Growth Dependence of Human Papillomavirus 16 DNA-positive Cervical Cancer Cell Lines and Human Papillomavirus 16-Transformed Human and Rat Cells on the Viral Oncoproteins

The dependence on human papillomavirus (HPV) Oncoproteins of the growth of cervical cancer cell lines [C4-1, HeLa (both containing HPV 18 DNA), CaSki and SiHa (both containing HPV 16 DNA)], HPV 16-transformed human embryonic kidney cells, and HPV 16-transformed rat brain and 3Y1 cells was examined by using antisense RNA approaches. The cells were transfected with plasmids expressing RNA antisense to the HPV 16 or 18 open reading frames E6E7, together with plasmids expressing the hygromycin B resistance gene, and drug-resistant colonies were scored three weeks later. In all the human cell lines, the efficiency of colony formation was lowered by RNA antisense to the resident HPV type. Some of the rat cell lines responded to the antisense plasmids, but some did not. From a nonresponding rat tumor line (3Y1HP-1T), cell clones with various levels of E7 protein were isolated after transaction with the antisense plasmid, and were examined for anchorageindependent growth in soft agar. The colonies formed by the clones with lower E7 levels tended to be smaller and fewer than those formed by the clones with higher E7 levels. These findings strongly suggest that some of the transformed or cancer phenotypes of cells in vitro are dependent, even after extensive passages and malignant changes, on expression of the oncoproteins of the resident HPV.     

Combined effects of human papillomavirus-18 and n-methyl-n-nitro-n-nitrosoguanidine on the transformation of normal human oral keratinocytes

We immortalized oral keratinocytes by transfecting them with recombinant human papillomavirus (HPV) type 18 DNA and established three cell lines. These lines were morphologically different from their normal counterpart, contained integrated entire HPV-18 DNA, and expressed the viral E6/E7 genes. The cells contained less p53 protein and more c-myc mRNA than normal cells. However, they proliferated only in keratinocyte growth medium (KGM) containing low calcium and were not tumorigenic in nude mice. To test the hypothesis that tumors result from the combined effect of a “high-risk” HPV and chemical carcinogens in the human oral cavity, we exposed the immortalized cells to the chemical carcinogen N-methyl-N′-nitro-N-nitrosoguanidine. Three chemically transformed cell colonies were isolated. These cells (a) proliferated well in both KGM and Dulbecco′s modified minimum essential medium containing physiological levels of calcium; (b) were capable of proliferating in nude mice; (c) contained intact, integrated HPV-18 sequences; (d) transcribed substantially more HPV-18 E6/E7, transforming growth factor-α, and c-myc than the immortalized counterpart; and (e) contained, like the immortalized counterpart, less wild-type p53 protein and DCC message. These data indicate that human oral keratinocytes can be transformed by sequential exposure of normal keratinocytes to a “high-risk” HPV and chemical carcinogens. © 1994 Wiley-Liss, Inc.     

Cloning and characterization of human papillomavirus type 52 from cervical carcinoma in Indonesia.

Human papillomavirus (HPV) type 52 from a cervical carcinoma in Indonesia was molecularly cloned and characterized. By hybridization with cervical carcinoma DNAs from Indonesian patients, HPV 52 was detected in 3 of 52 cases (6%), whereas HPV 16 and 18 were detected in 8 and 7 cases, respectively (15% and 13%). Sequence analysis revealed that the E6-E7 ORFs contained several DNA binding motifs (Cys-X-X-Cys) like previously sequenced HPVs. The E6 ORF also contained splice donor and acceptor signals, which may allow the expression of E6* protein. The E7 ORF encoded an amino acid sequence that is conserved in some DNA tumor viruses and is involved in binding to Rb protein and in cellular transformation. Transfection of a subgenomic fragment of HPV 52 under the control of a heterologous promoter showed that the E7 ORF alone induced anchorage-independent growth of established rodent cells and immortalized primary rat embryo fibroblasts (REF), and that in cooperation with activated ras, it induced malignant transformation of REF. The E6 ORF also induced, less efficiently, anchorage-independent growth. These results strongly suggest that HPV 52, like HPV 16 and 18, has oncogenic potential.     

Cytogenetic analysis of rat cells, transformed by human papillomavirus genes

Chromosomal analysis of immortalized rat embryo fibroblasts (IE5) and transformed by HPV18 E6+E7 genes (A4E5) or HPV16 E7 alone (trB4; trF8; trC2) variants have been done. Transformed cell lines represented heterogeneous cell populations containing neardiploid subpopulations with 41-44 chromosomes and also heterogeneous polyploid cells in contrast to immortal cells IE5 that contained normal number of chromosomes-42. In transformed cells the abnormalities of interphase nuclei (giant-, micro-, apoptotic nuclei) were observed which could reflect genomic instability of polyploid cells. Several chromosomal alterations were revealed in immortal IE5 cells, but only reciprocal translocation t(8; 10) (q22q12.3) was stable and kept in cells transformed by HPV18 E6+E7 genes or HPV16 E7 alone. We can conclude that genomic instability and clonal expansion of the cells with specific chromosomal alterations contribute to HPV-mediated transformation.     

Immortalization of human adult prostatic adenocarcinoma cells by human papilloma virus HPV16 and — 18 DNA

Primary prostate epithelial and prostate adenocarcinoma cells cultured in serum-free medium grew for up to 10 passages before senescence. Cells from prostate adenocarcinoma of a 55-year-old patient without lymph node involvement were transfected with plasmids containing recombinant human papilloma virus HPV16 or HPV18 DNA and the selectable neomycinresistance gene. After G-418 selection, cells underwent crisis, and surviving cells infected with retroviruses encoding the HPV18 E6/E7 genes (HPV-PAC1), transfected with a head-to-tail dimer of the complete HPV16 genome (HPV-PAC2), or transfected with HPV18 E6/E7 early genes (HPV-PAC3) were established. HPV-PAC1 and HPV-PAC2 cultures appeared morphologically similar to primary cultures even after 40 passages. However, HPV-PAC2 cultures had a clonal morphology. All lines were positive for cytokeratin 18, had acquired vimentin expression, and contained either HPV16 or HPV18 sequences integrated into host DNA. None was tumorigenic in nude mice or formed colonies in soft agar. These cells did not secrete prostate specific antigen nor respond to androgen although tamoxifen inhibited the growth of the cells. Immunohistochemistry showed no evidence of p53 overexpression. Further characterization of these cell lines and examination of their response to chemotherapeutic agents may provide relevant information for the study of hormone-independent PC.     

Correlation of modified human papilloma virus early gene expression with altered growth properties in C4-1 cervical carcinoma cells

In cervical carcinomas and cell lines derived from these tumors the DNA of specific types of human papilloma viruses (HPVs) is integrated into the host cell genome. The two viral open reading frames E6 and E7 are consistently transcribed in tumors and cell lines, and the respective proteins were detected in cells cultured in vitro. As shown here, modulation of HPV 18 E6 and E7 gene expression in C4-1 cervical carcinoma cells is accompanied by an altered cell growth. HPV 18 E6 and E7 expression can be enhanced by glucocorticoid treatment of C4-1 cells, and an increased cell proliferation is observed. In contrast, after introduction of complementary RNA to the HPV 18 E6 and E7 open reading frames, their expression is inhibited, and decreased cell growth is observed. These results support the hypothesis that expression of HPV E6 and E7 open reading frames is directly involved in growth regulation of cervical carcinoma cells.     

Tumorigenic Transformation of Primary Rat Embryonal Fibroblasts by Human Papillomavirus Type 8 E7 Gene in Collaboration with the Activated H-ras Gene

Particular types of human papillomavirus (HPV) are associated with skin cancer of epidermo-dysplasia verruciformis (EV) patients. Here, we show, for the first time, that the E7 gene of EV-associated HPV8 possesses a potential oncogenic transforming ability. The HPV8 E7 open reading frame (ORF) and the HPV16 E7 ORF were cloned under the SV40 promoter/enhancer to construct recombinant plasmids pcD2-8E7 and pcD2-16E7, respectively. Transfection of primary rat embryonal flbroblasts having an activated H- ras gene revealed that pcD2-8E7 as well as pcD2-16E7 induced transformation of cells In G418-resistant colonies at an efficiency of 12.3% and 42.9%, respectively. The resulting transformed cell lines induced by pcD2-8E7 and activated H- ras were tumorigenic when injected into syngeneic immunocompetent rats. The potential tumorigenicity of HPV8 E7 seemed to be higher than that of HPV16 E7.     

Tumorigenic transformation of primary rat embryonal fibroblasts by human papillomavirus type 8 E7 gene in collaboration with the activated H-ras gene.

Particular types of human papillomavirus (HPV) are associated with skin cancer of epidermodysplasia verruciformis (EV) patients. Here, we show, for the first time, that the E7 gene of EV-associated HPV8 possesses a potential oncogenic transforming ability. The HPV8 E7 open reading frame (ORF) and the HPV16 E7 ORF were cloned under the SV40 promoter/enhancer to construct recombinant plasmids pcD2-8E7 and pcD2-16E7, respectively. Transfection of primary rat embryonal fibroblasts having an activated H-ras gene revealed that pcD2-8E7 as well as pcD2-16E7 induced transformation of cells in G418-resistant colonies at an efficiency of 12.3% and 42.9%, respectively. The resulting transformed cell lines induced by pcD2-8E7 and activated H-ras were tumorigenic when injected into syngeneic immunocompetent rats. The potential tumorigenicity of HPV8 E7 seemed to be higher than that of HPV16 E7.     

Detection of human papillomavirus DNA in esophageal carcinoma in Japan by polymerase chain reaction.

BACKGROUND. Human papillomaviruses (HPV) have been implicated strongly in the pathogenesis of human squamous cell carcinomas, especially of anogenital carcinomas. Some pathologic changes of the esophagus may be one of the candidates for HPV etiology, but the role of HPV infections in the carcinogenesis of the esophagus remains to be clarified. METHODS. To elucidate the association of HPV with carcinogenesis of the esophagus, 45 biopsy samples of esophageal squamous cell carcinomas were examined for the presence of HPV DNA by polymerase chain reaction (PCR). Primers for PCR were (1) consensus primers (CP) for the simultaneous amplification of the E6-E7 regions of cancer-associated HPV types (HPV 16, 18, 31, 33, 52b, and 58), which have been shown to have transforming activities; (2) type-specific primers (SP16, SP18) for the E7 regions of HPV 16 and HPV 18, respectively; and (3) general primers (GP) for the simultaneous amplification of the L1 regions of HPV 6, 11, 16, 18, 31, and 33. RESULTS. PCR using CP first was done for screening and showed that 3 (6.7%) of 45 specimens contained HPV 16 or HPV 18 DNA, the oncogenic high-risk HPV types. This was confirmed by SP16 and SP18 PCR. However, no HPV DNA was detected by PCR using GP. These results suggested that the HPV DNA detected might be integrated into the cell genome with their transforming genes retained and their late regions deleted. CONCLUSIONS. Most oncogenic types of HPV (HPV 16 and HPV 18) were detected by PCR in carcinomas of the esophagus. Thus, HPV might play a role, although at a low frequency, in carcinogenesis of the esophagus.     

HPV-16 E7 functions at the G1 to S phase transition in the cell cycle

Previous reports have demonstrated that E7 is the major transforming gene of HPV-16 and that continued expression of the gene is required to maintain the transformed phenotype of primary baby rat kidney cells transformed by HPV-16 E7 and EJ-ras. To investigate the point of action of E7 in the cell cycle we have utilised a system of inducible expression of the E7 gene. The studies reported here show that stimulation of cellular DNA synthesis by E7 is distinct from that observed with calf serum. In combination with cytofluorimetric analyses these results indicate that E7 functions at the transition from G1 to S phase of the cell cycle. This adds further support to the hypothesis of a common pathway of transformation shared by the DNA tumour viruses HPV, SV40 and Adenovirus.     

Establishment and characterization of 12 uterine cervical-carcinoma cell lines: Common sequence variation in the E7 gene of HPV-16-positive cell lines

A total of 12 carcinoma cell lines of the human uterine cervix were established from 5 keratinizing and 5 non-keratinizing squamous-cell carcinomas, and 2 small-cell carcinomas. Of these, 10 lines grew as adherent cells and 2 as floating aggregates. All lines showed (i) similarity in morphology to the primary tumor from which they were derived; (ii) high viability with relatively long doubling times (48–96 hr); (iii) absence of Mycoplasma and other bacteria, apart from one Mycoplasma-contaminated line; (iv) genetic heterogeneity by DNA-fingerprinting analysis; (v) absence of p53 mutation from exon 4 through 9; and (vi) the presence of HPV DNA sequence. Among the lines, 7 were infected by HPV-16, 3 by HPV-18, 1 by HPV-31, and 1 by HPV-33; the 2 cell lines derived from small-cell carcinomas contained HPV-18. Interestingly, 6 of the 7 cell lines containing HPV-16-type DNA harbored the same alteration of E7 at nucleotide position 647 (amino acid 29, AAT |iO AGT, Asn |iO Ser), whereas the 3 HPV-18-positive lines did not; 3 cell lines proved to have intact E1/E2 of HPV, suggesting the presence of episomally replicating HPV DNA as well as the integrated form, whereas the other 9 lines were shown to have integrated HPV. Taken together, these cell lines would be very useful for studying the biology of uterine cervical carcinoma. Int. J. Cancer 72:313–320, 1997. © 1997 Wiley-Liss, Inc.     

The state of immortalized, transforming and suppressor genes in rat-cells, transfected by polyomavirus large-T and hpv18 e6+e7 genes

Several novel cell clones (A1-A6) derived from the rat embryo fibroblasts immortalized by the polyoma virus T-antigen (LT) gene and transformed by HPV18 E6 and E7 genes were explored. Using E6 or E7 peptide antisera we detected E6 and E7 proteins with approximate molecular masses of 16 kDa and 20 kDa, respectively. Monoclonal antibody to p53 PAb421 but not PAb240 precipitated different but appreciable amounts of p53 protein in all cell clones, indicating that wild-type p53 is expressed in these cells. So expression of HPV18 E6 protein in cells does not always lead to a complete reduction in p53 levels. The quantity of p53 protein in cell clones did not correlate with the level of polyoma virus large T-antigen expression. Variations in levels of p53 protein in clones did not influence on such cell biological properties as anchorage-independence and tumorigenicity for nude mice which were similar in all cell clones.     

Tumorigenicity of the E6 and E6-E7 gene constructions derived from human papillomavirus type 33.

Human papillomavirus type 33 (HPV33) belongs to the group of HPV types frequently found in severe cervical dysplasias and carcinomas. By analogy with HPV types 16 and 18 selectively expressing E6 and E7 genes in malignant tissues, we studied the HPV33 E6 and E7 open reading frames in various configurations with upstream promoter and noncoding region (NCR) known to contain a particular 78-bp tandem repeat. HPV DNA fragments were cloned into expression vectors between the SV40 or the mouse metallothionein I promoter and the neo gene, transfected into NIH3T3 cells, selected by neo resistance, and inoculated into nude mice. In these bioassays, a weak transforming activity was detected for E6 open reading frame, and could be significantly enhanced either by the NCR or the E7 open reading frame. No tumorigenicity could be detected for E7 alone or in configuration with the upstream NCR. Further analyses of tumor cells showed that HPV33-derived genes were not sufficient to induce an anchorage-independent phenotype and, interstingly, there was no requirement for virus-transfected tumor cells to retain HPV sequences during tumor progression. We concluded that the transformation function of HPV33 resides in E6 gene as assayed by tumorigenicity. An enhancer of the E6 promoter is located in the NCR. On the other hand, in the absence of the NCR, E6 tumorigenicity may be augmented by the E7.     

Selective cytotoxicity of phospholipids and diacylglycerols to rat 3Y1 fibroblasts transformed by adenovirus type 12 or its E1A gene

The colony-forming ability of rat 3Y1 fibroblasts transformed by adenovirus type 12 (Ad12) was drastically reduced when the cells were cultivated for 18 h in medium augmented with 300 micrograms/ml of liposomes composed of either phosphatidylcholine (PC) or phosphatidylinositol. In contrast, those of untransformed 3Y1 cells and simian virus 40-transformed and polyomavirus-transformed 3Y1 cells were not. The cytotoxicity of PC liposomes was also observed in 3Y1 cells transformed by plasmid DNA containing Ad12-E1A gene but not in those transformed by adenovirus type 2, Rous avian sarcoma virus, or plasmid DNA carrying v-Ha-ras oncogene. The extensive killing of Ad12-transformed and E1A-transformed 3Y1 cells occurred in liposomes of dioleoyl-PC and of dilinoleoyl PC but not those of dipalmitoyl PC, distearoyl-PC, or diarachidonyl PC, suggesting that the acyl groups of phospholipids play an important role in cytotoxicity. Dilinoleoylglycerol, 60 micrograms/ml, was also cytotoxic selectively to Ad12-transformed and E1A-transformed 3Y1 cells, although the toxicity of lysophosphatidylcholine or linoleic acid was not specific to these transformants. These results suggest that cell transformation by Ad12 is characterized by a high sensitivity to exogenously administered phospholipids and diacylglycerol that contain oleoyl or linoleoyl acyl groups and that the sensitivity is attributable to the expression of E1A gene of Ad12.     

Intratumor injection of small interfering RNA-targeting human papillomavirus 18 E6 and E7 successfully inhibits the growth of cervical cancer

Human papillomavirus (HPV) 18 is related not only to squamous cell carcinoma of the cervix, but also to adenocarcinoma and small cell carcinoma of the cervix, in which prognosis is known to be poor. Small interfering RNA (siRNA) that targets HPV18 E6 and E7 was tested in HPV18-positive cell lines to investigate its effect and investigate its mechanism of action. Nude mice were also tested in a combination of siRNA and atelocollagen to determine whether it might be useful as a new molecule-targeting therapy for cervical cancer. siRNAs targeting HPV18 E6 and E7 were transfected into cervical cancer cells in vitro and they were investigated for cell growth inhibition, expression of E6 and E7 mRNA, expression of retinoblastoma protein, and senescence-associated beta-galactosidase staining. Sequence-specific siRNA inhibited cell growth. Decreased expression of E6 and E7 mRNA followed with E7 protein was observed in the transfected cells, but the expression of retinoblastoma protein and the beta-galactosidase staining increased, suggesting cell growth inhibitory effect through senescence. Treatment of xenografts established from SKG-II cells with siRNA specific for E6 and E7 obviously suppressed tumor growth in vivo. These results indicate that atelocollagen-mediated delivery of siRNA HPV18 E6 and E7 can be used as a novel therapeutic approach for cervical cancer.     

Development of a replication‐deficient adenoviral vector‐based vaccine candidate for the interception of HPV16‐ and HPV18‐induced infections and disease

High‐risk Human papilloma virus (HPV) types are the causative agents of cervical cancer and several other anogenital malignancies. The viral proteins expressed in the (pre)malignant cells are considered ideal targets for immunological intervention. Many approaches have been evaluated for this purpose, mostly aiming at the induction of HPV16 E7‐ and/or E6‐specific cellular immunogenicity. As clinical success has so far been limited, novel approaches are required. We describe the development and pre‐clinical testing of a vaccine candidate consisting of replication‐deficient adenovirus type 26 and 35 based vectors for the interception of HPV16‐ and HPV18‐related disease. We developed HPV16‐ and HPV18‐specific antigens consisting of fusion proteins of E2, E6 and E7. The vaccine will be suitable for every disease stage, from incident and persistent infections where E2 is predominantly expressed up to late stages where E6 and E7 expression are upregulated. Importantly E6 and E7 are present as reordered fragments to abrogate the transforming activity of these two proteins. Loss of transforming activity was demonstrated in different in vitro models. Robust T‐cell immunogenicity was induced upon immunization of mice with the vaccine candidate. Finally, the developed vaccine vectors showed considerable therapeutic efficacy in the TC‐1 mouse model. The absence of transforming activity of the antigens and the favorable immunogenicity profile of the adenovirus based vectors along with the fact that these vectors can be readily produced on a large scale makes this approach attractive for clinical evaluation.