Normal Cervical Keratinocytes Research Articles

Inhibiting TLR9 Signaling Stimulates Apoptosis and Cell Cycle Arrest, and Alleviates Angiogenic Property in Human Cervical Cancer Cells.

The aim of the study was to assess the effect of blocking TLR9 signaling on the proliferation of cervical cancer cells and its angiogenic property. Toll-Like Receptors (TLRs) have been implicated for their crucial role in not only cervical cancer but also in other malignancies. TLR9 is expressed on an array of cells such as macrophages, dendritic cells, melanocytes, and keratinocytes and is reported to modulate oncogenesis along with tumorigenesis by augmenting NF-κB mediated inflammation within the tumor environment. TLR9 has also been reported to positively regulate oncogenesis within the cervix and as a marker to evaluate malignant remodeling of cervical squamous cells. Therefore, this study was designed to explore the functional relevance of blocking the TLR9signaling pathway in cervical cancer cells. The objective of the current study was to investigate the effect of human TLR9 antagonist, ODN INH-18, on apoptosis and cell cycle regulation, and angiogenic property of human cervical cancer Caski cells. MTT assay was performed to measure cell viability and flow cytometry analysis was performed to assess cell cycle arrest. Quantitative Real-Time PCR (qRT-PCR) analysis was performed to measure fold change in the gene expression of various markers of apoptosis, cell cycle regulation, and angiogenesis. The qRT-PCR results showed a higher expression level of TLR9 mRNA in Caski cervical cancer cells as compared to normal cervical keratinocytes. The apoptotic, angiogenic, and cell cycle regulatory factors were also deregulated in Caski cells in comparison to normal keratinocytes. The MTT assay demonstrated that treatment of TLR9 antagonist, ODN INH18, significantly reduced the proliferation of Caski cells in a dose-dependent manner. Treatment of ODN INH18 led to substantial cell cycle arrest in Caski cells at G0/G1 phase. Moreover, the qRT-PCR results demonstrated that ODN INH18 treatment led to suppressed mRNA expression of Bcl-2 and enhanced expression of Bax, signifying the induction of apoptosis in Caski cells. Moreover, the expression of cyclin D1, Cdk4, and Cdc25A was found to be reduced whereas expression of p27 was increased in ODN INH18-treated Caski cells; indicating G0/G1 phase arrest. Interestingly, expression of VEGF and VCAM-1 was found to be significantly inhibited in ODN INH18-treated Caski cells, substantiating alleviation of angiogenic property of cervical cancer cells. The results of our study suggest that inhibiting TLR9 signaling might be an interesting therapeutic intervention for the treatment of cervical cancer.

High resolution analysis of the intracellular proteome of cervical cancer cell lines unveils novel regulators of cervical carcinogenesis.

Cervical cancer remains the fourth most common and most lethal type of cancer in women, despite the applied regular screening and prevention strategies, while the available treatment schemes still pose a threat to fertility. Substantial understanding of the underlying mechanisms and development of novel diagnostic, prognostic and therapeutic approaches are critical steps for improving cervical cancer management. Towards this goal, a comparative proteomic analysis was conducted between three cervical cancer cell lines (HeLa: HPV18+, SiHa: HPV16+, C33A: HPV‑) and normal cervical keratinocytes (HCK1T). The total cell extract of each cell line was analyzed by liquid chromatography coupled to tandem mass spectrometry (LC‑MS/MS). Differential expression analysis revealed 919, 826 and 1,370 deregulated proteins in the comparisons of HeLa, SiHa and C33A with HCK1T cell lines, respectively. Pathway enrichment analysis of the differentially expressed proteins highlighted common cancer characteristics such as high metabolic demands and increased cell turnover, confirming the validity of the proteomic results. Extensive literature mining of the consistently differentially expressed proteins that resulted from the three comparisons was performed leading to a shortlist of 21 proteins that are potentially involved in cervical malignancy. The criteria for this shortlisting were the association of the proteins with various types of cancer, while there is no study as yet associating their expression to cervical cancer. Moreover, the expression trend of two of the shortlisted proteins was validated using western blot analysis. The proteomic datasets generated in this study can be utilized to enrich the current knowledge on cervical cancer pathology and unveil key molecular mechanisms of carcinogenesis. In conclusion, the shortlist of consistently deregulated proteins between cervical cancer cell lines and normal cervical keratinocytes can be used for validation in clinical samples and in functional investigation experiments that could ultimately lead to the discovery of novel disease biomarkers and drug targets.

Biological Characteristics of Cervical Precancerous Cell Proliferation.

High-grade squamous intraepithelial lesions (HSILs) are regarded as precancerous lesions that can progress to cervical carcinoma; however, it is very difficult to effectively differentiate these precancerous cells from cancerous cells based on morphology alone. Additionally, the difference between precancerous cells and cancerous cells in regard to biological behaviour remains unclear. We previously cultured primary normal uterine cervical keratinocytes from human normal cervical tissue and cervical precancerous cells that were naturally infected with human papillomavirus from small-sized neoplastic cervical tissues. Here, we extended our study to further observe the in vitro proliferative characteristics of cervical precancerous cells at the cellular and molecular levels. In this study, we found that the growth rate of precancerous cells was significantly faster than that of normal cervical cells and slower than that of Caski cells. However, the proliferative capacity of such precancerous cells was similar to that of cancerous cells of the cervix at the molecular level. These results suggest that the surrounding environment of the cells may play an important role in the development of cervical cancer, which provides an important basis for the further study of precancerous and cancerous lesions of the cervix.

Suppression of miR-22, a tumor suppressor in cervical cancer, by human papillomavirus 16 E6 via a p53/miR-22/HDAC6 pathway.

MicroRNAs (miRNAs) are small non-coding RNAs that function to down-regulate gene expression involving in various cellular processes related to carcinogenesis. Recently, miR-22 was identified as a tumor-suppressing miRNA in many human cancers. However, the regulatory mechanism and the specific function of this miRNA in cervical cancer remain unclear. In the present study, we carried out gene transfection, western blot and quantitative RT-PCR to explore the regulatory mechanism and the functional role of miR-22 in cervical cancer. We verified that miR-22 was down-regulated in cervical cancer tissues and cervical cancer cell lines relative to matched non-tumor tissues and normal human cervical keratinocyte line (HCK1T). By contrast, histone deacetylase 6 (HDAC6) was inversely correlated with miR-22 in both cervical tissues and cancer cell lines. Mechanically, HDAC6 was down-regulated by miR-22 at the post-transcriptional level, via a specific target site within the 3’UTR, identified by a luciferase reporter assay. Moreover, we also showed that the correlation between miR-22 and HDAC6 expression was regulated by an E6/p53 pathway in HCK1Ts expressing HPV16 E6. For functional study, an ectopic expression of miR-22 could inhibit cell proliferation and migration, and could induce apoptosis of cervical cancer cell lines. These findings demonstrated that miR-22 was down-regulated in cervical cancer and inversely collated with its downstream target HDAC6. MiR-22 acts as tumor suppressor by inhibiting proliferation and migration, and by inducing apoptosis of cervical cancer cell lines by targeting the 3’UTR of HDAC6. This newly identified E6/p53/miR-22/HDAC6 regulatory network might be a candidate therapeutic target for cervical cancer.

Inhibition of tumor cell growth by adenine is mediated by apoptosis induction and cell cycle S phase arrest.

Gekko swinhonis has a long standing history in Chinese traditional medicine recognized for its application in treating patients with terminal cancer.In order to discover novel anticancer drugs with high anti-tumor efficacy and low toxicity to normal cells, we aim to investigate the anti-tumor components from Gekko swinhonis. Four nucleosides from the extracted samples were enriched, namely adenosine, guanosine, thymidine and inosine. We evaluated the antitumor effect of the four nucleosides and found that adenosine possessed the strongest anti-tumor effect. Besides, adenine could inhibit the growth of Bel-7402 and Hela cells in a dose and time dependent manner, but not normal human cervical keratinocytes. Bel-7402 and Hela cells had undergone apoptosis 48 hours after treatment as evidenced by morphologic changes under TEM, while adenine blocked cell cycle of tumor cells at S phase and subsequently causing cell cycle exit and promoting apoptosis. Moreover, the pharmacokinetics of adenine was stable in cell culture medium for up to 72 hours. Combining its potency with stability, we conclude adenine makes a promising candidate for an anti-tumor drug.

Roles of the PDZ-binding motif of HPV 16 E6 protein in oncogenic transformation of human cervical keratinocytes.

The high‐risk human papillomavirus E6 proteins have been shown to interact with and lead to degradation of PDZ‐domain‐containing proteins through its carboxy‐terminal motif. This PDZ‐binding motif plays important roles in transformation of cultured cells and carcinogenesis of E6‐transgenic mice. However, its biological effects on the natural host cells have not been elucidated. We have examined its roles in an in vitro carcinogenesis model for cervical cancer, in which E6 and E7 together with activated HRAS (HRASG 12V) can induce tumorigenic transformation of normal human cervical keratinocytes. In this model, E6Δ151 mutant, which is defective in binding to PDZ domains, almost lost tumorigenic ability, whereas E6SAT mutant, which is defective in p53 degradation showed activity close to wild‐type E6. Interestingly, we found decreased expression of PAR3 in E6‐expressing cells independently of E6AP, which has not been previously recognized. Therefore, we knocked down several PDZ‐domain containing proteins including PAR3 in human cervical keratinocytes expressing E7, HRASG 12V and E6Δ151 to examine whether depletion of these proteins can restore the tumorigenic ability. Single knockdown of SCRIB, MAGI1 or PAR3 significantly but partially restored the tumorigenic ability. The combinatorial knockdown of SCRIB and MAGI1 cooperatively restored the tumorigenic ability, and additional depletion of PAR3 further enhanced the tumorigenic ability surpassing that induced by wild‐type E6. These data highlight the importance of the carboxy‐terminal motif of the E6 protein and downregulation of PAR3 in tumorigenic transformation of human cervical keratinocytes.

RETRACTED ARTICLE: miR-484 suppresses proliferation and epithelial–mesenchymal transition by targeting ZEB1 and SMAD2 in cervical cancer cells

BackgroundMicroRNAs (miRNAs) play important roles in cancer initiation and development. Epithelial–mesenchymal transition (EMT) is a form of cellular plasticity that is critical for embryonic development and metastasis. The purpose of the study was to determine the function and mechanism of miR-484 in initiation and development of cervical cancer (CC).MethodsWe determined the expression levels of miR-484 in cervical cancer tissues and cell lines with RT-qPCR. Prediction algorithms and EGFP reporter assay were performed to evaluate the targets for miR-484. MTT assay, colony formation assay, flow cytometric analysis, transwell cell migration and invasion assays, and detection of EMT markers were employed to investigate the roles of miR-484 and the targets in regulation of cell proliferation and EMT process. We also used rescue experiments to confirm the effect of miR-484 on CC cells through directly regulating the expression of its targets.ResultsFirstly we found miR-484 was down-regulated in cervical cancer tissues and cell lines compared with their matched non-cancerous tissues or normal cervical keratinocytes cells. Further studies revealed that overexpression of miR-484 suppressed the cell proliferation, while exacerbates apoptosis. Besides, miR-484 suppressed cellular migration, invasion and EMT process of CC cells. EGFP reporter assay showed that miR-484 binds to ZEB1 and SMAD2 3′UTR region and reduced their expression. The expression of miR-484 had reverse correlation with SMAD2/ZEB1, and SMAD2/ZEB1 had positive correlation with each other in cervical cancer tissues and cell lines. Furthermore, the ectopic expression of ZEB1 or SMAD2 could rescue the malignancies suppressed by miR-484, suggesting that miR-484 down-regulates ZEB1 and SMAD2 to repress tumorigenic activities.ConclusionWe found miR-484 inhibits cell proliferation and the EMT process by targeting both ZEB1 and SMAD2 genes and functions as a tumor suppressor, which may served as potential biomarkers for cervical cancer.

Gene expression changes in progression of cervical neoplasia revealed by microarray analysis of cervical neoplastic keratinocytes.

To evaluate the gene expression changes involved in neoplastic progression of cervical intraepithelial neoplasia. Using microarray analysis, large-scale gene expression profile was carried out on HPV16-CIN2, HPV16-CIN3, and normal cervical keratinocytes derived from two HPV16-CIN2, two HPV-CIN3 lesions, and two corresponding normal cervical tissues, respectively. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16-CIN2 keratinocytes and in HPV16-CIN2 keratinocytes compared with HPV16-CIN3 keratinocytes; 37 candidate genes with continuously increasing or decreasing expression during CIN progression were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of CIN toward cancer. Gene expression changes occurring during CIN progression were investigated using microarray analysis, for the first time, in CIN2 and CIN3 keratinocytes naturally infected with HPV16. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression.

Establishment of a novel method for primary culture of normal human cervical keratinocytes

Background Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum (FBS). However, it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor. Therefore, the culture of these cells is extremely difficult. In this study, we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro. Methods Normal cervical epithelial tissue pieces were obtained and digested with type I collagenase to dissociate the cells and a single cell suspension produced. The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type I from rat tail, with modified keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS. After attachment, the medium were replaced with K-SFM without FBS. The expression of basal keratins of the ectocervical epithelium, K5, K14 and K19 were assayed by immunofluorescence with monoclonal antibodies to identify the cell purity. Results Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5% FBS than in K-SFM alone, as well as to tissue culture plastic coated with collagen type I than plastic alone. The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type I from rat tail was the best method for culture of normal cervical epithelial cells. K5, K14 and K19 were assayed and keratinocyte purity was nearly 100%. Conclusion A novel, simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.

A critical role of MYC for transformation of human cells by HPV16 E6E7 and oncogenic HRAS

Human papillomaviruses (HPVs) are the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 is considered to contribute to disease initiation. Recently, we have demonstrated that transduction of oncogenic HRAS (HRAS(G12V)) and MYC together with HPV16 E6E7 is sufficient for tumorigenic transformation of normal human cervical keratinocytes (HCKs). Here, we show that transduction of HRAS(G12V) on the background of E6E7 expression causes accumulation of MYC protein and tumorigenic transformation of not only normal HCKs but also other normal primary human cells, including tongue keratinocytes and bronchial epithelial cells as well as hTERT-immortalized foreskin fibroblasts. Subcutaneous transplantation of as few as 200 HCKs expressing E6E7 and HRAS(G12V) resulted in tumor formation within 2 months. Dissecting RAS signaling pathways, constitutively active forms of AKT1 or MEK1 did not result in tumor formation with E6E7, but tumorigenic transformation was induced with addition of MYC. Increased MYC expression endowed resistance to calcium- and serum-induced terminal differentiation and activated the mammalian target of rapamycin (mTOR) pathway. An mTOR inhibitor (Rapamycin) and MYC inhibition a level not affecting proliferation in culture both markedly suppressed tumor formation by HCKs expressing E6E7 and HRAS(G12V). These results suggest that a single mutation of HRAS could be oncogenic in the background of deregulated expression of E6E7 and MYC plays a critical role in cooperation with the RAS signaling pathways in tumorigenesis. Thus inhibition of MYC and/or the downstream mTOR pathway could be a therapeutic strategy not only for the MYC-altered but also RAS-activated cancers.

Expression of Tissue factor in Adenocarcinoma and Squamous Cell Carcinoma of the Uterine Cervix: Implications for immunotherapy with hI-con1, a factor VII-IgGFcchimeric protein targeting tissue factor

BackgroundCervical cancer continues to be an important worldwide health problem for women. Up to 35% of patients who are diagnosed with and appropriately treated for cervical cancer will recur and treatment results are poor for recurrent disease. Given these sobering statistics, development of novel therapies for cervical cancer remains a high priority. We evaluated the expression of Tissue Factor (TF) in cervical cancer and the potential of hI-con1, an antibody-like-molecule targeted against TF, as a novel form of immunotherapy against multiple primary cervical carcinoma cell lines with squamous- and adenocarcinoma histology.MethodsBecause TF is a transmembrane receptor for coagulation factor VII/VIIa (fVII), in this study we evaluated the in vitro expression of TF in cervical carcinoma cell lines by immunohistochemistry (IHC), real time-PCR (qRT-PCR) and flow cytometry. Sensitivity to hI-con1-dependent cell-mediated-cytotoxicity (IDCC) was evaluated in 5-hrs-51chromium-release-assays against cervical cancer cell lines in vitro.ResultsCytoplasmic and/or membrane TF expression was observed in 8 out of 8 (100%) of the tumor tissues tested by IHC and in 100% (11 out of 11) of the cervical carcinoma cell lines tested by real-time-PCR and flow cytometry but not in normal cervical keratinocytes (p = 0.0023 qRT-PCR; p = 0.0042 flow cytometry). All primary cervical cancer cell lines tested overexpressing TF, regardless of their histology, were highly sensitive to IDCC (mean killing ± SD, 56.2% ± 15.9%, range, 32.4%-76.9%, p < 0.001), while negligible cytotoxicity was seen in the absence of hI-con1 or in the presence of rituximab-control-antibody. Low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (p = 0.025) while human serum did not significantly decrease IDCC against cervical cancer cell lines (p = 0.597).ConclusionsTF is highly expressed in squamous and adenocarcinoma of the uterine cervix. hI-con1 induces strong cytotoxicity against primary cervical cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of cervical cancer refractory to standard treatment modalities.

An In vitro Multistep Carcinogenesis Model for Human Cervical Cancer

Human papillomaviruses (HPV) are believed to be the primary causal agents for development of cervical cancer, and deregulated expression of two viral oncogenes E6 and E7 in basal cells, mostly by integration, is considered to be a critical event for disease progression. However, lines of evidence suggest that, besides expression of E6 and E7 genes, additional host genetic alterations are required for cancer development. To directly test this hypothesis, we first transduced HPV16 E6 and E7 with or without hTERT into several lines of normal human cervical keratinocytes (HCK) from independent donors and then searched for additional alterations required for carcinogenesis. Oncogenic Hras(G12V) (Hras) provided marked tumor forming ability in nude mice and ErbB2 or c-Myc (Myc) endowed weaker but significant tumor forming ability. Combined transduction of Myc and Hras to HCKs expressing E6 and E7 resulted in the creation of highly potent tumor-initiating cells. These results show that only one or two genetic changes occurring after deregulated expression of high-risk HPV oncogenes might be sufficient for development of cervical cancer.

Molecular basis of cervical carcinogenesis by high-risk human papillomaviruses

Over the last two decades since discovery of human papillomavirus (HPV) type 16 and 18 DNAs in cervical cancers by Dr. Harald zur Hausen, HPVs have been well characterized as causative agents for cervical cancer. Viral DNA from a specific group of HPVs can be detected in at least 90% of all cervical cancers and two viral genes, E6 and E7, are invariably expressed in HPV-positive cervical cancer cells. Their gene products are known to inactivate the major tumor suppressors, p53 and pRB, respectively. In addition, one function of E6 is to activate telomerase, and E6 and E7 cooperate to effectively immortalize human primary epithelial cells. Though expression of E6 and E7 is itself not sufficient for cancer development, it seems to be either directly or indirectly involved in every stage of multi-step carcinogenesis. Indeed, it has been shown that only one or two genetic alterations in addition to expression of E6 and E7 are experimentally sufficient to confer tumorigenicity to normal human cervical keratinocytes. Epidemiological and biological studies suggest the potential efficacy of prophylactic vaccines to prevent genital HPV infection as an anti-cancer strategy. However, given the widespread nature of HPV infection and unresolved issues about the duration and type specificity of the currently available HPV vaccines, it is crucial that molecular details of the natural history of HPV infection as well as the biological activities of the viral oncoproteins be elucidated in order to provide the basis for development of new therapeutic strategies against HPV-associated malignancies. This review highlights the novel functions of E6 and E7 as well as the molecular mechanisms of HPV-induced carcinogenesis.

Gene expression profiles of primary HPV16- and HPV18-infected early stage cervical cancers and normal cervical epithelium: identification of novel candidate molecular markers for cervical cancer diagnosis and therapy

With the goal of identifying genes with a differential pattern of expression between invasive cervical carcinomas (CVX) and normal cervical keratinocytes (NCK), we used oligonucleotide microarrays to interrogate the expression of 14,500 known genes in 11 primary HPV16 and HPV18-infected stage IB–IIA cervical cancers and four primary normal cervical keratinocyte cultures. Hierarchical cluster analysis of gene expression data identified 240 and 265 genes that exhibited greater than twofold up-regulation and down-regulation, respectively, in primary CVX when compared to NCK. Cyclin-dependent kinase inhibitor 2A (CDKN2A/p16), mesoderm-specific transcript, forkhead box M1, v-myb myeloblastosis viral oncogene homolog (avian)-like2 (v-Myb), minichromosome maintenance proteins 2, 4, and 5, cyclin B1, prostaglandin E synthase (PTGES), topoisomerase II alpha (TOP2A), ubiquitin-conjugating enzyme E2C, CD97 antigen, E2F transcription factor 1, and dUTP pyrophosphatase were among the most highly overexpressed genes in CVX when compared to NCK. Down-regulated genes in CVX included transforming growth factor beta 1, transforming growth factor alpha, CFLAR, serine proteinase inhibitors (SERPING1 and SERPINF1), cadherin 13, protease inhibitor 3, keratin 16, and tissue factor pathway inhibitor-2 (TFPI-2). Differential expression of some of these genes including CDKN2A/p16, v-Myb, PTGES, and TOP2A was validated by quantitative real-time PCR. Flow cytometry on primary CVX and NCK and immunohistochemical staining of formalin fixed paraffin-embedded tumor specimens from which primary CVX cultures were derived as well as from a separate set of invasive cervical cancers confirmed differential expression of the CDKN2A/p16 and PTGES markers on CVX versus NCK. These results identify several genes that are coordinately disregulated in cervical cancer, likely representing common signaling pathways triggered by HPV transformation. Moreover, these data obtained with highly purified primary tumor cultures highlight novel molecular features of human cervical cancer and provide a foundation for the development of new type-specific diagnostic and therapeutic strategies for this disease.

The serine protease stratum corneum chymotryptic enzyme (kallikrein 7) is highly overexpressed in squamous cervical cancer cells

Objective. To determine whether the Stratum Corneum Chymotryptic Enzyme (SCCE), a novel serine protease known to contribute to the cell shedding process by catalyzing the degradation of intercellular cohesive structures at the skin surface, is overexpressed in human cervical tumors. Methods. SCCE expression was evaluated in 18 cervical cancer cell lines (i.e., 10 primary and 8 established cell lines) as well as in 8 normal cervical keratinocyte cultures by RT-PCR. In addition, SCCE expression was evaluated by immunohistochemistry on paraffin-embedded tumor tissue. Results. Normal cervical keratinocytes did not express SCCE. In contrast, 50% of the primary and 50% of the established cervical cancer cell lines expressed SCCE by RT-PCR. Eighty percent (i.e., four of five) of primary squamous cervical tumors and 20% (i.e., one of five) of primary adenocarcinomas expressed SCCE. Five out of five (100%) of the patients harboring SCCE-positive tumors were found to have metastatic involvement of the pelvic tumor draining lymph nodes. Immunohistochemistry staining of paraffin-embedded cervical cancer specimens confirmed SCCE expression in tumor cells and its absence on normal cervical epithelial cells. Conclusion. Squamous cervical cancer expressed high levels of SCCE, suggesting that this protease may play an important role in invasion and metastasis. Because SCCE appears only in abundance in tumor tissue and contains a secretion signal sequence, suggesting that SCCE is secreted, it may prove to be a useful diagnostic/prognostic tool for the detection of metastatic or recurrent disease or as a novel molecular target for cervical cancer therapy.

The novel serine protease tumor-associated differentially expressed gene-14 (KLK8/Neuropsin/Ovasin) is highly overexpressed in cervical cancer

Objective Serine proteases are redundant enzymes implicated in the extracellular modulation required for tumor growth and invasion. Tumor-associated differentially expressed gene-14 (TADG-14) is a novel transmembrane serine protease recently reported by our group to be highly overexpressed in ovarian carcinomas. The goal of this study was to investigate the frequency of expression of the TADG-14 gene in human cervical tumors. Study design TADG-14 expression was evaluated in 19 cervical cancer cell lines (11 primary and 8 established cell lines) as well as in 8 normal cervical keratinocyte cultures by reverse transcriptase polymerase chain reaction. In addition, to validate gene expression data at the protein level, TADG-14 expression was evaluated by immunohistochemistry on paraffin-embedded tissue from which all 11 primary tumor cell lines were established. Results TADG-14 was found to be highly expressed in 82% (9/11) primary cervical cancer cell lines and in 87% (7/8) established cervical cancer cell lines by reverse transcriptase–polymerase chain reaction. Expression of TADG-14 by primary squamous cervical tumors was 100% (6/6), whereas 60% (3/5) of primary adenocarcinomas expressed TADG-14. In contrast, none of the normal cervical keratinocyte control cultures (n = 4) or flash frozen normal cervical biopsy specimens (n = 4) expressed TADG-14. Immunohistochemistry staining of paraffin-embedded cervical cancer specimens confirmed TADG-14 expression in tumor cells and its absence on normal cervical epithelial cells. Conclusion Cervical cancer expressed a high level of TADG-14, suggesting that this protease may play an important role in invasion and metastasis. Because TADG-14 appears only in abundance in tumor tissue and contains a secretion signal sequence, suggesting that TADG-14 is secreted, it may prove to be a useful diagnostic tool for the early detection of recurrent/persistent cervical cancer after standard treatment or as a novel molecular target for cervical cancer therapy.

The novel serine protease tumor-associated differentially expressed gene-15 (matriptase/MT-SP1) is highly overexpressed in cervical carcinoma.

Tumor-associated differentially expressed gene-15 (TADG-15/matriptase/MT-SP1) is a novel transmembrane serine protease involved in numerous biologic processes, including activation of growth and angiogenic factors and degradation of extracellular matrix components. To assess the value of TADG-15 as a possible marker for tumor detection and/or as a target for therapeutic intervention, the authors investigated the frequency of expression of TADG-15 in human cervical tumors. TADG-15 expression was evaluated in 19 cervical carcinoma cell lines (i.e., 11 primary tumor cell lines and 8 established cell lines) and in 8 normal cervical keratinocyte control cultures using reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, to validate gene expression data at the protein level, TADG-15 expression was evaluated by immunohistochemistry on paraffin embedded tissue from which all 11 primary tumor cell lines were established. TADG-15 was expressed at high levels in 8 of 11 (73%) primary cervical carcinoma cell lines and in 6 of 8 (75%) established cervical carcinoma cell lines by RT-PCR. Expression of TADG-15 was found in 6 of 6 (100%) primary squamous cell cervical carcinomas, whereas 2 of 5 (40%) primary adenocarcinomas expressed TADG-15. In contrast, none of the normal cervical keratinocyte control cultures (n = 4) or flash-frozen normal cervical biopsy specimens (n = 4) expressed TADG-15. Immunohistochemistry staining of paraffin embedded cervical carcinoma specimens confirmed TADG-15 expression in tumor cells and its absence on normal cervical epithelial cells. Cervical carcinoma cells expressed high levels of TADG-15, suggesting that this protease may play an important role in invasion and metastasis. Because TADG-15 appears only in abundance in squamous tumor tissue and contains a proteolytic cleavage site, suggesting that the TADG-15 protease domain is released, it may prove to be a useful diagnostic tool for the early detection of recurrent/persistent cervical carcinoma after standard treatment or as a novel molecular target for therapy in patients with cervical carcinoma.

HPV 16-E6-mediated degradation of intrinsic p53 is compensated by upregulation of p53 gene expression in normal cervical keratinocytes

In this study we investigated the relationship between the development of either HSILs (high-grade squamous intraepithelial lesions) or invasive cancers of the uterine cervix and the p53 codon 72 polymorphisms consisting of arginine (Arg)- or proline (Pro)-encoded allele in Japanese populations. Furthermore, we determined if intrinsic p53 is affected by the p53-degradation activities of human papillomavirus type 16 (HPV 16)-E6 in normal cervical keratinocytes with each p53 codon 72 genotype. Using PCR with p53 codon 72 polymorphic allele-specific primers, p53 genotypes of 112 normal cervical specimens and 88 cervical lesions including 44 HSILs and 44 invasive cancers were determined. The expression levels of p53 proteins and mRNAs in keratinocytes following the expression of HPV 16-E6 from an HPV 16-E6-expressing recombinant adenovirus were analyzed. The frequencies of p53 genotypes, Pro/Pro, Arg/Pro and Arg/Arg were, respectively, 12, 49 and 39% in controls; and 18, 55 and 27% in HSILs; and 11, 41 and 48% in invasive cancers. The expression levels of the p53 proteins in normal human keratinocytes of each p53 codon 72 genotype were not affected by the introduction of HPV 16-E6, whereas the p53 mRNAs were found to be upregulated regardless of the p53 genotypes when HPV 16-E6 was expressed. In statistical analysis, no relationships between p53 genotypes and the development of cervical neoplasias were found. Furthermore, we demonstrated that p53 protein expression levels in normal cervical keratinocytes is not affected by the p53-degradation activities of HPV E6, probably due to a tight transcriptional regulation of p53.

Radiosensitizing Effect of Administration of 13-cis-Retinoic Acid and Interferon-Alpha-2a in Normal Cervical Keratinocyte and Cervical Cancer Cell Lines

Radiosensitizing Effect of Administration of 13-cis-Retinoic Acid and Interferon-Alpha-2a in Normal Cervical Keratinocyte and Cervical Cancer Cell Lines

Tumor necrosis factor-alpha promotes human papillomavirus (HPV) E6/E7 RNA expression and cyclin-dependent kinase activity in HPV-immortalized keratinocytes by a ras-dependent pathway.

Tumor necrosis factor-alpha (TNF-alpha) inhibits growth of normal cervical keratinocytes but stimulates proliferation of human papillomavirus (HPV)-immortalized and cervical carcinoma-derived cell lines when mitogens such as epidermal growth factor (EGF) or serum are depleted. Current work identifies the mechanism of growth stimulation. TNF-alpha promoted cell cycle progression by increasing expression of HPV-16 E6/E7 RNAs and enhancing activity of cyclin-dependent kinase (cdk)2 and cdc2 after 3 d. Increased kinase activity was mediated by upregulation of cyclins A and B and decreases in cdk inhibitors p21(waf) and p27(kip). TNF-alpha stimulated these changes in part by increasing transcription and stabilization of RNA for amphiregulin, an EGF receptor ligand, and amphiregulin directly increased HPV-16 E6/E7 and cyclin A RNAs. To define which components of the EGF receptor signaling pathway were important, HPV-immortalized cells were transfected with activated or dominant negative mutants of Ha-ras, raf, or MAPKK. Expression of activated Ha-ras maintained HPV-16 and cyclin gene expression and promoted rapid growth in the absence of EGF. Furthermore, ras activation was necessary for TNF-alpha mitogenesis as transfection with a dominant negative ras mutant (Asn-17) strongly inhibited growth. Thus, activation of ras promotes expression of HPV-16 E6/E7 RNAs, induces cyclins A and B, and mediates growth stimulation of immortal keratinocytes by TNF-alpha. These studies define a pathway by which ras mutations, which occur in a subset of cervical cancers, may contribute to pathogenesis. Mol. Carcinog. 27:97-109, 2000. Published by Wiley-Liss, Inc.