Repression of the human papillomavirus E6 gene initiates p53-dependent, telomerase-independent senescence and apoptosis in HeLa cervical carcinoma cells

Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins. When both HPV oncogenes are repressed in HeLa cervical carcinoma cells, the dormant p53 and retinoblastoma (Rb) tumor suppressor pathways are activated, and the cells undergo senescence in the absence of apoptosis. When the E6 gene is repressed in cells that continue to express an E7 gene, the p53 pathway, but not the Rb pathway, is activated, and both senescence and apoptosis are triggered. To determine the role of p53 signaling in senescence or apoptosis after repression of HPV oncogenes, we introduced a dominant-negative allele of p53 into HeLa cells. Dominant-negative p53 prevented senescence and apoptosis when E6 alone was repressed but did not inhibit senescence when both E6 and E7 were repressed. To determine whether reduced telomerase activity was involved in senescence or apoptosis after E6 repression, we generated HeLa cells stably expressing an exogenous hTERT gene, which encodes the catalytic subunit of telomerase. Although these cells contained markedly elevated telomerase activity and elongated telomeres, hTERT expression did not prevent senescence and apoptosis when E6 alone was repressed. These results demonstrate that when the Rb tumor suppressor pathway is inactivated by the E7 protein, E6 repression activates p53 signaling, which in turn is required for growth inhibition, senescence, and apoptosis. Thus, sustained inactivation of the p53 pathway by the E6 protein is required for maintenance of the proliferative phenotype of HeLa cervical carcinoma cells.     

Stranglehold on the spindle assembly checkpoint: the human papillomavirus E2 protein provokes BUBR1-dependent aneuploidy

The Human Papillomavirus (HPV) E2 protein, which inhibits the E6 and E7 viral oncogenes, is believed to have anti-oncogenic properties. Here, we challenge this view and show that HPV-18 E2 over-activates the Spindle Assembly Checkpoint (SAC) and induces DNA breaks in mitosis followed by aneuploidy. This phenotype is associated with interaction of E2 with the Mitotic Checkpoint Complex (MCC) proteins Cdc20, MAD2 and BUBR1. While BUBR1 silencing rescues the mitotic phenotype induced by E2, p53 silencing or presence of E6/E7 (inactivating p53 and increasing BUBR1 levels respectively) both amplify it. This work pinpoints E2 as a key protein in the initiation of HPV-induced cervical cancer and identifies the SAC as a target for oncogenic pathogens. Moreover, our results suggest a role of p53 in regulating the mitotic process itself and highlight SAC over-activation in a p53-negative context as a highly pathogenic event.     

Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation,senescence, and apoptosis in HeLa cervical carcinoma cells

Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins, and repression of HPV gene expression causes the cells to cease proliferation and undergo senescence. However, it is not known whether both HPV proteins are required to maintain the proliferative state of cervical cancer cells, or whether mutations that accumulate during carcinogenesis eliminate the need for one or the other of them. To address these questions, we used the bovine papillomavirus E2 protein to repress the expression of either the E6 protein or the E7 protein encoded by integrated HPV18 DNA in HeLa cervical carcinoma cells. Repression of the E7 protein activated the Rb pathway but not the p53 pathway and triggered senescence, whereas repression of the E6 protein activated the p53 pathway but not the Rb pathway and triggered both senescence and apoptosis. Telomerase activity, cyclin-dependent kinase activity, and expression of c-myc were markedly inhibited by repression of either E6 or E7. These results demonstrate that continuous expression of both the E6 and the E7 protein is required for optimal proliferation of cervical carcinoma cells and that the two viral proteins exert distinct effects on cell survival and proliferation. Therefore, strategies that inhibit the expression or activity of either viral protein are likely to inhibit the growth of HPV-associated cancers.     

Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells

Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways, respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.     

Human Papillomavirus Oncoproteins E6 and E7 Independently Abrogate the Mitotic Spindle Checkpoint

The E6 and E7 genes of the high-risk human papillomavirus (HPV) types encode oncoproteins, and both act by interfering with the activity of cellular tumor suppressor proteins. E7 proteins act by associating with members of the retinoblastoma family, while E6 increases the turnover of p53. p53 has been implicated as a regulator of both the G₁/S cell cycle checkpoint and the mitotic spindle checkpoint. When fibroblasts from p53 knockout mice are treated with the spindle inhibitor nocodazole, a rereplication of DNA occurs without transit through mitosis. We investigated whether E6 or E7 could induce a similar loss of mitotic checkpoint activity in human keratinocytes. Recombinant retroviruses expressing high-risk E6 alone, E7 alone, and E6 in combination with E7 were used to infect normal human foreskin keratinocytes (HFKs). Established cell lines were treated with nocodazole, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Cells infected with high-risk E6 were found to continue to replicate DNA and accumulated an octaploid (8N) population. Surprisingly, expression of E7 alone was also able to bypass this checkpoint. Cells expressing E7 alone exhibited increased levels of p53, while those expressing E6 had significantly reduced levels. The p53 present in the E7 cells was active, as increased levels of p21 were observed. This suggested that E7 bypassed the mitotic checkpoint by a p53-independent mechanism. The levels of MDM2, a cellular oncoprotein also implicated in control of the mitotic checkpoint, were significantly elevated in the E7 cells compared to the normal HFKs. In E6-expressing cells, the levels of MDM2 were undetectable. It is possible that abrogation of Rb function by E7 or increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the mitotic checkpoint.     

The p53/retinoblastoma-mediated repression of testicular orphan receptor-2 in the rhesus monkey with cryptorchidism

Whereas the linkage of infertility to cryptorchidism, the failure of the testis to descend into the scrotum at birth, has been well documented, the detailed molecular mechanism remains unclear. Here we report that the testicular orphan receptor-2 (TR2) expression, which modulates many signal pathways, was completely repressed in the surgery-induced cryptorchidism of the rhesus monkey. Further studies link TR2 repression to the induction of p53 and results suggest that induced p53 could repress TR2 expression via the p53-->p21-->CDK-->Rb-->E2F signal pathway. In return, TR2 could also control the expression of p53 and Rb through the regulation of human papillomavirus 16 E6/E7 genes. Together, our data suggest a feedback control mechanism between TR2 and p53/Rb tumor suppressors, which might play important roles in male infertility associated with cryptorchidism.     

Apoptosis induced by an antagonist peptide against HPV16 E7 in vitro and in vivo via restoration of p53

Human papilloma virus type 16 (HPV16) E7 is a viral oncoprotein that is believed to play a major role in cervical neoplasia. A novel antagonist peptide against HPV16 E7 was previously selected by phage display screening and the selected peptide was found to have anti-tumor efficacy against HPV16-positive cervical carcinoma through induction of cell cycle arrest. In the current study, to further elucidate the mechanisms of the antagonist peptide, the effects of the peptide on apoptosis are investigated by RT-PCR, Western blotting, MTT assay, TUNEL staining, Annexin V apoptosis assay, flow cytometry, and animal experiments. The antagonist peptide showed obvious anti-tumor efficacy through apoptosis induction, both in HPV16-positive cervical cancer cell lines and tumor xenografts. Our results also revealed that the peptide induced accumulation of cellular p53 and p21, and led to HPV16 E7 protein degradation. In the case of mRNA levels, it resulted in unaltered p53 and HPV16 E7 expression, but increased expression of p21. In contrast, the induction of apoptosis and p53 reactivation effects by the selected peptide were abolished after E7 knocked down with siRNA. These results demonstrate that the selected peptide can induce E7 degradation and lead to marked apoptosis in HPV16-related cancer cells by activating cellular p53 and its target genes, such as p21. Furthermore, the evident therapeutic efficacy obtained from the subcutaneous tumor model experiments in nude mice suggests a therapeutic potential for HPV16-related cancers of the selected peptide. Therefore, this specific peptide may be used to create specific biotherapies for the treatment of HPV 16-positive cervical cancers.     

Induction of senescence-like state and suppression of telomerase activity through inhibition of HPV E6/E7 gene expression in cells immortalized by HPV16 DNA

The E6 and E7 oncoproteins of human papillomavirus (HPV) play a major role in the development of cervical carcinoma. In this study, a recombinant adenovirus that expresses the bovine papillomavirus (BPV) E2, which has been shown to inhibit HPV early gene expression, was delivered to two HPV-immortalized cell lines as well as CaSki, a cervical carcinoma cell line. We tested whether the carcinoma and the immortal cells were equally affected by the expression of BPV E2. In all cell lines, BPV E2-mediated inhibition of HPV E6/E7 expression caused a dramatic suppression of cell growth, being preceded by the activation of the p53-Rb growth-inhibitory pathway, and a decrease in hTERT mRNA expression and telomerase activity. This suggests that the HPV E6 and E7 proteins are required not only for induction of the proliferative phenotype and telomerase activity, but also for their maintenance. In both the carcinoma and the immortal lines, the number of cells with enlarged cytoplasm and senescence-associated beta-galactosidase activity, which are markers for cellular senescence, was significantly increased. These results suggest that a senescence program exists in cells immortalized by HPV DNA as well as in cervical carcinoma cells.     

Restoration of p53 tumor suppressor pathway in human cervical carcinoma cells by sodium arsenite

In most cervical cancer cells, p53 and Rb are disrupted by human papillomaviruses (HPVs) E6 and E7, respectively. Restoration of p53 or Rb function by blocking E6/p53 or E7/Rb pathway might be a potential therapeutic purpose for these cancer cells. Treatment with sodium arsenite (SA) resulted in significant repression of E6 and E7 mRNA levels in SiHa cells. After E6 and E7 repression, p53 was dramatically induced and accumulated in cellular nuclei and Rb was also induced. Two p53-responsive genes, p21(waf1/cip1) and mdm2, were induced after SA treatment. Furthermore, SA also reduced the expressions of Cdc25A and cyclin B, blocked cell cycle progression at G2/M phase, and induced apoptosis in SiHa cells. SA-induced apoptosis was greatly reduced by expression of a dominant-negative mutated p53. In this study, we have first demonstrated that SA did repress E6 and E7 oncogenes, restore the p53 tumor suppressor pathway and induce apoptosis in SiHa cells. Therefore, it would be a potential strategy to promote SA as therapeutic purpose for HPV-positive cancer cells.     

Molecular screening of compounds to the predicted Protein-Protein Interaction site of Rb1-E7 with p53- E6 in HPV

Cervical cancer is malignant neoplasm of the cervix uteri or cervical area. Human Papillomaviruses (HPVs) which are heterogeneous groups of small double stranded DNA viruses are considered as the primary cause of cervical cancer, involved in 90% of all Cervical Cancers. Two early HPV genes, E6 and E7, are known to play crucial role in tumor formation. E6 binds with p53 and prevents its translocation and thereby inhibit the ability of p53 to activate or repress target genes. E7 binds to hypophosphorylated Rb and thereby induces cells to enter into premature S-phase by disrupting Rb-E2F complexes. The strategy of the research work was to target the site of interaction of Rb1 -E7 & p53-E6. A total of 88 compounds were selected for molecular screening, based on comprehensive literature survey for natural compounds with anti-cancer activity. Molecular docking analysis was carried out with Molegro Virtual Docker, to screen the 88 chosen compounds and rank them according to their binding affinity towards the site of interaction of the viral oncoproteins and human tumor suppressor proteins. The docking result revealed that Nicandrenone a member of Withanolides family of chemical compounds as the most likely molecule that can be used as a candidate drug against HPV induced cervical cancer. ABBREVIATIONS: HPV - Human Papiloma Virus, HTSP - Human Tumor Suppressor Proteins, VOP - Viral oncoproteins.     

Mechanisms underlying the pro-survival pathway of p53 in suppressing mitotic death induced by adriamycin

The p53 tumor suppressor responds to chemotherapeutic stress by triggering apoptosis or eliciting pro-survival pathway through arresting cell cycle progression for DNA damage repair. Here we examined the pro-survival activity of p53 on the adriamycin-induced stress using H1299 cells stably expressing tsp53 V143A, a temperature-sensitive mutant activating only the subset of p53 target genes related to growth arrest and DNA repair, but not apoptosis. At 38 degrees C, cells evaded from adriamycin-induced G2 arrest and died of apoptosis and mitotic catastrophe, which could be inhibited by Cdk inhibitors. Activation of functional tsp53 V143A at 32 degrees C led to suppression of Cdk1/2 activities and Cyclin B1/Cdk1 expression, cells exhibited prolonged G2 arrest, regained reproductive potential and were protected from mitotic catastrophe induced by adriamycin. Inhibition of mitotic catastrophe and Cyclin B1/Cdk1 expression was ablated upon silencing p21 Waf1 expression in tsp53 V143A-H1299 cells or in HCT116 cells. Together we show that p21 Waf1 is a key component of G2 checkpoint necessary and sufficient for protecting tumor cells against adriamycin-induced mitotic catastrophe.