Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines.

Human cervical carcinoma cell lines that harbor human papillomavirus (HPV) have been reported to retain selectively and express HPV sequences which could encode viral E6 and E7 proteins. The potential importance of HPV E6 to tumors is suggested further by the observation that bovine papillomavirus (BPV) E6 can induce morphologic transformation of mouse cells in vitro. To identify HPV E6 protein, a polypeptide encoded by HPV-16 E6 was produced in a bacterial expression vector and used to raise antisera. The antisera specifically immunoprecipitated the predicted 18-kd protein in two human carcinoma cell lines known to express HPV-16 RNA and in mouse cells morphologically transformed by HPV-16 DNA. The 18-kd E6 protein was distinct from a previously identified HPV-16 E7 protein. The HPV-16 E6 antibodies were found to be type specific in that they did not recognize E6 protein in cells containing HPV-18 sequences and reacted weakly, if at all, to BPV E6 protein. The results demonstrate that human tumors containing HPV-16 DNA can express an E6 protein product. They are consistent with the hypothesis that E6 may contribute to the transformed phenotype in human cervical cancers that express this protein.     

Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV-16 plus EJ-ras.

The close association between HPV-16 and cervical cancer implies some role for the virus in development of this cancer. Recent studies have shown that the HPV-16 E7 gene encodes the major transforming activity of the virus in baby rat kidney (BRK) cell transformation assays. To investigate the requirement for continued E7 expression in BRK cells transformed by HPV-16 E7 plus EJ-ras, we have developed a system for inducible expression of the E7 gene. The studies reported here show that continued expression of the HPV-16 E7 gene is required for maintenance of the transformed phenotype in these cells. The implications these observations bear on the role of the E7 gene in cervical carcinoma are discussed.     

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.     

Inhibition of apoptosis in human laryngeal cancer cells by E6 and E7 oncoproteins of human papillomavirus 16

The carcinogenesis of human papillomaviruses type 16 (HPV-16) is mainly due to its two oncoproteins, E6 and E7. Their carcinogenic features in term of their relationship with Bcl-2 family are still unclear. We thus aimed to analyze the expression of Bcl-2 family members, Bcl-2, Bax, and Bak in laryngeal cancer cells transfected with the E6 or E7 and to determine the sensitivity of these cells to apoptotic stimuli. We employed two human laryngeal cancer cell lines, UMSCC12 and UMSCC11A in this study. These two cell lines were stably transfected with HPV16 E6, E7 or empty vector, pcDNA3.1. We found that E6 and E7 inhibited apoptosis induced by TNF-alpha/CHX in both UMSCC11A and UMSCC12 cells, enhanced the stability of Bcl-2 protein and increased the degradation of Bak protein. Furthermore, it was found that HPV-16 E7 statistically enhanced the expression of Bcl-2 in laryngeal cancer. The alteration of Bak by E6 and E7 was not through the influence on the Bak promoter, as the luciferase assay showed that neither E6 nor E7 changed the Bak promoter activity. We conclude that the evasion of apoptosis mediated by HPV-16 E6 and E7 is associated with increased Bcl-2 and decreased Bak in laryngeal carcinogenesis and that the decreased level of Bak by E6 and E7 is not caused by the regulation of the Bak promoter but by reducing its protein stability.     

Induction of apoptosis in cervical carcinoma cells by peptide aptamers that bind to the HPV-16 E7 oncoprotein.

Human papillomaviruses (HPV) of the high-risk type are causally involved in human tumors, in particular cervical carcinoma. Expression of the viral oncogenes E6 and E7 is maintained in HPV-positive tumors, and it was shown that E6 and E7 of HPV-16 can immortalize human keratinocytes, the natural host cells of the virus. Expression of the viral genes is also required for maintenance of the transformed phenotype. The oncogenic activity of the E6 and E7 oncoproteins is mediated by their physical and functional interaction with cellular regulatory proteins. To knock out the function of the E7 protein in living cells, we have developed peptide aptamers with high specific binding activity for the E7 protein of HPV-16. We show here that E7-binding peptide aptamers induce programmed cell death (apoptosis) in E7-expressing cells, whereas E7-negative cells are not affected. Furthermore, E7-binding peptide aptamers induce apoptosis in HPV-16-positive tumor cells derived from cervical carcinoma. The data suggest that E7-binding peptide aptamers may be useful tools to specifically eliminate HPV-positive tumors.     

Normal Human Fibroblasts Immortalized by Introduction of Human Papillomavirus Type 16 (HPV-16) E6-E7 Genes

This report demonstrates that normal human fibroblasts can be immortalized by the introduction of HPV-16 E6-E7 genes. We designed zinc-inducible expression plasmids with HPV-16 E6, E7 or both. Each plasmid was introduced into normal human fibroblasts (TIG-3 cells) using lipofection methods. Only trans-fectants with the HPV-16 E6-E7 zinc-inducible expression plasmid, which were cultured in medium supplemented with 100 μM ZnSO₄, overcame crisis and could be cultured over 200 population doubling levels (PDLs). These cell lines showed the reactivation of telomerase after crisis, and morphological alterations were also observed.     

Genomic and proteomic expression patterns in HPV-16 E6 gene transfected stable human carcinoma cell lines

cDNA microarray and proteomics studies were performed to analyze the genomic and proteomic expression patterns in HPV-16 E6 gene transfected stable human carcinoma cell lines. Among 1024 known genes and ESTs tested by cDNA microarray, we found 50 upregulated and 35 downregulated genes in RC10.1 HPV-16 E6 transfected human colon adenocarcinoma cells compared to RKO cells, and 27 upregulated and 43 downregulated genes in A549E6 HPV-16 E6 transfected human lung adenocarcinoma cells compared to A549 cells. Employing two dimensional gel electrophoresis and MALDI-TOF-MS, the global pattern of protein expressions in RC10.1 human colon adenocarcinoma and A549E6 human lung adenocarcinoma cell lines stably expressing the HPV 16-E6 gene were compared with those of RKO and A549 cell lines to generate a differential protein expression catalog. We found 13 upregulated and 13 downregulated proteins in RC10.1 (E6-expressing RKO) cells compared to RKO cells and 12 upregulated and 14 downregulated proteins in A549E6 (E6-expressing A549) cells compared to A549 cells. The identified genes and proteins were classified into several groups according to the subcellular function. Expressing pattern of three genes and proteins (CDK5, Bak, and I-TRAF) were matched in both analyses of cDNA microarray and proteomics. These powerful approaches using cDNA microarray and proteomics could provide in-depth information on the impact of HPV-16 E6-related genes and proteins. Differential gene and protein expression patterns by transfection of HPV-16 E6 will provide the nucleus of valuable resource for investigation of the biochemical basis of cervical carcinogenesis. Further understanding of this data base may provide valuable resources for developing novel diagnostic markers and therapeutic targets of cervical cancer.     

Both conserved region 1 (CR1) and CR2 of the human papillomavirus type 16 E7 oncogene are required for induction of epidermal hyperplasia and tumor formation in transgenic mice.

High-risk human papillomavirus type 16 (HPV-16) and HPV-18 are associated with the majority of human cervical carcinomas, and two viral genes, HPV E6 and E7, are commonly found to be expressed in these cancers. The presence of HPV-16 E7 is sufficient to induce epidermal hyperplasia and epithelial tumors in transgenic mice. In this study, we have performed experiments in transgenic mice to determine which domains of E7 contribute to these in vivo properties. The human keratin 14 promoter was used to direct expression of mutant E7 genes to stratified squamous epithelia in mice. The E7 mutants chosen had either an in-frame deletion in the conserved region 2 (CR2) domain, which is required for binding of the retinoblastoma tumor suppressor protein (pRb) and pRb-like proteins, or an in-frame deletion in the E7 CR1 domain. The CR1 domain contributes to cellular transformation at a level other than pRb binding. Four lines of animals transgenic for an HPV-16 E7 harboring a CR1 deletion and five lines harboring a CR2 deletion were generated and were observed for overt and histological phenotypes. A detailed time course analysis was performed to monitor acute effects of wild-type versus mutant E7 on the epidermis, a site of high-level expression. In the transgenic mice with the wild-type E7 gene, age-dependent expression of HPV-16 E7 correlated with the severity of epidermal hyperplasia. Similar age-dependent patterns of expression of the mutant E7 genes failed to result in any phenotypes. In addition, the transgenic mice with a mutant E7 gene did not develop tumors. These experiments indicate that binding and inactivation of pRb and pRb-like proteins through the CR2 domain of E7 are necessary for induction of epidermal hyperplasia and carcinogenesis in mouse skin and also suggest a role for the CR1 domain in the induction of these phenotypes through as-yet-uncharacterized mechanisms.     

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.     

Human papillomavirus type 16 E6 and HPV-16 E6/E7 sensitize human keratinocytes to apoptosis induced by chemotherapeutic agents: roles of p53 and caspase activation

We and others have previously reported that human papillomavirus (HPV)-16 E6 protein expression sensitizes certain cell types to apoptosis. To confirm that this sensitization occurred in HPV's natural host cells, and to explore the mechanism(s) of sensitization, we infected human keratinocytes (HKCs) with retroviruses containing HPV-6 E6, HPV-16 E6, HPV-16 E7, or HPV-16 E6/E7. Apoptosis was monitored by DNA fragmentation gel analysis and direct observation of nuclei in cells stained with DAPI. Exposure of HKCs to etoposide, cisplatin, mitomycin C (MMC), atractyloside, and sodium butyrate, resulted in a time and dose-dependent induction of apoptosis. Expression of HPV-16 E6 and HPV-16 E6/E7, but not HPV-6 E6 or HPV-16 E7, enhanced the sensitivity of HKCs to cisplatin-, etoposide- and MMC-, but not atractyloside- or sodium butyrate-induced apoptosis. Expression of both HPV-16 E6 and HPV-16 E6/E7 decreased, but did not abolish, p53 protein levels relative to normal HKCs, and resulted in cytoplasmic localization of wt p53. P53 induction occurred in HPV-16 E6 and HPV-16 E6/E7 expressing cells after exposure to cisplatin or MMC, though never to levels found in normal untreated HKCs. P21 levels were decreased in HPV-16 E6 and HPV-16 E6/E7 expressing HKCs, and no induction of p21 was seen in these cells following exposure to cisplatin or MMC. Caspase-3 activity was found to be elevated in HPV-16 E6-expressing HKCs following exposure to cisplatin and MMC as documented by fluorometric and Western Blot analysis. Expression of wt CrmA or treatment of HPV-16 E6 expressing HKCs with the caspase-3 inhibitor DEVD.fmk prevented HPV-16 E6-induced sensitization in HKCs. These results suggest that HPV-16 E6 and HPV-16 E6/E7 expression sensitizes HKCs to apoptosis caused by cisplatin, etoposide and MMC, but not atractyloside or sodium butyrate. The data also suggest that wt p53 and caspase-3 activity are required for HPV-16 E6 and HPV-16 E6/E7-induced sensitization of HKCs to DNA damaging agents.     

Immortalization of fetal mouse brain glial cells by human papillomavirus type 16 E7 genes.

Fetal mouse brain glial cells in primary cultures were immortalized by recombinant retroviruses containing human papillomavirus type 16 E7 genes, and named VR-2g cells. The presence and expression of E7 genes in VR-2g cells were demonstrated by the Southern and the Northern blot analyses. VR-2g cells did not form colonies in soft agar culture, indicating that VR-2g cells have no transforming phenotypes. By the karyotype analysis, VR-2g cells consisted of two cell populations, the pseudo-diploid and the pseudo-tetraploid. VR-2g cells were positive in immunostaining with mono- and polyclonal antibodies against glial fibrillary acidic proteins (glial-specific intermediate filaments). In addition, VR-2g cells secreted neurotrophic factors as assayed with primary cultures of fetal rat striatum neurons, although molecular characterization of the factor(s) was not yet determined. These results indicate that the present method for cell immortalization will be useful for establishing untransformed cell lines from primary cultures of fetal brain cells.     

Immortalization of rat kidney glomerular mesangial cell and its coculture with glomerular epithelial cell

Mesangial cell has several key roles in the control of glomerular function: it participates in the regulation of glomerular filtration rate, macromolecular clearance, and as both a source and target of numerous hormones and autocrines. Many of these insights into mesangial cell function have been obtained by studying mesangial cells in culture. However, no suitable cell lines have been established yet. We here reported the immortalization of rat kidney glomerular mesangial cell by transfection of E6 and E7 genes of human papillomavirus type 16 (HPV-16) via electroporation and lipofection. The results showed that only electroporation could transfect the genes to mesangial cells and the transfected cells maintained the viability for longer than 6 months. Fluorescence microscopic observation showed that cellular contractility and phagocytosis, which are the two main phenotypes of mesangial cells, are well maintained after transfection. The coculture of transfected mesangial cells with rat glomerular epithelial cells showed that the growth of mesangial cells was suppressed by epithelial cell, but the growth of epithelial cells was enhanced by mesangial cells. Moreover, an enhancing effect on the phagocytosis of mesangial cell was also observed in coculture. Such results may imply that the glomerular cell-cell interaction plays an important role in the regulation of cell proliferation and differentiation.     

The E7 proteins of the nononcogenic human papillomavirus type 6b (HPV-6b) and of the oncogenic HPV-16 differ in retinoblastoma protein binding and other properties.

The E7 early viral protein of the oncogenic human papillomavirus type 16 (HPV-16) has been strongly implicated in the maintenance of the malignant phenotype in cervical cancers and cancer-derived cell lines. HPV-16 E7 is a nuclear phosphoprotein that can cooperate with ras to transform baby rat kidney cells, transactivates the adenovirus E2 promoter, and binds to the retinoblastoma (RB) protein. The E7 phosphoprotein of the nononcogenic HPV-6b, which is generally associated with benign genital warts, is similar to the HPV-16 E7 in amino acid sequence but differs dramatically in migration in sodium dodecyl sulfate-polyacrylamide gels, sedimentation in nondenaturing glycerol gradients, and the ability to bind the RB protein. Our results indicate that the RB protein preferentially binds the phosphorylated form of HPV-6b E7, which comprises a minor fraction of the total E7 expressed in transiently transfected COS-7 cells. These characteristics may help to explain the difference in the oncogenic potential of the oncogenic and nononcogenic types of genital papillomaviruses.     

Mutation of the endogenous p53 gene in cells transformed by HPV-16 E7 and EJ c-ras confers a growth advantage involving an autocrine mechanism.

Rat embryo fibroblasts transformed with the HPV-16 E7 gene and the activated c-H-ras gene fall into two distinct phenotypic classes. At high cell density, clones of one class form colonies in methylcellulose supplemented with low serum; at low cell density, these cells display responsiveness to mitogenic factors present in serum-free conditioned medium from rat embryo fibroblasts. In contrast, clones of the second class exhibit an absolute dependency on growth factors present in serum at all cell densities in the methylcellulose colony assay and fail to respond to conditioned medium. We find that the status of the endogenous p53 gene is tightly correlated with these two classes of clones. Clones of the first class contain missense mutations in the p53 gene and have lost the wild-type allele. Clones of the second class express wild-type p53 protein. The importance of mutant p53 expression in reducing the growth factor dependency of transformed clones was confirmed in a separate series of experiments in which rat embryo fibroblasts were transformed with three genes, E7 + ras + mutant p53. The growth behaviour of these triply transfected clones was similar to that of the E7 + ras clones expressing endogenous mutant p53. We demonstrate that the enhanced proliferation of E7 + ras clones expressing mutant p53 protein involves an autocrine mechanism.     

The E5 oncoprotein of human papillomavirus type 16 transforms fibroblasts and effects the downregulation of the epidermal growth factor receptor in keratinocytes.

To determine the function of the E5 open reading frame (ORF) of the human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts were transformed to colony formation in soft agar, and the transformation frequency was increased by epidermal growth factor (EGF) but not by platelet-derived growth factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-16 were mitogenic in primary human foreskin epithelial cells (keratinocytes) and acted synergistically with EGF. Investigation of keratinocytes expressing HPV-16 E5 showed that the number of endogenous EGF receptors (EGFRs) per cell was increased two- to fivefold. Immunofluorescence microscopy of HPV-16 E5-expressing keratinocytes indicated that there was an apparent delay in the internalization and degradation of EGFRs compared with controls. Kinetic studies with [125I]EGF showed that the ligand underwent normal internalization and degradation in both HPV-16 E5-expressing and control keratinocytes, but in E5-expressing cells, a greater number of receptors recycled back to the cell surface within 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphorylation of the EGFR on tyrosine, an indication of receptor kinase activity, was of greater magnitude in the HPV-16 E5-expressing keratinocytes than in control cells, although the basal level of receptor phosphorylation was similar.