Effect of silencing E6-associated protein on level of p53 protein in human papilloma virus negative cervical cancer cells / 中国生物制品学杂志

@#Objective To investigate the effect of silencing E6-associated protein(E6AP)on the level of p53 protein in human papilloma virus(HPV)negative cervical cancer cells(C33A cells).Methods The siRNA sequence silencing E6AP(siE6AP)and silencing control disordered siRNA sequence(siControl)were transfected into C33A cells with the mediation of LipofectamineTM2000 transfection reagent respectively.The silencing effect of siRNA on E6AP and the expression of p53and cleaved-caspase-3 proteins were detected by Western blot.Results The levels of E6AP protein in C33A cells of siE6AP group were significantly lower(t =-4.597,P<0.05),while the levels of p53 and cleaved-caspase-3 proteins were significantly higher than those of siControl group(t = 4.533 and 7.099 respectively,each P<0.05).Conclusion Silencing of E6AP significantly increased the expression of p53 protein in C33A cells,indicating that silencing of E6AP may restore the activity and function of p53 protein in C33A cells.

Degradation of p53 by HPV16-E6 variants isolated from cervical cancer specimens of Moroccan women.

Cervical cancer is the second most diagnosed cancer in Moroccan women. The main etiological factor for developing cervical cancer is the persistent infection with HPV16. Genetic studies have reported the occurrence of amino acid variations within the E6 oncoprotein that promotes host cell transformation by targeting p53 for degradation. To verify the biological effects of E6 polymorphisms towards p53 degradation, HPV16-E6 prototype and 7 variants isolated from cervical cancer biopsies of Moroccan women were evaluated for their activities by transient expression assays using pcDNA3.1-E6 constructs in C33A cell line. Expression of E6 genes in transfected cells was detected with reverse transcription PCR (RT-PCR), then, p53 levels were evaluated by western blot analysis. Significant dissimilarities in p53 degradation activities of HPV16-E6 prototype and intratypic variants were noticed. As compared to the prototype, the highest p53 degradation were exhibited by the African variants Af2-a/r, Af1-d/G295 and Af2-a/G285 (p < 0.001), followed by the European variants E- C442/G350 and E-G350/r (p < 0.01), then, the North American variant NA1-b/r (p < 0.05). The inter-variant differences were statistically significant between Af2-a/r variant and the North American variants NA1-b/r and NA1 (p < 0.05). Thus, the Af2-a/r variant was significantly more active in degrading p53 in our in vitro experiments (p < 0.0001). Our findings support the fact that HPV16-E6 variations have a biological impact on degrading p53, and so, represent a significant carcinogenic potential for developing cervical cancer.

Effects and Mechanism of Oncolytic Virus M 1 Inducing the Apoptosis of Cervical Cancer C- 33A Cells / 中国药房

OBJECTIVE：To study the effects and mechanism of oncolytic virus M 1（called M 1 virus for short ）inducing the apoptosis of cervical cancer C-33A cells. METHODS ：MTT assay was used to detect survival rate of C- 33A cells that were treated with different titers （0，0.001，0.01，0.1，1，10 PFU/cell）of M 1 virus. C- 33A cells were divided into control group （0 PFU/cell）， low-dose，medium-dose and high-dose groups of M 1 virus（0.001，0.01，0.1 PFU/cell）. After treated with corresponding titers of M1 virus for 48 h，flow cytometry was used to detect the apoptotic rate and infection rate of cells；Western blot was performed to detect the protein expression of C/EBP homologous proteins （CHOP），caspase-12，caspase-3 and cleaved-caspase- 3. RESULTS ： After treated with different titers of M 1 virus，the survival rate of C- 33A cells decreased significantly （P＜0.01），and showed a dose-dependent tr end. Compared with control group ，the apoptotic rate and infection rate of cells in M 1 virus groups as well as the protein expression of CHOP ，caspase-12 and cleaved-caspase- 3（except for medium-dose group ）in M 1 virus medium-dose and high-dose groups were increased significantly （P＜0.01）. CONCLUSIONS ：M1 virus can induce the apoptosis of cervical cancer C-33A cells ，and its mechanism may be related to the activation of endoplasmic reticulum stress pathway.

ALOX12B promotes carcinogenesis in cervical cancer by regulating the PI3K/ERK1 signaling pathway.

Cervical cancer is a malignant disease and a threat to women's health worldwide. Surgical resection followed by radiotherapy or chemotherapy is the main treatment strategy for cervical cancer; however, patients with cervical cancer, especially those with late-stage disease, may not benefit from these traditional therapies, which results in poor clinical outcome. ALOX12B is a gene encoding lipoxygenase, and a mutation in ALOX12B was detected in lung and breast cancer. Furthermore, ALOX12B is essential to the proliferation of epidermoid carcinoma cells; however, the role of ALOX12B in cervical cancer has not been studied thus far, to the best of our knowledge. In the present study, the expression levels of ALOX12B were reduced in cervical cancer cells by lentiviral transfection, and it was found that both cell proliferation and clone formation ability were significantly reduced, and the cell cycle was blocked at G1 phase. Tumor growth was also suppressed in vivo in a xenograft tumor model, but the migration of tumor cells was not affected by ALOX12B. Subsequently, using western blotting, it was demonstrated that knockdown of ALOX12B decreased the expression levels of PI3K, MEK1, ERK1, C-fos and cdc25. Meanwhile, overexpression of ALOX12B increased the expression levels of these five molecules. Conclusively, ALOX12B promoted cell proliferation in cervical cancer via regulation of the PI3K/ERK1 signaling pathway. The present study may improve our understanding of the molecular mechanisms underlying the function of ALOX12B in cervical cancer and inform new therapeutic strategies.

C33-A cells transfected with E6*I or E6*II the short forms of HPV-16 E6, displayed opposite effects on cisplatin-induced apoptosis.

The HPV-16 E6/E7 bicistronic immature transcript produces 4 mature RNAs: the unspliced HPV-16 E6/E7pre-mRNA product and 3 alternatively spliced mRNAs. The 3 spliced mRNAs encode short forms of the E6 oncoprotein, namely E6*I, E6*II and E6^E7. In this study we showed that transfection of C-33A cells with monocistronic constructs of these cDNAs fused to GFP, produced different effects on apoptosis, after the treatment with cisplatin. Transfection of C-33A cells with the full-length E6-GFP oncoprotein resulted in a 50% decrease in cell death, while the transfection with the E6*I-GFP construct showed only a 25% of diminution of cell death, compared to the control cells. Transfection with the E6^E7-GFP or E7-GFP construct had no effect on the number of the apoptotic cells, compared with control cells. Conversely, transfection with the E6*II construct resulted in higher cell death than the control cells. Taken together, these results suggested that E6*I or E6*II, the short forms of HPV-16 E6, displayed opposite effects on cisplatin-induced apoptosis, when transfected in C-33A cells.

The Deacetylase SIRT1 Regulates the Replication Properties of Human Papillomavirus 16 E1 and E2.

Human papillomaviruses (HPV) replicate their genomes in differentiating epithelium using the viral proteins E1 and E2 in association with host proteins. While the roles of E1 and E2 in this process are understood, the host factors involved and how they interact with and regulate E1-E2 are not. Our previous work identified the host replication and repair factor TopBP1 as an E2 partner protein essential for optimal E1-E2 replication and for the viral life cycle. The role of TopBP1 in host DNA replication is regulated by the class III deacetylase SIRT1; activation of the DNA damage response prevents SIRT1 deacetylation of TopBP1, resulting in a switch from DNA replication to repair functions for this protein and cell cycle arrest. Others have demonstrated an essential role for SIRT1 in regulation of the HPV31 life cycle; here, we report that SIRT1 can directly regulate HPV16 E1-E2-mediated DNA replication. SIRT1 is part of the E1-E2 DNA replication complex and is recruited to the viral origin of replication in an E1-E2-dependent manner. CRISPR/Cas9 was used to generate C33a clones with undetectable SIRT1 expression and lack of SIRT1 elevated E1-E2 DNA replication, in part due to increased acetylation and stabilization of the E2 protein in the absence of SIRT1. The results demonstrate that SIRT1 is a member of, and can regulate, the HPV16 replication complex. We discuss the potential role of this protein in the viral life cycle.IMPORTANCE HPV are causative agents in a number of human diseases, and currently only the symptoms of these diseases are treated. To identify novel therapeutic approaches for combating these diseases, the viral life cycle must be understood in more detail. This report demonstrates that a cellular enzyme, SIRT1, is part of the HPV16 DNA replication complex and is brought to the viral genome by the viral proteins E1 and E2. Using gene editing technology (CRISPR/Cas9), the SIRT1 gene was removed from cervical cancer cells. The consequence of this was that viral replication was elevated, probably due to a stabilization of the viral replication factor E2. The overall results demonstrate that an enzyme with known inhibitors, SIRT1, plays an important role in controlling how HPV16 makes copies of itself. Targeting this enzyme could be a new therapeutic approach for combating HPV spread and disease.

Changes in global gene expression profiles induced by HPV 16 E6 oncoprotein variants in cervical carcinoma C33-A cells.

We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E-Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 387 different genes in comparison with E-Prototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such as adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants.

Analysis of JC virus DNA replication using a quantitative and high-throughput assay.

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.