Bovine papillomavirus E2 protein activates a complex growth-inhibitory program in p53-negative HT-3 cervical carcinoma cells that includes repression of cyclin A and cdc25A phosphatase genes and accumulation of hypophosphorylated retinoblastoma protein.

The bovine papillomavirus E2 protein can inhibit the proliferation of HT-3 cells, a p53-negative cervical carcinoma cell line containing integrated human papillomavirus type 30 DNA. Here, we analyzed HT-3 cells to explore the mechanism of p53-independent E2-mediated growth inhibition. Expression of the E2 protein repressed expression of the endogenous human papillomavirus type 30 E6/E7 genes. This was accompanied by hypophosphorylation and increased accumulation of p105Rb and repression of E2F1 expression. The E2 protein also caused reduced cyclin-dependent kinase (cdk) 2 activity, but this did not appear to be due to increased expression of cdk inhibitors. Rather, expression of cyclin A, which regulates cdk2 activity, and the cdc25A and cdc25B phosphatases, which are thought to activate cdk2, was significantly reduced at both the RNA and protein levels in response to E2 expression. The E2 protein reduced expression of cdc25A and cdc25B in both HT-3 and HeLa cells, but not in cells that were not growth-inhibited by the E2 protein. E2 point mutants unable to inhibit cell growth did not repress cdc25A and cdc25B expression, nor did the cell cycle inhibitors hydroxyurea and mimosine. Based on these results and the known properties of cell cycle components, we propose a model to account for E2-induced growth inhibition of cervical carcinoma cell lines.     

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.     

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.     

Inhibition of cervical carcinoma cell line proliferation by the introduction of a bovine papillomavirus regulatory gene.

Human papillomavirus (HPV) E6 and E7 oncogenes are expressed in the great majority of human cervical carcinomas, whereas the viral E2 regulatory gene is usually disrupted in these cancers. To investigate the roles of the papillomavirus E2 genes in the development and maintenance of cervical carcinoma, the bovine papillomavirus (BPV) E2 gene was acutely introduced into cervical carcinoma cell lines by infection with high-titer stocks of simian virus 40-based recombinant viruses. Expression of the BPV E2 protein in HeLa, C-4I, and MS751 cells results in specific inhibition of the expression of the resident HPV type 18 (HPV18) E6 and E7 genes and in inhibition of cell growth. HeLa cells, in which HPV gene expression is nearly completely abolished, undergo a dramatic and rapid inhibition of proliferation, which appears to be largely a consequence of a block in progression from the G1 to the S phase of the cell cycle. Loss of HPV18 gene expression in HeLa cells is also accompanied by a marked increase in the level of the cellular p53 tumor suppressor protein, apparently as a consequence of abrogation of HPV18 E6-mediated destabilization of p53. The proliferation of HT-3 cells, a human cervical carcinoma cell line devoid of detectable HPV DNA, is also inhibited by E2 expression, whereas two other epithelial cell lines that do not contain HPV DNA are not inhibited. Thus, a number of cervical carcinoma cell lines are remarkably sensitive to growth inhibition by the E2 protein. Although BPV E2-mediated inhibition of HPV18 E6 and E7 expression may contribute to growth inhibition in some of the cervical carcinoma cell lines, the BPV E2 protein also appears to exert a growth-inhibitory effect that is independent of its effects on HPV gene expression.     

E2 represses the late gene promoter of human papillomavirus type 8 at high concentrations by interfering with cellular factors.

The late gene promoter P7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is regulated by the viral E2 protein. Transfection experiments performed with the human skin keratinocyte cell line RTS3b and P7535 reporter plasmids revealed transactivation at low amounts and a repression of basal promoter activity at high amounts of E2 expression vector. This repression was promoter specific and correlated with the amount of transiently expressed E2 protein. Mutational analyses revealed that the negative regulation of P7535 activity is mediated by the low-affinity E2 binding site P2, which is separated by one nucleotide from the P7535 TATA box. Biochemical and genetic analyses suggested that repression is due to a displacement of the TATA-box binding protein by E2 and an interference of E2 with promoter-activating cellular factors that specifically recognize the P2 sequence. The high conservation of the P2 sequence among several papillomaviruses (epidermodysplasia verruciformis-associated HPVs, HPV1, cottontail rabbit papillomavirus, and bovine papillomavirus type 1) in the vicinity of the late gene promoter cap site suggests that an interplay of E2 and cellular factors at this sequence element is important for the expression of structural proteins.     

Visualizing dynamic E2F-mediated repression in vivo

Although many E2F target genes have been identified recently, very little is known about how any single E2F site controls the expression of an E2F target gene in vivo. To test the requirement for a single E2F site in vivo and to learn how E2F-mediated repression is regulated during development and tumorigenesis, we have constructed a novel series of wild-type and mutant Rb promoter-LacZ transgenic reporter lines that allow us to visualize the activity of a crucial E2F target in vivo, the retinoblastoma tumor suppressor gene (Rb). Two mutant Rb promoter-LacZ constructs were used to evaluate the importance of a single E2F site or a nearby activator (Sp1/Ets) site that is found mutated in low-penetrance retinoblastomas. The activity of the wild-type Rb promoter is dynamic, varying spatially and temporally within the developing nervous system. While loss of the activator site silences the Rb promoter, loss of the E2F site stimulates its activity in the neocortex, retina, and trigeminal ganglion. Surprisingly, E2F-mediated repression of Rb does not act globally or in a static manner but, instead, is a highly dynamic process in vivo. Using neocortical extracts, we detected GA-binding protein alpha (GABPalpha, an Ets family member) bound to the activator site and both E2F1 and E2F4 bound to the repressor site of the Rb promoter in vitro. Additionally, we detected binding of both E2F1 and E2F4 to the Rb promoter in vivo using chromatin immunoprecipitation analysis on embryonic day 13.5 brain. Unexpectedly, we detect no evidence for Rb promoter autoregulation in neuroendocrine tumors from Rb+/-; RbP-LacZ mice that undergo loss of heterozygosity at the Rb locus, in contrast to the situation in human retinoblastomas where high RB mRNA levels are found. In summary, this study provides the first demonstration that loss of an E2F site is critical for target gene repression in vivo and underscores the complexity of the Rb and E2F family network in vivo.     

Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells

The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of "high-risk" HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Here, we show that bovine papillomavirus (BPV) E2-induced growth arrest of HeLa cells requires the repression of the E6 and E7 promoter. This repression is specific for E2TA and not E2TR, a BPV E2 variant that lacks the N-terminal transactivation domain. We demonstrate that expression of HPV16 E6 and E7 from a heterologous promoter that is not regulated by E2 rescues HeLa cells from E2-mediated growth arrest. Our data indicate that the pathway of E2-mediated growth arrest of HeLa cells requires repression of E6 and E7 expression through an activity specified by the transactivation domain of E2TA.     

Human papillomavirus type 16 mutant E7 protein induces oncogenic transformation via up-regulation of cyclin A and cdc25A

A new mutant human papiUomavirus type 16 E7 gene, termed HPV16 HBE7, was isolated from cervical carcinoma biopsy samples from patients in an area with high incidence of cervical cancer (Hubei province, China). A previous study showed that the HPVI6 HBE7 protein was primarily cytoplasmic while wild-type HPV16 E7 protein, termed HPV16 WET, was concentrated in the nucleus. With the aim of studying the biological functions of HPV16 HBE7, the transforming potential of HPV16 HBE7 in NIH/3T3 cells was detected through observation of cell morphology, cell proliferation assay and anchorage-independent growth assay. The effect of HPVI6 HBE7 on cell cycle was examined by flow cytometry. Dual-luciferase reporter assay and RT-PCR were used to investigate the influence of HPVI6 HBE7 protein on the expression of regulation factors associated with GI/S checkpoint. The results showed that HPV16 HBE7 protein, as well as HPV16 WE7 protein, held transformation activity. NIH/3T3 cells expressing HPV16 HBE7 could easily transition from G1 phase into S phase and expressed high level of cyclin A and cdc25A. These results indicated HPV16 mutant E7 protein, located in the cytoplasm, induces oncogenic transformation of NIH/3T3 cells via up-regulation of cyclin A and cdc25A.