Primary Cervical Cancer Tissues Research Articles

The regulating role of miR-494 on HCCR1 in cervical cancer cells.

This study focused on miR-494 inhibiting the proliferation, migration and invasion of cervical cancer cells by regulating HCCR1. During the study, 30 pairs of primary cervical cancer tissues and adjacent tissues diagnosed by pathology were collected, and then placed in a cryopreservation tube, immediately placed in liquid nitrogen to freeze, and then moved to a -80 °C refrigerator for storage until use. They were dipped with crystal violet staining solution for 20 minutes, washed 3 times with PBS, and dried at room temperature. The polycarbonate film on the chamber was gently cut, placed on a glass slide, and covered with neutral resin. The number of cells in 5 random fields was counted under a microscope, and the differences between the groups were compared. The qRT-PCR results showed that compared with the normal cervical tissue cell line HCCR1, the expression levels of miR-494 in cervical cancer cell lines HCCR1 and C-33A were significantly reduced. Compared with the transfection of mimics NC, the level of HCCR1mRNA and protein decreased significantly when transfected with miRNA-494mimics (P <0.05); compared with the transfection of inhibitor NC, the level of HCCR1 mRNA and protein increased significantly when transfected with miRNA-494 inhibitor (P <0.05).

Circular RNA circNFATC3 acts as a miR-9-5p sponge to promote cervical cancer development by upregulating SDC2.

Circular RNAs (circRNAs) constitute a class of regulatory RNAs that are thought to play important roles in tumor initiation and progression. Several studies have reported that circRNAs may be involved in various biological processes via networks of competing endogenous RNAs (ceRNAs). However, the regulatory roles and underlying mechanisms of circRNAs in cervical cancer (CC) still largely remain to be resolved. CircNFATC3 (hsa_circ_0005615) expression was assessed in CC cell lines (SiHa, H8) using circRNA microarray analysis, whereas qRT-PCR was used to detect circNFATC3 and miR-9-5p expression in primary human CC tissues and cell lines. The tumor promoting role of circNFATC3 was verified in CC cells using a series of functional assays, and interactions between circNFATC3, miR-9-5p and syndecan-2 (SDC2) were investigated using dual-luciferase reporter assays. SDC2 protein expression was detected using Western blotting and immunohistochemistry. The tumor promoting role of circNFATC3 was confirmed in vivo using a CC xenograft model. We found that circNFATC3 expression was upregulated in primary CC tissues and positively correlated with CC tumor size and stromal invasion. In addition, we found that exogenous circNFATC3 overexpression enhanced the proliferation, migration and invasion of HeLa cells, while its knockdown reduced the malignancy of SiHa cells. We also found that circNFATC3 may act directly as a miR-9-5p sponge to regulate SDC2 expression and its downstream signaling pathways, thereby enhancing CC development. Our data indicate that circNFATC3 sponges miR-9-5p to regulate SDC2 expression and, thereby, to promote CC tumor development.

Identification of lysine acetylome in cervical cancer by label-free quantitative proteomics

BackgroundLysine acetylation is a post-translational modification that regulates a diversity of biological processes, including cancer development.MethodsHere, we performed the quantitative acetylproteomic analysis of three primary cervical cancer tissues and corresponding adjacent normal tissues by using the label-free proteomics approach.ResultsWe identified a total of 928 lysine acetylation sites from 1547 proteins, in which 495 lysine acetylation sites corresponding to 296 proteins were quantified. Further, 41 differentially expressed lysine acetylation sites corresponding to 30 proteins were obtained in cervical cancer tissues compared with adjacent normal tissues (Fold change > 2 and P < 0.05), of which 1 was downregulated, 40 were upregulated. Moreover, 75 lysine acetylation sites corresponding to 58 proteins were specifically detected in cancer tissues or normal adjacent tissues. Motif-X analysis showed that kxxxkxxxk, GkL, AxxEk, kLxE, and kkxxxk are the most enriched motifs with over four-fold increases when compared with the background matches. KEGG analysis showed that proteins identified from differently and specifically expressed peptides may influence key pathways, such as Notch signaling pathway, viral carcinogenesis, RNA transport, and Jak-STAT, which play an important role in tumor progression. Furthermore, the acetylated levels of CREBBP and S100A9 in cervical cancer tissues were confirmed by immunoprecipitation (IP) and Western blot analysis.ConclusionsTaken together, our data provide novel insights into the role of protein lysine acetylation in cervical carcinogenesis.

Abstract 1710: Role of Nurr1 in cervical cancer pathogenic pathway

Abstract Aims: To study the oncogenic role of Nurr1 in cervical cancer and elucidate the underlying mechanism. Background: Orphan nuclear receptor related-1 protein (Nurr1) belonging to the NR4A family is important for carcinogenesis in multiple biological processes such as cell proliferation and apoptosis. It has been reported that the expression of Nurr1 is significantly correlated with various types of cancer, such as prostate and breast cancer. However, the significance of Nurr1 in cervical cancer etiology remains poorly understood. Methods: Primary cancer cells were established from tumor tissues resected from cervical cancer patients. Six pairs of attached and spheroid cells derived from primary cervical cancer tissues were established namely CP-1A/CP-1S, CP-2A/CP-2S, CP-3A/CP-3S, CP-4A/CP-4S, CP-5A/CP-5S and CP-6A/CP-6S. A microarray was performed to identify differentially expressed genes in two pairs of attached and sphere cells, followed by validation with qPCR. Stable clones of cervical cancer cell lines with Nurr1 overexpression and knock-down were generated. The role of the selected gene was then functionally characterized by XTT assay, migration assay, spheroid formation assay, apoptosis assay, in vitro and in vivo tumorigenicity assay in cervical cancer cell lines. Downstream proteins of Nurr1 were detected by western blot. Results: Microarray analysis showed that Nurr1 was highly expressed in sphere cells as compared with attached cells. Relative expression of Nurr1 mRNA in sphere cells was approximately 200-fold more than that in attached cells in qPCR validation. Cancer cell lines, in which Nurr1 was transiently knocked down by siRNA demonstrated an attenuation in proliferation rate, migration, spheroid formation and colony formation. By flow cytometric analysis, more cells were detected in early apoptotic stage with Nurr1 siRNA transfection as compared with NTC siRNA transfected cells, suggesting the role of Nurr1 in preventing cervical cancer cells from apoptosis. Western blotting also demonstrated that there was an increased expression of Bim and p21 in Nurr1 silenced cells. This may indicate the involvement of Nurr1 in modulating the transcription of pro-apoptotic and tumor suppressor genes. Conclusion: Nurr1 is a potential oncogenic target in cervical cancer pathogenic pathway. Suppression of Nurr1 expression attenuated cancer progression and promoted apoptosis. Citation Format: Kok Ting Wan, Thomas Ho Yin Leung, Michelle Kwan Yee Siu, Hextan Yuen Sheung Ngan. Role of Nurr1 in cervical cancer pathogenic pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1710.

PO-296 Elucidating the potential role of CD109 as a biomarker for cancer stem-like cells in cervical cancer

IntroductionCervical cancer is a common genital tract cancer. Radiotherapy is the mainstay of management for advanced cervical cancer. Response to radiation varies widely which may be explained by the existence of cancer stem-like cells (CSCs). Since CSCs is implicated in cervical cancer and demonstrated a high degree of resistance to radiation, the identification of novel CSC markers could be critical to specifically target the cervical CSC. In our pilot study, we established attached and spheroid cells from primary cervical tumour tissue. Multiple ‘stemness’ genes were detected in spheroid cells which indicated primary cervical cancer tissue harboured CSCs population. cDNA microarray analysis was performed to compare cDNA expression profile and CD109 was significantly up-regulated in spheroid cells. Therefore, we hypothesise that CD109 may serve as a potential cervical CSCs marker.Material and methodsFlow cytometry was performed to analyse the CD109 expression and isolate the CD109 positive and negative sub-populations in cervical cancer cells. Following the sorting, cell proliferation and migration assay were performed. In order to investigate the effect on the blockade of CD109, SiHa, Caski and C4-1 cells were transfected with CD109 siRNAs. XTT assay, migration and invasion assay and spheroid formation assay were performed. Immunohistochemistry (IHC) was performed for the detection of CD109 expression in cervical cancer tissue microarray (TMA).Results and discussionsThe post-sorted CD109 (+) cells grew remarkably faster and have stronger migration capability than CD109(-) cells in Caski and C4-1. The CD109 knockdown cells with siRNA exhibited a slower cell growth, decreasing migration and self-renewal ability, as compared with the control group in SiHa, Caski and C4-1 cells. IHC of TMA indicated that CD109 was highly expressed in cancer cases than that in normal/benign cases.ConclusionCD109 increased cell proliferation rate and migration ability in post-sorted cervical cancer cell lines Caski and C4-1. On the contrary, CD109 knockdown reduced cell growth, migration and self-renewal capability in cervical cancer SiHa, Caski and C4-1 cells. Cervical carcinoma showed high expression of CD109 protein by IHC. Further in vivo and in vitro functional assays are essential to characterise the CD109-positive sub-population in cervical cancer which may provide more information of cervical CSCs-related properties and resistance of radiation therapy, with the underlying molecular mechanism involved.

MicroRNA-204 inhibits cell migration and invasion in human cervical cancer by regulating transcription factor 12.

Deregulated microRNAs (miRs) and their roles in carcinogenesis have attracted great attention in recent years. Although miR-204 was reportedly dysregulated in various types of cancer, its function and mechanism in cervical cancer remain unknown. The present study focused on the expression and mechanisms of miR-204 in cervical cancer development. Expression of miR-204 in cervical cancer tissues and non-tumor tissues was measured using PCR analysis. The effect of ectopic expression of miR-204 on cell motility was evaluated using wound-healing and Transwell invasion assays. Luciferase activity and western blot assays were used to verify the regulatory effect of miR-204 on its target gene. It was demonstrated that miR-204 was significantly decreased in primary cervical cancer tissues, and that downregulated miR-204 was associated with lymph node metastasis and poor survival. In addition, it was revealed that ectopic expression of miR-204 significantly inhibited the migratory and invasive ability of cervical cancer cells in vitro. In addition, bioinformatic prediction and experimental validation demonstrated that transcription factor 12 (TCF12) was a direct target of miR-204. Overexpression of TCF12 attenuated the inhibitory effect of miR-204 on cell motility. Taken together, the present data indicated that miR-204 is a metastasis-associated gene and may contribute to the progression of cervical cancer by regulating TCF12, providing novel insights, including that miR-204/TCF12 may be an important mechanism for cervical cancer metastasis.

MiR-9, miR-21, and miR-155 as potential biomarkers for HPV positive and negative cervical cancer

BackgroundCervical cancer is the second leading cause of death among female patients with cancer in the world. High risk human papillomavirus has causal roles in cervical cancer initiation and progression by deregulating several cellular processes. However, HPV infection is not sufficient for cervical carcinoma development. Therefore, other genetic and epigenetic factors may be involved in this complex disease, and the identification of which may lead to better diagnosis and treatment. Our aim was to analyze the expression of microRNAs in cervical cancer cases positive or negative for HPV E6/E7 mRNA, and to assess their diagnostic usefulness and relevance.MethodsThe expression of three different microRNAs (miR-9, miR-21, and miR-155) in 52 formalin-fixed paraffin-embedded (FFPE) primary cervical cancer tissue samples and 50 FFPE normal cervical tissue samples were evaluated.ResultsMiR-9, miR-21, and miR-155 were significantly overexpressed in cervical cancer tissues compared to normal tissues (P < 0.001). MiR-21 and miR-155 expression combined with the HPV E6/E7 mRNA assay in HPV E6/E7 negative cervical cancer showed increased AUC of 0.7267 and 0.7000, respectively (P = 0.01, P = 0.04), demonstrating their potential as diagnostic tools. Moreover, miR-21 and miR-155 were predictors showing a 7 fold and 10.3 fold higher risk for HPV E6/E7 negative patients with cervical cancer (P = 0.024 and P = 0.017, respectively) while miR-155 was a predictor showing a 27.9 fold higher risk for HPV E6/E7 positive patients with cervical cancer (P < 0.0001).ConclusionsThere is a strong demand for additional, alternative molecular biomarkers for diagnosis and management of precancer patients. MiR-21 and miR-155 may be helpful in the prediction of both HPV positive and HPV negative cases of cervical cancer.

Long Noncoding RNA GAS5, Which Acts as a Tumor Suppressor via microRNA 21, Regulates Cisplatin Resistance Expression in Cervical Cancer.

ObjectivesThe aims of this study were to investigate the functions of GAS5 as a tumor suppressor in cervical cancer and explore the mechanism.MethodsThe expression of GAS5 and microRNA 21 (miR-21) was detected in primary cervical cancer tissue specimens, as well as in cervical cancer cell lines. We identified the interaction of GAS5 and miR-21 by quantitative polymerase chain reaction, Western blot, and dual-luciferase reporter assay. We also studied the functions of GAS5 in proliferation, apoptosis, migration, and invasion in cervical cancer cells in vitro and vivo. Finally, the impact of GAS5 on cisplatin resistance and its mechanism in cervical cancer cells was also identified.ResultsThe expression of GAS5 and miR-21 was detected in primary cervical cancer tissue specimens, as well as in cervical cancer cell lines. GAS5, which is a tumor suppressor playing roles in inhibiting the malignancy of cervical cancer cells, including proliferation in vivo and vitro, migration, and invasion, has a low expression in cervical cancer tissue and cervical cancer cell lines, whereas miR-21 expression is high. GAS5 significantly decreased the expression of miR-21, and there is a reciprocal repression of gene expression between GAS5 and miR-21. Besides, most importantly, we found that high expression of GAS5 and low expression of miR-21 can enhance the sensitivity of SiHa/cDDP cancer cells to cisplatin. A further experiment for identifying the mechanism of cisplatin resistance by GAS5 showed that GAS5 can not only regulate phosphatase and tensin homolog through miR-21 but also influence the phosphorylation of Akt.ConclusionsOur results indicate that GAS5 is a direct target of miR-21 and can predict the clinical staging of cervical cancer. Most importantly, GAS5 can also influence cisplatin resistance in cervical cancer via regulating the phosphorylation of Akt. All of these suggest that GAS5 may be a novel therapeutic target for treating cervical cancer.

Feasibility of miR-9, miR-21, and miR-155 as alternative biomarkers in both HPV-positive and HPV-negative cervical cancers.

52 Background: Cervical cancer is the second leading cause of death among female patients with cancer in the world. High risk human papillomavirus has causal roles in cervical cancer initiation and progression by deregulating several cellular processes. However, HPV infection is not sufficient for cervical carcinoma development. Therefore, other genetic and epigenetic factors may be involved in this complex disease, and the identification of which may lead to better diagnosis and treatment. Our aim was to analyze the expression of microRNAs in cervical cancer cases positive or negative for HPV E6/E7 mRNA, and to assess their diagnostic usefulness and relevance. Methods: The expression of three different microRNAs (miR-9, miR-21, and miR-155) in 52 formalin-fixed paraffin-embedded (FFPE) primary cervical cancer tissue samples and 50 FFPE normal cervical tissue samples were evaluated. Results: MiR-9, miR-21, and miR-155 were significantly overexpressed in cervical cancer tissues compared to normal tissues ( P &lt; 0.001). MiR-9, miR-21, and miR-155 expression combined with the HPV E6/E7 mRNA assay showed sensitivities of 82.7 %, 92.3 %, and 84.6 % respectively, demonstrating their potential as diagnostic tools. Moreover, miR-21 and miR-155 were predictors showing a 7 fold and 10.3 fold higher risk for HPV E6/E7 negative patients with cervical cancer ( P = 0.024 and P = 0.017, respectively) while miR-155 was a predictor showing a 27.9 fold higher risk for HPV E6/E7 positive patients with cervical cancer ( P &lt; 0.0001). Conclusions: There is a strong demand for additional, alternative molecular biomarkers for diagnosis and management of precancer patients. MiR-9, miR-21, and miR-155 may be helpful in the prediction of both HPV positive and HPV negative cases of cervical cancer.

MicroRNA-195 inhibits proliferation of cervical cancer cells by targeting cyclin D1a.

Cervical cancer is one of the most frequent gynecological malignancies in women worldwide. MicroRNA-195 (miR-195) was recently found highly expressed in cervical cancer. However, the role of miR-195 in the pathology of cervical cancer remains poorly understood. In this study, we first confirmed the downregulation of miR-195 in primary cervical cancer tissues. For the functional study, we introduced the sequences of miR-195 or miR-195 inhibitor into Hela and SiHa cervical cancer cell lines. Overexpression of miR-195 inhibited the proliferation of both Hela and SiHa cells. In contrast, reducing the endogenous miR-195 level by miR-195 inhibitor promoted the proliferation of cervical cancer cells. Flow cytometric assay showed that overexpression of miR-195 induced G1 phase arrest, whereas miR-195 inhibitor shortened G1 phase of cervical cancer cells. In addition, the suppressive role of miR-195 in cell cycle was also demonstrated by the western blot results of various cell cycle indicators, such as phosphorylated retinoblastoma (p-Rb) and proliferating cell nuclear antigen (PCNA), in the gain and loss of function experiments. Furthermore, Dual-Luciferase Reporter Assay revealed that miR-195 targeted the 3'-untranslated region of cyclin D1a transcript, such as to regulate cyclin D1 expression. In summary, our results suggest that miR-195 acts as a suppressor in the proliferation and cell cycle of cervical cancer cells by directly targeting cyclin D1a mRNA.

Over Expression of Minichromosome Maintenance Genes is Clinically Correlated to Cervical Carcinogenesis

Minichromosome Maintenance (MCM) proteins play important roles in cell cycle progression by mediating DNA replication initiation and elongation. Among 10 MCM homologues MCM 2–7 form a hexamer and assemble to the pre-replication complex acting as replication licensing factors. Binding and function of MCM2-7 to pre-replication complex is regulated by MCM10 mediated binding of RECQL4 with MCM2-7. The purpose of this study is to explore the role of MCMs in cervical cancer and their correlation with the clinical parameters of cervical cancer. We have investigated sixty primary cervical cancer tissue samples, eight cervical cancer cell lines and thirty hysterectomised normal cervical tissue. The expression profiling of MCMs was done using semi-quantitative RT-PCR, immunoblotting and immunohistochemistry. MCM2, 4, 5, 6, 7, 10 and RECQL4 are significantly over-expressed in cervical cancer. Among these, MCM4, 6 and 10 show increased frequency of over expression along with advancement of tumor stages. MCM4, 5 and 6 also show differential expression in different types of lesion, while MCM2 and MCM10 are over expressed in cervical cancer irrespective of clinico-pathological parameters. Our data indicates the role of MCM4, MCM5, MCM6, MCM10 and RECQL4 in the progression of cervical cancer.

Correlation of TWIST2 up-regulation and epithelial–mesenchymal transition during tumorigenesis and progression of cervical carcinoma

ObjectiveGlobally, cervical cancer is the second most common cancer among women, and determining potential targets involved in tumor progression is necessary. This study investigated the clinic-pathological significance of twist homolog 2 (TWIST2), a basic helix–loop–helix transcription factor, and correlated TWIST2 and E-cadherin expression in cervical cancer. MethodsA series of 142 samples, including 14 cases of normal cervical tissues, 58 cases of cervical intraepithelial neoplasia (CIN) and 70 cases of squamous cell carcinoma (SCC), were examined TWIST2 and E-cadherin immunohistochemical staining and statistical analysis. ResultsIncreased cytoplasmic and nuclear expression levels of TWIST2 were associated with the malignant transformation of cervical epithelium and the histological progression of cervical cancer. A logistic test showed that TWIST2 was a relatively independent predictor of lymph node metastasis of SCC. Further, increased levels of TWIST2 were also associated with aberrant expression of E-cadherin, an important EMT indicator. ConclusionsThe present data suggest that TWIST2 overexpression was significantly linked to cervical cancer progression, which makes it a promising marker for determining the metastatic potential of cervical cancer, and up-regulation of TWIST2, in combination with aberrant E-cadherin expression in primary cervical cancer tissues, may predict the malignant transformation and distal metastasis of carcinomas.

Shank-interacting protein–like 1 promotes tumorigenesis via PTEN inhibition in human tumor cells

Inactivation of phosphatase and tensin homolog (PTEN) is a critical step during tumorigenesis, and PTEN inactivation by genetic and epigenetic means has been well studied. There is also evidence suggesting that PTEN negative regulators (PTEN-NRs) have a role in PTEN inactivation during tumorigenesis, but their identity has remained elusive. Here we have identified shank-interacting protein-like 1 (SIPL1) as a PTEN-NR in human tumor cell lines and human primary cervical cancer cells. Ectopic SIPL1 expression protected human U87 glioma cells from PTEN-mediated growth inhibition and promoted the formation of HeLa cell-derived xenograft tumors in immunocompromised mice. Conversely, siRNA-mediated knockdown of SIPL1 expression inhibited the growth of both HeLa cells and DU145 human prostate carcinoma cells in vitro and in vivo in a xenograft tumor model. These inhibitions were reversed by concomitant knockdown of PTEN, demonstrating that SIPL1 affects tumorigenesis via inhibition of PTEN function. Mechanistically, SIPL1 was found to interact with PTEN through its ubiquitin-like domain (UBL), inhibiting the phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase activity of PTEN. Furthermore, SIPL1 expression correlated with loss of PTEN function in PTEN-positive human primary cervical cancer tissue. Taken together, these observations indicate that SIPL1 is a PTEN-NR and that it facilitates tumorigenesis, at least in part, through its PTEN inhibitory function.

Tumor carbonic anhydrase 9 expression is associated with the presence of lymph node metastases in uterine cervical cancer

Tumor hypoxia has a pronounced effect on malignant progression and metastatic spread of human tumors. As carbonic anhydrases (CA) 9 and 12 are induced by the low-oxygen environment within tumors, we investigated the relationship between the expression of these two CA and the presence of metastatic lymph nodes (LN) in uterine cervical cancer. CA9/CA12 expression was evaluated histochemically in primary cervical cancer tissues of 73 patients who underwent laparoscopic LN staging and two patients with clinical staging before definitive radiotherapy at the National Cancer Center, Korea. We also evaluated CA9 expression in 33 patients with pathologically confirmed metastatic LN. CA9 expression in the primary tumors was significantly associated with LN metastasis (P = 0.03) and poorer disease-free survival (relative risk, 6.1; 95% confidence interval, 1.3-28.3, P = 0.02, multivariate analysis), whereas CA12 expression did not show such a relationship. In addition, 21 of 24 metastatic LN revealed similar CA9 expression (P = 0.001), suggesting that CA9-expressing tumor cells had a higher metastatic potential. CA9 was expressed in 45 of 75 (60%) primary tumors, with positive tumor cells observed predominantly in the area away from the blood vessels. In contrast, CA12 expression was observed in only 29 of 74 primary tumors (39%), without a specific pattern. These findings indicate that expression of CA9, but not CA12, in tumors is associated with the presence of LN metastases and poorer prognosis. Selective application of new treatment modalities based on CA9 expression to prevent LN metastases may improve overall treatment outcome in patients with uterine cervical cancer.

Clusterin confers paclitaxel resistance in cervical cancer

To measure clusterin expression in cervical cancer tissues and cell lines and to evaluate whether clusterin confers resistance to paclitaxel in cervical cancer cells. Immunohistochemical staining for clusterin was performed on 15 normal cervical tissues and 32 primary cervical cancer tissues, and clusterin expression in cervical cancer cell lines was quantified by Western blotting. The correlation between clusterin expression level and paclitaxel IC50 in cervical cancer cell lines was evaluated. The effect of clusterin siRNA on paclitaxel resistance was evaluated by XTT assays. Cervical cancer tissues expressed significantly higher levels of clusterin than did normal cervical tissues (4.08 vs. 1.35, P<0.05). Clusterin expression levels were correlated with paclitaxel resistance in cervical cancer cell lines, and transfection of clusterin siRNA into HeLaS3 cells significantly decreased their resistance to paclitaxel (P<0.05). Our finding that clusterin expression was significantly higher in cervical cancer than in normal cervical tissues suggests that clusterin may confer paclitaxel resistance in cervical cancer cells.

Functional Analysis of Bladder Cancer-Related Protein Gene: A Putative Cervical Cancer Tumor Suppressor Gene in Cervical Carcinoma

Our previous study has suggested thatthe bladder cancer-associated protein gene (BLCAP) was among the differentially expressed genes in cervical cancer. We confirm here that BLCAP is expressed in all noncancerous cervical tissues (10/10), but it is greatly lost in primary cervical cancer tissue (31/39). In order to further investigate the functional roles of BLCAP, we stably transfected BLCAP cDNA into HeLa cells. The HeLa cells expressing BLCAP show reduced cell growth and clone genicity compared to the vector-transfected cognate cells. BLCAP expression in HeLa cells leads to growth arrest and significantly enhanced apoptosis in vitro and reduced tumor formation in vivo. Thus, BLCAP might be a potential tumor suppressor gene in cervical carcinoma.

Human Secreted Frizzled-Related Protein Is Down-regulated and Induces Apoptosis in Human Cervical Cancer

To identify genes involved in cervical carcinogenesis, the mRNA differential display method was used. A 220-bp cDNA fragment called CA11 was present in normal cervical tissue but not in primary cervical cancer tissue or cervical cancer cell lines. CA11 exhibited 98% homology with the recorded human secreted frizzled-related protein (hsFRP) sequence. A dominant hsFRP mRNA transcript of approximately 4.6 kb was present in three normal cervical tissues examined. Expression of the transcript was nearly absent from three cervical cancer tissues and from five human cervical cancer-derived cell lines. Results from in situ hybridization showed that the hsFRP transcript was confined to the normal cervical epithelial layer. When hsFRP-transfected HeLa and CUMC-6 cervical cancer cells were cultured in serum-free medium, most of the cells died within 8 days. This effect is associated with the apoptotic process. The caspase-3 inhibitor 1, Ac-DEVD-CHO, blocked hsFRP-induced apoptotic cell death. Additionally, cleavage of poly (ADP-ribose) polymerase in hsFRP-transfected cells was confirmed by colorimetric assay. These results indicate that the hsFRP gene probably functions as a tumor suppressor in normal cervical epithelium and down-regulation of hsFRP contributes to development of cervical cancer.

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 β-catenin was up-regulated. These suggest that overexpressions of MMP-2 and MT1-MMP, loss of TIMP-2 expression, and up-regulation of β-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.

Absence ofp15INK4Bandp16INK4AGene Alterations in Primary Cervical Carcinoma Tissues and Cell Lines with Human Papillomavirus Infection

Objective.Alterations of thep15INK4Bandp16INK4Agene which are separated by 25 kb on chromosome 9p21 have been reported in various tumor-derived cell lines and primary tumors, but the role of these genes in cervical cancer is unknown. To determine the frequency of deletions and point mutations of these genes in human cervical cancer, we examined for alterations of thep15INK4Bandp16INK4Agenes in cervical carcinomas.Methods.We examined 57 primary tumors and matched normal tissues and 3 cervical cancer-derived cell lines. All the tumor tissues and cell lines were human papillomavirus (HPV) positive. Deletions or point mutations of exon 2 of thepINK4Bgene and exons 1, 2, and 3 of thep16INK3Agene were examined by polymerase chain reaction and direct sequencing, respectively.Results.Our data indicate no evidence for intragenic homozygous deletion or point mutation in the primary cervical cancer tissues or cancer-derived cell lines.Conclusion.Deletions or point mutations in thep15INK4Borp16INK4Agene may not be required for the development of HPV-positive cervical cancer or for establishment of cervical cancer cell lines.