Human Papillomavirus Type 16 E6 Gene Research Articles

Resistance of MCF7 human breast carcinoma cells to TNF-induced cell death is associated with loss of p53 function.

We have investigated the relationship between the development of tumor resistance towards the cytotoxic action of tumor necrosis factor-alpha (TNF) and p53 function, using the TNF-sensitive MCF7 human breast adenocarcinoma cell line and two TNF-resistant sublines, MCF7/R-A1 and MCF7/Adr. Use of single-strand conformation polymorphism (SSCP) analysis and DNA sequencing shows that MCF7 has a wild-type p53 gene, whereas both TNF-resistant sublines exhibit mutant p53. This includes a point mutation R280K in MCF7/R-A1 cells, and a point mutation at the splicing acceptor site on the upstream border of exon 5 resulting in a 21 pb deletion in MCF7/Adr cells. These mutations result in loss of p53 capacity to transactivate FASAY (functional assay in yeast). In contrast to what is observed for parental MCF7 cells, treatment of resistant sublines with TNF or gamma-irradiation fails neither to induce the expression of the p53-regulated gene products p21waf1/CIP1 and MDM2, nor to arrest the cells in the G1 phase of the cell cycle. Disruption of p53 wild-type function in MCF7 cells by transfection with human papillomavirus type-16 E6 gene, leads to abrogation of the cytotoxic, but not the cytostatic activity of TNF. Altogether, our results strongly suggest that wild-type p53 is involved in cytotoxic action of TNF, and point out that loss of p53 function contributes to resistance of tumor cell to TNF-induced killing.

Cells lacking CIP1/WAF1 genes exhibit preferential sensitivity to cisplatin and nitrogen mustard.

We have previously shown that p53 disruption sensitizes certain cancer cell types to cisplatin (CDDP) (Fan et al., 1995). In the present study we investigated the role of the p53 downstream effector, p21CIP1/WAF1 (p21), in this sensitization. Studies were performed in human colon cancer HCT-116 cells and murine embryonic fibroblasts (MEF) with intact versus disrupted p21 genes. For comparison, HCT-116 cells lacking p53 function were also prepared through stable transfection with the human papillomavirus type-16 E6 gene. HCT-116/E6 cells were found to be more sensitive than control transfectants to CDDP and another DNA crosslinking agent, nitrogen mustard (HN2). HCT-116 cells with disrupted p21 genes also exhibited greater CDDP and HN2-sensitivity than parental HCT-116 cells. In contrast, the clonogenic survival of HCT-116 cells exposed to ionizing radiation, adriamycin, taxol or vincristine was not affected by p53 or p21 disruption. Sensitization of HCT-116/p21-/- cells to CDDP and HN2 was not limited to the HCT-116 cell background since MEF from p21 knockout mice were also more sensitive to these DNA crosslinking agents. Investigations into a possible cause of this enhanced sensitivity revealed that HCT-116 cells lacking p53 or p21 function exhibited a reduced ability to repair cisplatin-damaged CAT-reporter plasmids transfected into the cells. In addition, we found that HCT-116/p21-/- cells were much more susceptible to HN2-induced cell cycle delay than parental cells. Our results suggest that p21 disruption preferentially sensitizes at least some cell types to DNA crosslinking agents.

Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7 oncogene

The human papillomavirus type 16 (HPV-16) genome is commonly present in human cervical carcinoma, in which a subset of the viral genes, E6 and E7, are expressed. The HPV-16 E6 and E7 gene products can associated with and inactivate the tumor suppressor proteins p53 and Rb (the retinoblastoma susceptibility gene product), and in tissue culture cells, these viral genes display oncogenic properties. These findings have led to the hypothesis that E6 and E7 contribute to cervical carcinogenesis. This hypothesis has recently been tested by using transgenic mice as an animal model. HPV-16 E6 and E7 together were found to induce cancers in multiple tissues in which they were expressed, including squamous cell carcinoma, the cancer type most commonly associated with HPV-16 in the human cervix. We have extended these studies to investigate the in vivo activities of HPV-16 E7 when expressed in squamous epithelia of transgenic mice. Grossly, E7 transgenic mice had multiple phenotypes, including wrinkled skin that was apparent prior to the appearance of hair on neonates, thickened ears, and loss of hair in adults. In lines of mice expressing higher levels of E7, we observed stunted growth and mortality at an early age, potentially caused by an incapacity to feed. Histological analysis demonstrated that E7 causes epidermal hyperplasia in multiple transgenic lineages with high penetrance. This epithelial hyperplasia was characterized by an expansion of the proliferating compartment and an expansion of the keratin 10-positive layer of cells and was associated with hyperkeratosis. Hyperplasia was found at multiple sites in the animals in addition to the skin, including the mouth palate, esophagus, forestomach, and exocervix. In multiple transgenic lineages, adult animals developed skin tumors late in life with low penetrance. These tumors arose from the squamous epithelia and from sebaceous glands and were characterized histologically to be highly differentiated, locally invasive, and aggressive in their growth properties. On the basis of these phenotypes, we conclude that HPV-16 E7 can alter epithelial cell growth parameters sufficiently to potentiate tumorigenesis in mice.

Cloning and sequencing of the murine farnesyltransferase α-encoding cDNA from a cell line which expresses the human papillomavirus type-16 E6 gene

Using a differential hybridization technique, the murine farnesyltransferase α (FTA)-encoding cDNA was cloned from a mouse 10Tl/2 cell line which expresses the human papillomavirus type 16 (HPV16) E6 gene. Sequence analysis revealed that the murine 1647-bp FTA cDNA encoded 377 amino acids (aa). The murine and human sequences showed 83.2% nucleotide and 92.6% aa sequence identity.

POLYMORPHISM OF THE HPV-16 E6 GENE OF CERVICAL-CARCINOMA

Polymorphism of the human papillomavirus type 16 (HPV-16) genome has been reported to occur within the noncoding regulatory long control region (LCR) and in the E7 and L1 genes. The current study focuses on the HPV-16 E6 oncogene which interacts with the antioncogenic regulator p53. Seventy-eight HPV-16-positive DNA samples derived from cervical carcinomas were screened for the presence of polymorphism in the HPV-16 E6 gene by polymerase chain reaction (PCR) linked single stranded conformational polymorphism (SSCP) analysis. Nine DNA samples had heterozygous mutations within the same region of the E6 gene 3' terminus; T to C transitions at HPV-16 position 511 (silent) and one of the nine also had a 513 mutation (Met to Thr). These mutations correlated with the clinical aggressiveness of the tumor, suggesting that the presence of these mutations may be due to genomic instability of advanced cervical carcinoma.

Tumorigenicity by human papillomavirus type 16 E6 and E7 in transgenic mice correlates with alterations in epithelial cell growth and differentiation.

The human papillomavirus type 16 (HPV-16) E6 and E7 oncogenes are thought to play a role in the development of most human cervical cancers. These E6 and E7 oncoproteins affect cell growth control at least in part through their association with and inactivation of the cellular tumor suppressor gene products, p53 and Rb. To study the biological activities of the HPV-16 E6 and E7 genes in epithelial cells in vivo, transgenic mice were generated in which expression of E6 and E7 was targeted to the ocular lens. Expression of the transgenes correlated with bilateral microphthalmia and cataracts (100% penetrance) resulting from an efficient impairment of lens fiber cell differentiation and coincident induction of cell proliferation. Lens tumors formed in 40% of adult mice from the mouse lineage with the highest level of E6 and E7 expression. Additionally, when lens cells from neonatal transgenic animals were placed in tissue culture, immortalized cell populations grew out and acquired a tumorigenic phenotype with continuous passage. These observations indicate that genetic changes in addition to the transgenes are likely necessary for tumor formation. These transgenic mice and cell lines provide the basis for further studies into the mechanism of action of E6 and E7 in eliciting the observed pathology and into the genetic alterations required for HPV-16-associated tumor progression.

Transforming growth factors beta 1 and 2 transcriptionally regulate human papillomavirus (HPV) type 16 early gene expression in HPV-immortalized human genital epithelial cells

Human papillomavirus type 16 (HPV16) early proteins E6 and E7 have been implicated in maintenance of the malignant phenotype in cervical cancer. Transforming growth factors beta one and two (TGF betas 1 and 2), polypeptides that regulate cellular growth and differentiation, reversibly inhibited expression of the HPV16 E6 and E7 genes in several immortal genital epithelial cell lines. Loss of E6 and E7 protein expression followed a dramatic time- and dose-dependent decrease in E6 and E7 RNA levels and was accompanied by cessation of cell proliferation. TGF betas 1 and 2 inhibited HPV16 RNA expression at the transcriptional level; inhibition was dependent upon ongoing protein synthesis. TGF betas 1 and 2 also induced a six- to sevenfold increase in TGF beta 1 RNA. Cells became partially resistant to the inhibitory effects of TGF beta 1 on cell growth and HPV early gene expression after prolonged cultivation in vitro or after malignant transformation. Thus, TGF beta 1 may function as an autocrine regulator of HPV gene expression in infected genital epithelial cells.