Differential early viral gene expression in two stages of human papillomavirus type 16 DNA-induced malignant transformation.

Human papillomavirus (HPV) type 16 DNA induces progressive transformation in NIH 3T3 cells. Two types of cell lines, PM3T3G0 and PM3T3Fo, were isolated by G418 or focus selection, respectively, after transfection of cells by a recombinant HPV 16 DNA carrying the neo gene. These cell lines exhibited distinct phenotypes compared with controls. Saturation densities of PM3T3G0 and PM3T3Fo lines were two- to three- and five- to sevenfold greater than that of control NIH 3T3 cells, respectively. Neither cell type required high serum for growth, in contrast to NIH 3T3 cells. PM3T3G0 lines were premalignant, whereas PM3T3Fo lines manifested tumorigenicity within 2 weeks. Subpopulations of three PM3T3G0 lines underwent progressive transformation as reflected by focus formation. Analysis of HPV 16-specific mRNA species demonstrated that high levels of early and late gene expression were detected in premalignant PM3T3G0 lines, whereas relatively low quantities of selected gene messages were expressed in malignant transformants. Thus, high levels of viral gene expression are not crucial for malignant transformation.     

Progression of the phenotype of transformed cells after growth stimulation of cells by a human papillomavirus type 16 gene function.

Alteration of the growth properties of the established murine fibroblast cell lines NIH 3T3 and 3Y1 was studied in monolayer cultures and in cells suspended in semisolid medium after introduction of a cloned human papillomavirus type 16 (HPV16) DNA. HPV 16 DNA stimulated both cell lines to grow beyond their saturation densities in monolayer cultures without any apparent morphological changes or tendency to pile up. These cells were also stimulated to grow in soft agar. Since essentially all the cells that received the viral gene were stimulated to grow, the growth-stimulatory activity of HPV16 appeared to be due to the direct effect of a viral gene function. The NIH 3T3 cells showed an additional change in growth properties upon prolonged incubation of dense monolayers of cells containing the HPV16 DNA; morphologically recognizable dense foci appeared at a frequency of about 10(-3). These cells, when cloned from the foci, grew more rapidly in soft agar than the parental cells and were morphologically transformed. In other words, there were two sequential steps in cell transformation induced by HPV16. Practically all the viral DNAs were present in the cells as large rearranged multimers and were integrated into host chromosomal DNA. There was no obvious difference in the state of viral DNA in the cells of the original clone or the three subclones derived from it as dense foci. There was no difference in the amount or the number of viral RNA species expressed in the cells at these two stages. The secondary changes in the growth properties of NIH 3T3 cells appear to be due to some cellular alterations.     

Involvement of human papillomavirus type 8 genes E6 and E7 in transformation and replication.

We investigated the transforming activity of human papillomavirus type 8 (HPV8) by expressing all early open reading frames from a heterologous promoter in different rodent fibroblast lines. Morphological transformation was observed only in G418-selected mouse C127 and Rat 1 cells containing an intact E6-coding region. E6 of HPV8 did not transform NIH 3T3 cells as did E6 of bovine papillomavirus type 1. C127 cells transformed by E6 were anchorage independent and had a reduced serum requirement but did not form tumors in nude mice. E7 of HPV8 showed no transforming potential, in contrast to E7 of HPV18 and HPV16. It was, however, able to complement an E7 mutant of bovine papillomavirus type 1 with a defect in high-copy-number DNA maintenance. The data indicate that the expression of the HPV8 E6 open reading frame is sufficient to induce morphological transformation in rodent fibroblasts, whereas E7 is involved in the replication of the viral DNA.     

Mutant p53 can substitute for human papillomavirus type 16 E6 in immortalization of human keratinocytes but does not have E6-associated trans-activation or transforming activity.

Human papillomavirus type 16 (HPV16) E6 and E7 are selectively retained and expressed in HPV16-associated human genital tumors. E6 is active in several cell culture assays, including transformation of NIH 3T3 cells, trans activation of the adenovirus E2 promoter, and cooperation with E7 to immortalize normal human keratinocytes. Biochemically, the HPV16 E6 protein has been shown to bind to tumor suppressor protein p53 in vitro and induce its degradation in a rabbit reticulocyte lysate. To examine the relationship between the various biological activities of E6 and inactivation of p53, we tested the abilities of dominant negative mutants of p53 to substitute functionally for E6 in the three cell culture assays. While wild-type p53 inhibited keratinocyte proliferation, both mouse and human mutant p53s, in conjunction with E7, increased proliferation of the keratinocytes, resulting in generation of immortalized lines. However, in contrast to E6, mutant p53 was unable to induce transformation or trans activate the adenovirus E2 promoter in NIH 3T3 cells. These results suggest that inactivation of wild-type p53 is necessary for HPV-induced immortalization of human keratinocytes and that different or additional activities are required for E6-dependent transformation and trans activation of NIH 3T3 cells.     

Conservation of E6 and E7 regions of human papillomavirus types 16 and 18 present in cervical cancers

The E6 and E7 regions of human papillomavirus (HPV) type 16 were present in the DNA samples from cervical cancer cell lines, SKG-IIIa and SKG-IIIb, and those from cervical cancer tissues of three different patients. T601 cells, an NIH3T3 transformant obtained by transfection of DNA from a surgical specimen of a cervical cancer, also contained the E6 and E7 regions. The E6 region of HPV type 16 was expressed as mRNA in SKG-IIIa, SKG-IIIb and T601 cells. The E6 and E7 regions of HPV type 18 were present in the DNA samples from cervical cancer cell lines, SKG-I and SKG-II, and those from cervical cancer tissues of two different patients. SKG-I and SKG-II cells expressed the E6 region of HPV type 18 as mRNAs. These results strongly suggest that the E6 and E7 regions or the sequence surrounding these regions are important for maintaining malignant phenotype of cervical cancer cells.     

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.     

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.     

Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y1 cells.

Human papillomavirus type 16 (HPV 16) DNA is capable of morphologically transforming rat 3Y1 cells. The expression plasmids, constructed from the simian virus 40-based expression vector pSV2-0 and specific DNA fragments from the putative early region of the HPV 16 genome, were tested for their transforming capacity. Among the various pSV2 plasmids, only those containing the intact E7 coding region were found to produce foci of the transformed rat cells which could grow in a soft-agar medium. The data indicate that expression of the HPV 16 E7 open reading frame is sufficient to induce focal transformation of rat cells.     

人乳头瘤病毒16型亚基因DNA体外转化功能的细胞学研究

利用HZIP16和HZIP16K(见材料和方法)质粒,将人乳头瘤病毒16型(HPV-16)的全早期区基因及其开放读码框架(ORF)E6-E7分别转入ψ2细胞,所产生的重组病毒能诱导NIH3T3细胞发生转化。转化细胞具有恶性细胞的生物学和形态学特征,可在0.3％软琼脂中形成集落,可使裸鼠致瘤。Southern blot证明,HPV-16 E6-E7 ORFs序列以整合形式存在于转化细胞和裸鼠肿瘤细胞DNA中,表明HPV-16 DNA具有体外诱导NIH3T3细胞恶性转化的作用,E6-E7 ORFs是诱导细胞转化的关键基因。     

Identification of a transforming gene of human papillomavirus type 16.

Previously, we observed sequential two-step alteration, growth stimulation, and progression to a more malignant state in NIH 3T3 cells transfected by human papillomavirus type 16 (HPV-16) DNA. In this study, we prepared a cDNA library from RNA extracted from cells transfected with the HPV-16 DNA and isolated cDNA clones which had growth-stimulating activity. Analysis of these cDNA clones indicated that the E7 open reading frame alone is responsible for inducing both steps of this cell transformation.     

Tumorigenic transformation of murine keratinocytes by the E5 genes of bovine papillomavirus type 1 and human papillomavirus type 16.

To examine the biological properties of the bovine papillomavirus type 1 (BPV) and human papillomavirus type 16 (HPV16) E5 genes, each was cloned separately into a retroviral expression vector and helper-free recombinant viruses were generated in packaging cell lines. The BPV E5 retroviruses efficiently caused morphologic and tumorigenic transformation of cultured lines of murine fibroblasts, whereas the HPV16 E5 viruses were inactive in these assays. In contrast, infection of the p117 established line of murine epidermal keratinocytes with either the BPV or the HPV16 E5 retrovirus resulted in the generation of tumorigenic cells. Pam212 murine keratinocytes were also transformed to tumorigenicity by the HPV16 E5 gene but not by the gene carrying a frameshift mutation. These results establish that the HPV16 E5 gene is a transforming gene in cells related to its normal host epithelial cells.     

Image analysis of Feulgen-stained NIH/3T3 cells transformed with DNA of 4-nitroquinoline 1-oxide treated human breast epithelial cells.

The nuclear phenotypes of Feulgen-stained NIH/3T3 cells transformed with 4-nitroquinoline 1-oxide (4NQO) treated, human breast epithelial cell (HBEC) DNA were studied by scanning microspectrophotometry and image analysis and compared with data obtained for nontransformed cells and for NIH/3T3 cells under ras oncogene transfecting situations. The Feulgen-DNA content of the individual nuclei (NQ1, NQ2, and NQ3 phenotypes) of the transformed cells was found not to be deeply affected, although presence of chromatin structures resembling double minutes could be verified in part of the metaphases of the transformed cells. On the other hand, the chromatin supraorganization of these cells showed some changes involving increased (NQ2, NQ3) or decreased (NQ1) levels of condensation. The changes in chromatin packing states, however, were of small magnitude compared with those reported for NIH/3T3 cells transfected with a c-H-ras oncogene or an N-ras-containing MCF-7 cell DNA. It was assumed that the transformation of the NIH/3T3 cells is not always necessarily accompanied by high levels of chromatin condensation. The transformation of the NIH/3T3 cells induced by the 4NQO-treated HBEC DNA and particularly the changes in chromatin condensation in these transformed cells could not be attributed merely to a ras activation elicited by the carcinogen. It is suggested that a more complex transforming mechanism is involved, probably owing to the fact that a whole genomic DNA of the 4NQO-treated HBEC has been used for transfection.     

In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype.

We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific.     

The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein.

The human papillomavirus (HPV) E5 proteins are predicted from DNA sequence analysis to be small hydrophobic molecules, and the HPV type 6 (HPV-6) and HPV-11 E5 proteins share several structural similarities with the bovine papillomavirus type 1 (BPV-1) E5 protein. Also similar to the BPV-1 E5 protein, the HPV-6 and HPV-16 E5 proteins exhibit transforming activity when assayed on NIH 3T3 and C127 cells. In this study, we expressed epitope-tagged E5 proteins from both the "low-risk" HPV-6 and the "high-risk" HPV-16 in order to permit their immunologic identification and biochemical characterization. While the HPV-6 and HPV-16 E5 proteins fail to form disulfide-linked dimers and oligomers, they did resemble the BPV-1 E5 protein in their intracellular localization to the Golgi apparatus, endoplasmic reticulum, and nuclear membranes. In addition, the HPV E5 proteins also bound to the 16-kDa pore-forming protein component of the vacuolar ATPase, a known characteristic of the BPV-1 E5 protein. These studies reveal a common intramembrane localization and potential cellular protein target for both the BPV and HPV E5 proteins.     

Regions of human papillomavirus type 16 E7 oncoprotein required for immortalization of human keratinocytes.

Binding of the retinoblastoma gene product (pRB) by viral oncoproteins, including the E7 of human papillomavirus type 16 (HPV 16), is thought to be important in transformation of cells. One of the steps in transformation is the immortalization process. Here we show that mutations in E7 within the full-length genome which inhibit binding of pRB do not abrogate the ability of the HPV 16 DNA to immortalize primary human epithelial (keratinocyte) cells. A mutation in one of the cysteines of a Cys-X-X-Cys motif which is contained in the carboxy half of the E7 and is part of a zinc finger arrangement completely eliminates the ability of HPV 16 DNA to immortalize cells. The results indicate the importance of E7 in the immortalization of primary keratinocytes but suggest that the binding of pRB is not essential.