HPV-negative Cervical Cancer Cell Lines Research Articles

The Mechanism of HPV-mediated DNA Methylation in the Promoter Region of Long-chain Non-coding RNA MAGI2-AS3 in the Occurrence and Development of Cervical Cancer

Background: Cervical cancer, a malignancy of gynecological origin, typically necessitates a therapeutic approach combining surgery and chemoradiotherapy as the primary intervention. However, the 5-year survival rate remains suboptimal, prompting researchers to explore novel strategies for the early diagnosis and treatment of cervical cancer. This study delves into the investigation of human papillomavirus (HPV)-mediated DNA methylation modifications within the promoter region of the long non-coding RNA MAGI2-AS3 during cervical cancer development. This paper is an experimental study, laboratory based, using cell lines. Methods: A lentivirus overexpression vector encoding HPV16 E6/E7 proteins was established for transfecting cervical epithelial cells. The methylation status of DNA in the MAGI2-AS3 promoter region was assessed using MassARRAY, and the MAGI2-AS3 gene expression was measured through quantitative real time polymerase chain reaction (qRT-PCR). Subsequently, the correlation between methylation levels and gene expression in cervical cancer was analyzed. Results: (1) Relative to the HPV-negative cervical cancer cell line C33A, MAGI2-AS3 expression significantly decreased in the HPV-positive cervical cancer cell line Siha. (2) The methylation rate of 16 CpG sites in the HPV-positive cervical cancer cell line Siha was notably higher compared to the HPV-negative cervical cancer cell line C33A. (3) To further substantiate the regulatory impact on DNA methylation and expression within the MAGI2-AS3 promoter region, we silenced the expression of HPV16 E6 in the HPV-positive cervical cancer cell line Siha using HPV16 E6 siRNA. The ensuing qRT-PCR analysis revealed a significant up-regulation of MAGI2-AS3 expression in the HPV16 E6 siRNA group when contrasted with the negative control group. MassARRAY analysis was employed to gauge the DNA methylation levels in the promoter region of the MAGI2-AS3 gene in HPV-positive cervical cancer cells (Siha) following HPV16 E6 silencing. The results demonstrated a significantly lower methylation rate at the CpG_29 site in the HPV16 E6 siRNA group compared to the HPV16 E6 siRNA group. Conclusions: The study establishes a close association between HPV infection and elevated methylation levels coupled with diminished expression of MAGI2-AS3. The influence of HPV infection on the malignant transformation of cervical epithelial cells is potentially mediated through the regulatory modulation of MAGI2-AS3 expression via DNA methylation.

Complementary Targeting of Rb Phosphorylation and Growth in Cervical Cancer Cell Cultures and a Xenograft Mouse Model by SHetA2 and Palbociclib.

Cervical cancer is caused by high-risk human papillomavirus (HPV) types and treated with conventional chemotherapy with surgery and/or radiation. HPV E6 and E7 proteins increase phosphorylation of retinoblastoma (Rb) by cyclin D1/cyclin dependent kinase (CDK)4/6 complexes. We hypothesized that cyclin D1 degradation by the SHetA2 drug in combination with palbociclib inhibition of CDK4/6 activity synergistically reduces phosphorylated Rb (phospho-Rb) and inhibits cervical cancer growth. The effects of these drugs, alone, and in combination, were evaluated in SiHa and CaSki HPV-positive and C33A HPV-negative cervical cancer cell lines using cell culture, western blots and ELISA, and in a SiHa xenograft model. Endpoints were compared by isobolograms, ANOVA, and Chi-Square. In all cell lines, combination indexes documented synergistic interaction of SHetA2 and palbociclib in association SHetA2 reduction of cyclin D1 and phospho-Rb, palbociclib reduction of phospho-Rb, and enhanced phospho-Rb reduction upon drug combination. Both drugs significantly reduced phospho-Rb and growth of SiHa xenograft tumors as single agents and acted additively when combined, with no evidence of toxicity. Dilated CD31-negative blood vessels adjacent to, or within, areas of necrosis and apoptosis were observed in all drug-treated tumors. These results justify development of the SHetA2 and palbociclib combination for targeting phospho-Rb in cervical cancer treatment.

Knockdown of METTL14 inhibits the growth and invasion of cervical cancer

Background: Increasing evidence has revealed that N6-methyladenosine (m6A) modification is implicated in multiple biological functions in mammals. Methyltransferase-like 14 (METTL14), an important component of m6A modification, has been reported to play important roles in the pathogenesis of acute myeloid leukaemia and hepatocellular carcinoma metastasis. However, its role in cervical cancer remains unclear. Methods: Expression of METTL14 was knocked down by shRNA-METTL14 interference in HPV-positive and HPV-negative cervical cancer cell lines SiHa and C33a. CCK8, colony formation, wound-healing, and Transwell assays were performed to evaluate the effects of METTL14 knockdown on the proliferation, migration and invasion abilities of SiHa and C33a cells. Flow cytometry analysis was utilized to detect cell cycle distribution, and the expression of related proteins was examined by western blot analysis. Results: Bioinformatics analysis demonstrated that up-regulation of METTL14 acted as an adverse prognostic factor for overall survival in cervical cancer patients. We demonstrated that down-regulation of METTL14 inhibited the proliferation, migration and invasion abilities of SiHa and C33a cells. Moreover, silencing METTL14 induced cell cycle arrest in cervical cancer cells. METTL14 knockdown suppressed the PI3K/Akt/mTOR signaling pathway by decreasing the phosphorylation of Akt and mTOR, and the expression of downstream apoptosis-related proteins was also impacted. Conclusions: In conclusion, these data suggest an important oncogenic role of METTL14 in the growth and invasion of both HPV-positive and HPV-negative cervical cancer cells.

Knockdown of METTL14 inhibits the growth and invasion of cervical cancer.

BackgroundIncreasing evidence has revealed that N6-methyladenosine (m6A) modification is implicated in multiple biological functions in mammals. Methyltransferase-like 14 (METTL14), an important component of m6A modification, has been reported to play important roles in the pathogenesis of acute myeloid leukaemia and hepatocellular carcinoma metastasis. However, its role in cervical cancer remains unclear.MethodsExpression of METTL14 was knocked down by shRNA-METTL14 interference in HPV-positive and HPV-negative cervical cancer cell lines SiHa and C33a. CCK8, colony formation, wound-healing, and Transwell assays were performed to evaluate the effects of METTL14 knockdown on the proliferation, migration and invasion abilities of SiHa and C33a cells. Flow cytometry analysis was utilized to detect cell cycle distribution, and the expression of related proteins was examined by western blot analysis.ResultsBioinformatics analysis demonstrated that up-regulation of METTL14 acted as an adverse prognostic factor for overall survival in cervical cancer patients. We demonstrated that down-regulation of METTL14 inhibited the proliferation, migration and invasion abilities of SiHa and C33a cells. Moreover, silencing METTL14 induced cell cycle arrest in cervical cancer cells. METTL14 knockdown suppressed the PI3K/Akt/mTOR signaling pathway by decreasing the phosphorylation of Akt and mTOR, and the expression of downstream apoptosis-related proteins was also impacted.ConclusionsIn conclusion, these data suggest an important oncogenic role of METTL14 in the growth and invasion of both HPV-positive and HPV-negative cervical cancer cells.

HPV16 oncogenes E6 or/and E7 may influence the methylation status of RASSFIA gene promoter region in cervical cancer cell line HT-3

Both human papillomavirus (HPV) infection and the aberrant Ras associated domain family gene 1A (RASSF1A) promoter methylation status participate in the pathogenesis of cervical cancer. Some studies suggest that E6, and E7 are involved in the pathogenetic mechanisms of RASSF1A. We mainly explored a possible involvement of HPV16 oncogenes E6 or/and E7 in RASSF1A promoter methylation status and possible roles of RASSF1A gene methylation in cervical cancer. Bisulfite genomic sequencing (BGS) PCR combined with TA clone, methylation-specific PCR (MSP) were used to analyze methylation status of the RASSF1A gene promoter in HPV16/18-positive and HPV-negative cervical cancer cell lines; ectopically expressed HPV16 E6, E7 and E6/E7 cervical cancer cell lines; normal cervical and cervical cancer tissues. The mRNA and protein expression of RASSF1A was detected by RT-PCR and western blotting. Re-expression and downregulated promoter methylation status were detected in the ectopically expressed HPV16 E6 and E7 cervical cancer cell line HT-3. The methylation status and expression of RASSF1A could be downregulated or reactivated by 5-Aza-dc in HT-3 and C33A cells. Additionally, statistics showed significant hypermethylation of RASSF1A in cervical cancer samples compared to that in normal cervical samples (P<0.05). The false negative rate (FNR) was 6.25% by HC2 method, when reconfirmed by HPV detection combining the MY09/11, GP5+/6+ and SPF1/2 methods. The ectopic expression of HPV16 E6 and/or E7 may be involved in aberrant methylation and expression of the RASSF1A gene. RASSF1A gene expression could be regulated by its promoter methylation status. Additionally, the false negativity of the HPV detection may contribute to the uncertain relationship between HPV infection and aberrant RASSF1A promoter methylation.

Cross-talk between Human Papillomavirus Oncoproteins and Hedgehog Signaling Synergistically Promotes Stemness in Cervical Cancer Cells.

Viral oncoproteins E6/E7 play key oncogenic role in human papillomavirus (HPV)-mediated cervical carcinogenesis in conjunction with aberrant activation of cellular signaling events. GLI-signaling has been implicated in metastasis and tumor recurrence of cervical cancer. However, the interaction of GLI-signaling with HPV oncogenes is unknown. We examined this relationship in established HPV-positive and HPV-negative cervical cancer cell lines using specific GLI inhibitor, cyclopamine and HPVE6/E7 siRNAs. Cervical cancer cell lines showed variable expression of GLI-signaling components. HPV16-positive SiHa cells, overexpressed GLI1, Smo and Patch. Inhibition by cyclopamine resulted in dose-dependent reduction of Smo and GLI1 and loss of cell viability with a higher magnitude in HPV-positive cells. Cyclopamine selectively downregulated HPVE6 expression and resulted in p53 accumulation, whereas HPVE7 and pRb level remained unaffected. siRNA-mediated silencing of HPV16E6 demonstrated reduced GLI1 transcripts in SiHa cells. Cervical cancer stem-like cells isolated by side population analysis, displayed retention of E6 and GLI1 expression. Fraction of SP cells was reduced in cyclopamine-treated cultures. When combined with E6-silencing cyclopamine resulted in loss of SP cell’s sphere-forming ability. Co-inhibition of GLI1 and E6 in cervical cancer cells showed additive anti-cancer effects. Overall, our data show existence of a cooperative interaction between GLI signaling and HPVE6.

Mechanism of antiproliferative action of a new d-secoestrone-triazole derivative in cervical cancer cells and its effect on cancer cell motility

Cervical cancer is the fourth most frequently diagnosed tumor and the fourth leading cause of cancer death in females worldwide. Cervical cancer is predominantly related with human papilloma virus (HPV) infection, with the most oncogenic types being HPV-18 and -16. Our previous studies demonstrated that some d-secoestrone derivatives exert pronounced antiproliferative activity. The aim of the current investigation was to characterize the mechanism of action of d-secoestrone-triazole (D-SET) on three cervical cancer cell lines with different pathological backgrounds.The growth-inhibitory effects of D-SET were determined by a standard MTT assay. We have found that D-SET exerts a pronounced growth-inhibitory effect on HPV 18-positive HeLa and HPV-negative C-33 A cells, but it has no substantial inhibitory activity on HPV 16-positive SiHa or on intact fibroblast MRC-5 cell lines. After 24h incubation, cells showed the morphological and biochemical signs of apoptosis determined by fluorescent double staining, flow cytometry and caspase-3 activity assay. Besides the elevation of the ratio of cells in the subG1 phase, flow cytometric analysis revealed a cell cycle arrest at G2/M in both HeLa and C-33 A cell lines. To distinguish the G2/M cell population immunocytochemical flow cytometric analysis was performed on HeLa cells. The results show that D-SET significantly increases the ratio of phosphorylated histone H3, indicating cell accumulation in the M phase. Additionally, D-SET significantly increased the maximum rate of microtube formation measured by an in vitro tubulin polymerization assay. Besides its direct antiproliferative activity, the antimigratory property of D-SET has been investigated. Our results demonstrate that D-SET significantly inhibits the migration and invasion of HeLa cells after 24h incubation.These results suggests that D-SET is a potent antiproliferative agent against HPV 16+ and HPV-negative cervical cancer cell lines, with an efficacious motility-inhibiting activity against HPV 16+ cells. Accordingly D-SET can be regarded as a potential drug candidate with a promising new mechanism of action among the antiproliferative steroids, potentially allowing for the design of novel anticancer agents.

Serine/threonine kinases 31(STK31) may be a novel cellular target gene for the HPV16 oncogene E7 with potential as a DNA hypomethylation biomarker in cervical cancer.

BackgroundCervical cancer (CC) is a leading cause of mortality in females, especially in developing countries. The two viral oncoproteins E6 and E7 mediate the oncogenic activities of high-risk human papillomavirus (hrHPV), and hrHPV, especially HPV16 or/and HPV18 (HPV16/18) play critical roles in CC through different pathways. STK31 gene of which the expression has been proven to be regulated by the methylation status of its promoter, is one of the novel cancer/testis (CT) genes and plays important roles in human cancers. Reasearches have indicated that viral infection is correlated to the methylation statuses of some genes. Herein, we detected methylation status of the STK31 gene in cervical tumors and explored its interaction with HPV16 or/and HPV18 (HPV16/18) infection.MethodsBisulfite genomic sequencing PCR (BGS) combined with TA clone, methylation-specific PCR (MSP) were used to analyze methylation statuses of the STK31 gene promoter/exon 1 region in HPV16/18-positive, HPV-negative CC cell lines; ectopically expressed HPV16 E6, -E7, and -E6/E7 CC cells; normal cervical tissues and cervical tumor tissues of different stages. The mRNA and protein expressions of STK31 were detected by RT-PCR and western blotting.ResultsThe STK31 gene promoter/exon 1 was hypomethylated in the HPV16/18-positive cell lines HeLa, SiHa and CaSki, and the mRNA and protein expression were detected. In contrast, the STK31 gene exhibited hypermethylation and silenced expression in the HPV-negative CC cells C33A and HT-3. Compared with the primary HPV-negative CC cell lines, the STK31 methylation was downregulated, and STK31 expression was induced in the HPV16E7/E67 transfected cells. The methylation statuses and expressions of STK31 were verified in the cervical tumor samples at different stages. Additionally, chemotherapy treatment may influence STK31 expression by regulating its methylation status.ConclusionsSTK31 may be a novel cellular target gene for the HPV16 oncogeneE7. The HPV16 oncogene E7 may affect STK31 expression through a methylation-mediated mechanism. The aberrant methylation of the STK31 promoter/exon 1 region may be a precursor of human cervical carcinogenesis and a potential DNA aberrant methylation biomarker of conditions ranging from precancerous disease to invasive cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0515-5) contains supplementary material, which is available to authorized users.

Differential expression of Oct4 in HPV-positive and HPV-negative cervical cancer cells is not regulated by DNA methyltransferase 3A

The colony-forming ability of cervical cancer is affected by many factors. Oct4, an important transcription factor, is highly expressed in several tumors and promotes the colony-forming ability of cancer cells. Thus, it is considered a potential target for the treatment of cancer. However, we know little about the expression level of Oct4 and its epigenetic regulatory mechanism in cervical cancer cells. In this study, we are the first to observe that human papillomavirus (HPV)-positive cervical cancer cell lines (HeLa, Caski) have a stronger colony-forming ability than HPV-negative cervical cancer cell lines (C-33A). Moreover, the expression level of Oct4 in both HeLa and Caski cells was also higher than that in C-33A cells. We then confirmed that there was a negative correlation between the expression of Oct4 and DNMT3A in these three types of cervical cancer cells, whereas DNA methyltransferase 1 and 3B had no differences among the cell lines. However, after DNA methylation in both key regulatory regions of the Oct4 gene and the genomic levels were analyzed, we found that DNA methyltransferase 3A could neither regulate the expression of Oct4 nor affect the whole level of genomic DNA methylation. These results suggest three points: (1) Oct4 might be treated as a new target for the treatment of cervical cancer, (2) we could not inhibit the expression of Oct4 by DNA demethylation, and (3) HPV virus might initiate cervical carcinogenesis by upregulation of Oct4 expression.

CD1d, a Sentinel Molecule Bridging Innate and Adaptive Immunity, Is Downregulated by the Human Papillomavirus (HPV) E5 Protein: a Possible Mechanism for Immune Evasion by HPV

CD1d and CD1d-restricted natural killer T (NKT) cells serve as a natural bridge between innate and adaptive immune responses to microbes. CD1d downregulation is utilized by a variety of microbes to evade immune detection. We demonstrate here that CD1d is downregulated in human papillomavirus (HPV)-positive cells in vivo and in vitro. CD1d immunoreactivity was strong in HPV-negative normal cervical epithelium but absent in HPV16-positive CIN1 and HPV6-positive condyloma lesions. We used two cell lines for in vitro assay; one was stably CD1d-transfected cells established from an HPV-negative cervical cancer cell line, C33A (C33A/CD1d), and the other was normal human vaginal keratinocyte bearing endogenous CD1d (Vag). Flow cytometry revealed that cell surface CD1d was downregulated in both C33A/CD1d and Vag cells stably transfected with HPV6 E5 and HPV16 E5. Although the steady-state levels of CD1d protein decreased in both E5-expressing cell lines compared to empty retrovirus-infected cells, CD1d mRNA levels were not affected. Confocal microscopy demonstrated that residual CD1d was not trafficked to the E5-expressing cell surface but colocalized with E5 near the endoplasmic reticulum (ER). In the ER, E5 interacted with calnexin, an ER chaperone known to mediate folding of CD1d. CD1d protein levels were rescued by the proteasome inhibitor, MG132, indicating a role for proteasome-mediated degradation in HPV-associated CD1d downregulation. Taken together, our data suggest that E5 targets CD1d to the cytosolic proteolytic pathway by inhibiting calnexin-related CD1d trafficking. Finally, CD1d-mediated production of interleukin-12 from the C33A/CD1d cells was abrogated in both E5-expressing cell lines. Decreased CD1d expression in the presence of HPV E5 may help HPV-infected cells evade protective immunological surveillance.

E6AP-Dependent Degradation of DLG4/PSD95 by High-Risk Human Papillomavirus Type 18 E6 Protein

In most cervical cancers, DNAs of high-risk mucosotropic human papillomaviruses (HPVs), such as types 16 and 18, are maintained so as to express two viral proteins, E6 and E7, suggesting that they play important roles in carcinogenesis. The carboxy-terminal PDZ domain-binding motif of the E6 proteins is in fact essential for transformation of rodent cells and induction of hyperplasia in E6-transgenic mouse skin. To date, seven PDZ domain-containing proteins, including DLG1/hDLG, which is a human homologue of the Drosophila discs large tumor suppressor (Dlg), have been identified as targets of high-risk HPV E6 proteins. Here, we describe DLG4/PSD95, another human homologue of Dlg, as a novel E6 target. DLG4 was found to be expressed in normal human cells, including cervical keratinocytes, but only to a limited extent in both HPV-positive and HPV-negative cervical cancer cell lines. Expression of HPV18 E6 in HCK1T decreased DLG4 levels more strongly than did HPV16 E6, the carboxy-terminal motif of the proteins being critical for binding and degradation of DLG4 in vitro. DLG4 levels were restored by expression of either E6AP-specific short hairpin RNA or bovine papillomavirus type 1 E2 in HeLa but not CaSki or SiHa cells, reflecting downregulation of DLG4 mRNA as opposed to protein by an HPV-independent mechanism in HPV16-positive cancer lines. The tumorigenicity of CaSki cells was strongly inhibited by forced expression of DLG4, while growth in culture was not inhibited at all. These results suggest that DLG4 may function as a tumor suppressor in the development of HPV-associated cancers.

Antiproliferative and antiviral mechanisms of ursolic acid and dexamethasone in cervical carcinoma cell lines

The chemical structure of ursolic acid is very similar to that of dexamethasone, a synthetic glucocorticoid. Herein, we investigated the antiproliferative and antiviral effects of ursolic acid and dexamethasone in human papillomavirus (HPV)-associated cervical cancer cells. We performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide assay to measure antiproliferative activity, and also characterized apoptosis by DNA fragmentation, 4'-6-diamidino-2-phenylindole (DAPI) staining, and flow cytometry (FACS) analysis. We investigated apoptosis-related proteins using western blots. After in vitro treatment, we used reverse transcription-polymerase chain reaction for the expression of the HPV E6/E7 gene to observe the antiviral effects. Ursolic acid suppressed the growth of HPV-positive cervical carcinoma cells (HeLa, CaSki, and SiHa) in a dose- and time-dependent manner, but not the HPV-negative cervical cancer cell line (C33A). Ursolic acid-treated HeLa cells showed typical apoptosis characteristics in DNA fragmentation, DAPI staining, and FACS analysis. The expression of Fas protein was induced, and caspase-8, caspase-3, and poly ADP-ribose polymerase (PARP) proteins were cleaved after ursolic acid treatment. HPV-18 E6/E7 gene expression decreased after ursolic acid treatment in HeLa cells, but the levels of p53 and Rb proteins did not change. In contrast, dexamethasone, which has a similar structure, did not inhibit proliferation. Our findings may offer new insight into the mechanism of antiproliferative and antiviral effect of ursolic acid. Also, these results suggest that ursolic acid might be a useful anticancer drug in treatment of HPV-associated cervical neoplasia.

Human papillomavirus genotyping by oligonucleotide microarray and p16INK4A expression in uterine cervical intraepithelial neoplasm and in invasive carcinoma in Korean women

For evaluating the diagnostic significance of p16(INK4A) over-expression in the uterine cervical intraepithelial neoplasm and in invasive carcinoma, human papillomavirus (HPV) was detected and genotyped by oligonucleotide microarray in archival tissues of 117 cervical specimens, including 47 invasive squamous cell carcinomas (SCC), 30 cases of cervical intraepithelial neoplasia (CIN), 20 adenocarcinomas, and 20 cases of non-neoplastic cervix. The expression of p16(INK4A) protein was immunohistochemically studied in these cases and in five HPV-positive and one HPV-negative cervical cancer cell lines. HPV was detected in 50% of CIN, 61.7% of SCC, and 45.5% of adenocarcinomas. p16(INK4A) expression was seen in all 20 cases of adenocarcinoma, 78.7% (37/47) of SCC, and 96.7% (29/30) of CIN, but not in any cases of the non-neoplastic cervix. There was no difference in p16(INK4A) expression between the HPV-positive and HPV-negative cervical lesions. All HPV-positive and -negative cervical cancer cell lines expressed p16(INK4A) protein. In conclusion, the presence of p16(INK4A) expression in cervical squamous and glandular epithelium indicates the existence of dysplasia or malignancy in the uterine cervix, regardless of HPV infection.

Protein profiling and identification of modulators regulated by the E7 oncogene in the C33A cell line by proteomics and genomics.

Human papillomaviruses (HPVs) have been recognized as the primary cause of cervical cancer. HPV 16 E7 binds to tumor suppressor retinoblastoma protein, and interferes with its function, causing release of the transcription factor E2F, which influences expression of cell cycle-related genes. This study was performed to identify the genes and proteins modulated by the HPV E7 oncogene. An HPV-negative cervical cancer cell line (C33A) was prepared to establish a stable cell line expressing E7. In order to analyze the target molecules modulated by E7 expression, we used two approaches: matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and DNA microarrays. Forty-seven spots were identified in C33A/E7 by two-dimensional electrophoresis and MALDI/TOF MS. Protein disulfide isomerase A3, integrase interactor 1 protein, growth inhibitory protein, glutathione S-transferase P, and vav proto-oncogene were down-regulated, whereas heat shock 60 kDa protein 1, Ku70 binding protein, alpha enolase, 26S proteasome subunit were up-regulated. A genomic approach using a microarray kit showed that IL-12R beta 1, cytochrome c, and tumor necrosis factor receptor II were induced by the E7 oncogene. These results suggest that E7 can evade immune surveillance by suppressing or inducing these cell signaling factors, cell cycle regulators, and chaperones.

Down modulation of IL-18 expression by human papillomavirus type 16 E6 oncogene via binding to IL-18

To understand modulation of a novel immune-related cytokine, interleukin-18, by human papillomavirus type (HPV) 16 oncogenes, HaCaT, normal keratinocyte cell line, and C-33A, HPV-negative cervical cancer cell line, were prepared to establish stable cell lines expressing E6, E6 mutant (E6m), E6E7, or E7 constitutively. Expressions of various HPV oncogene transcripts were identified by RT-PCR. Expression of HPV oncogene E6 was reversely correlated to the expression of interleukin-18, a novel pro-inflammatory cytokine. The expression of E6 in C-33A, independent of E6 splicing, resulted in decreased IL-18 expression and that of IL-18 was also significantly reduced in HaCaT cells expressing E6. The level of p53 was reduced in C-33A cells expressing E6 whereas not altered in HaCaT cells expressing E6, suggesting that E6 downregulated IL-18 expression via an independent pathway of p53 degradation in HaCaT cells which have a mutated p53 form. However, E7 did not affect IL-18 expression significantly in both C-33A and HaCaT cells. Cotransfection experiments showed that E6 oncogene did not inhibit the activities of IL-18 promoter P1 and P2, suggesting that E6 oncogene indirectly inhibited IL-18 expression. Taken together, E6, E6m and E6/E7 inhibited IL-18 expression with some variation, assuming that cells expressing E6 oncogene can evade immune surveillance by downregulating the expression of immune stimulating cytokine gene, IL-18, and inhibiting the cascade of downstream effects that follow activation of the IL-18 receptor.

Altered Growth and Viral Gene Expression in Human Papillomavirus Type 16-Containing Cancer Cell Lines Treated with Progesterone

This study explores interactions between high-risk human papillomavirus type 16 (HPV-16) and the female sex hormone progesterone in the growth of tumor cells and viral oncogene expression. For both the cervical cancer cell line CaSki containing integrated HPV-16 DNA and the laryngeal carcinoma cell line HEp-2 transfected with HPV-16 DNA, prolonged progesterone treatment enhances their colony formation efficiency both on plastic surface and in soft agar. In contrast, progesterone has no effect on the HPV-negative cervical cancer cell line C-33A or the un-transfected HEp-2 parental cells. Progesterone increases HPV-16 E6/E7 oncogene transcription in both HPV-16-containing cell lines. A detectable increase requires at least 3 days of treatment, and this delayed response may be due, at least in part, to increased stability of viral transcripts as determined by actinomycin D treatment. The progesterone antagonist RU 486 and nuclease-resistant oligomers containing HPV-16 progesterone response element are able to abrogate the enhancement by progesterone on cell growth and E6/E7 gene transcription. Taken together, these results support the notion that progesterone can be a cofactor in HPV-related malignancies.

Up-regulation of Epidermal Growth Factor-Receptors (EGF-R) by EGF in an HPV-negative cervical cancer cell line

Up-regulation of Epidermal Growth Factor-Receptors (EGF-R) by EGF in an HPV-negative cervical cancer cell line

Ischaemic heart disease in young women.

The mortality rate from ischaemic heart disease (I.H.D.) has increased in young women by about 50% in 12 years, and it is now possible to report the findings in 150 women who developed symptoms and signs of I.H.D. under the age of 45. Data obtained from 145 of these women form the basis of this report: 81 presented with myocardial infarction and 64 with angina. In the remaining five there was a definite nonatherosclerotic cause for the premature onset of I.H.D.Hypercholesterolaemia, hypertension, or excessive cigarette smoking each occurred in a large minority, and more than one of these major risk factors was present in most patients. Hypercholesterolaemia was the commonest factor. In women in whom lipoprotein typing was undertaken the type II pattern was more frequent than type IV. The prevalence of hypercholesterolaemia and hypertension was the same in those with myocardial infarction and in those with angina.Excessive cigarette smoking was more common in women with myocardial infarction than in those with angina. The latter did not differ in their cigarette smoking habits from the normal population.A premature menopause had occurred in 20% of these women, but there was no relation between the early onset of I.H.D. with age at menarche, parity, or the incidence of abortion. Oral contraceptives did not increase the risk of myocardial infarction unless one of the major risk factors was also present.Altogether 75% of patients with angina or myocardial infarction survived 12 years. Coexisting hypertension worsened the prognosis. The prognosis after myocardial infarction was similar in these women to that previously described for men under the age of 40.