besides, no specific subgroup was found to most benefit from the combined strategy. The value of adding HPV test to conventional Pap smear has to be evaluated after longer-term follow-up of this population-based cohort. ' 2008 Wiley-Liss, Inc.Key words: cervical cancer screening; Pap smear; human papillomavirus; polymerase chain reaction (PCR); prevalence Cervical cancer continues to be one of the leading female genital cancers worldwide. 1 The cervical cancer burden in Taiwan is still heavy with the incidence of invasive cancer being 24.5/ 100,000, which ranked the fifth place of cancer death in women. 2 Traditionally, primary screening of cancer precursors remains based on cytological classification of cervical smears. Conventional Papanicolaou (Pap) test or monolayer cytology provided sensitivity rates ranged from 30% to 87% and specificity rates from 86% to 100%. 3 Since human papillomavirus (HPV) has been established as a well-recognized etiologic agent of cervical neoplasia, HPV DNA testing is a promising alternative or complementary test to improve the efficacy of cervical screening and probably cost-effective, depending on screening interval. 4,5 Cervical cancer screening programs integrating HPV testing have been advocated in United Kingdom, United States, South Africa, Thailand and Germany. [6][7][8][9][10] Hybrid Capture assay (HCII) (Digene Corporation, Gaithersburg, MD) is the most widely used method of HPV DNA testing. HCII utilizes liquid hybridization of RNA probes against HPV DNA followed by signal amplification, has been validated for its reproducibility. 11 Methods using PCR is more sensitive and quality assays were generally consistent for...

The role of Epstein-Barr viruses (EBVs) in lymphoepithelioma-like carcinoma (LELC) of the uterine cervix is controversial. We aimed to investigate the existence of EBV and human papillomavirus (HPV) in LELC of the cervix. Nine patients of LELC of the cervix, treated at Chang Gung Memorial Hospital between 1996 and 2000, with complete clinicopathologic findings and follow-up data were studied. Twenty-five patients with squamous cell carcinoma were recruited as controls. The EBV genome was measured by real-time quantitative polymerase chain reaction (PCR) and EBV-encoded RNA in situ hybridization from formalin-fixed, paraffin-embedded tissues. HPV genotyping was carried out by SPF1/GP6+ PCR and hybridization with a GeneChip. Type-specific E6 PCR of the 18 most commonly found HPV genotypes in Taiwan was also performed. HPV-16 was found in 3 cases, HPV-18, HPV-31, and HPV-35, and HPV-58 in 1 case each. One case showed positive for both HPV-16 and HPV-58. Low copy number of EBV DNA was found in 9 cases of LELC (1-14.7 copies/microg) and 7 cases of squamous cell carcinoma (3.8-1586 copies/microg) using real-time quantitative PCR Bam H1 W fragment probe, but EBV-encoded RNA-in situ hybridization was negative in tumor cells. Therefore, positive rates for EBV and HPV were 0% and 88.9% (8/9) in LELC of the cervix, respectively. All patients with LELC of the cervix had no evidence of disease for more than 5 years from diagnoses. Our results suggest that EBV is not involved in the carcinogenesis of so-called LELC of the cervix but the EBV sequences might exist in a florid inflammatory stromal component.

A systematic analysis of the genes involved in taxol resistance (txr) has never been performed. In the present study, we created txr ovarian carcinoma cell lines to identify the genes involved in chemoresistance. Transcriptome analysis revealed 1,194 overexpressed genes in txr cells. Among the upregulated genes, more than 12 cryptic transcription factors were identified using MetaCore analysis (including AR, C/EBPβ, ERα, HNF4α, c-Jun/AP-1, c-Myc, and SP-1). Notably, individual silencing of these transcription factors (except HNF4`)sensitized txr cells to taxol. The androgen receptor (AR) and its target genes were selected for further analysis. Silencing AR using RNA interference produced a 3-fold sensitization to taxol in txr cells, a response similar to that produced by silencing abcb1. AR silencing also downregulated the expression of prominent txr gene candidates (including abcb1, abcb6, abcg2, bmp5, fat3, fgfr2, h1f0, srcrb4d, and tmprss15). In contrast, AR activation using the agonist DHT upregulated expression of the target genes. Individually silencing seven out of nine (78%) AR-regulated txr genes sensitized txr cells to taxol. Inhibition of AKT and JNK cellular kinases using chemical inhibitors caused a dramatic suppression of AR expression. These results indicate that the AR represents a critical driver of gene expression involved in txr.

Two cis-diamminedichloroplatinum (II) (cisplatin)-inducible proteins [b 130 (approximately 130 kDa) and b95 (approximately 95 kDa]) in HeLa cells that recognize both the cisplatin-modified and u.v.-modified DNA were identified in this study. These damage-recognition proteins were overexpressed in cisplatin-resistant HeLa cells. The results suggest that the damage-recognition proteins are regulated in the cells in response to DNA damage, and they may be important for DNA repair and probably the emergence of cisplatin resistance.

We report here that toll-like receptor 4 (TLR4) and ABCB1 are upregulated in SKOV3 ovarian carcinoma cells that acquired resistance to the anticancer drug taxol. Silencing of TLR4 using short-hairpin RNA sensitized taxol-resistant SKOV3 cells to taxol (4.6 fold), whereas ectopic expression of TLR4 in parental, taxol-sensitive SKOV3 cells or TLR4-null HEK293 cells induced taxol resistance (∼2 fold). A sub-lethal dose of taxol induced ABCB1 protein expression in taxol-resistant SKOV3 cells. Inactivation of TLR4 using chemical inhibitors (CLI-095 and AO-I) downregulated ABCB1 protein expression and enhanced the cytotoxic activity of taxol in taxol-resistant SKOV3 cells. While the sensitization effect of TLR4 inactivation was also detected in TOV21G ovarian cancer cells, which express moderate level of TLR4, ectopic expression of ABCB1 prevented the sensitization effect in these cells. Notably, the NFκB pathway was significantly activated by taxol, and inhibition of this pathway suppressed TLR4-regulated ABCB1 expression. Furthermore, taxol-induced NFκB signaling was reduced following TLR4 silencing in taxol-resistant SKOV3 cells. Consistent with these results, ectopic expression of TLR4 in taxol-sensitive SKOV3 cells enhanced ABCB1 expression and conferred resistance to taxol. The protective effect of exogenous TLR4 expression against taxol was reduced by treatment with NFκB inhibitor in these cells. These results demonstrate that taxol activates the TLR4-NFκB pathway which in turn induces ABCB1 gene expression. This cellular pathway thus represents a novel target to limit resistance to taxol in ovarian cancer cells.