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.     

Tetrasomy is induced by human papillomavirus type 18 E7 gene expression in keratinocyte raft cultures.

We have demonstrated previously that oncogenic human papillomaviruses (HPVs) induce basal cell tetrasomy in low-grade squamous intraepithelial lesions of the cervix. To identify HPV genes and growth conditions involved in this process, we analyzed: (a) organotypic raft cultures of primary human keratinocytes transfected with whole HPV-18 genomes; and (b) organotypic raft cultures acutely infected with recombinant retroviruses expressing the HPV-18 E6, E7, or E6/E7 genes from the differentiation-dependent HPV-18 enhancer-promoter. Cultures were examined for HPV DNA by in situ hybridization and for karyotype by interphase cytogenetics. Tetrasomy occurred in the suprabasal strata of raft cultures expressing E7 and E6/E7 but not in those expressing E6 alone or in a control culture. These data indicate that suprabasal tetrasomy occurs in association with expression of the E7 gene alone. Basal cell tetrasomy was additionally observed in the raft culture transfected with whole HPV-18 genomes, consistent with observations in low-grade squamous intraepithelial lesions. The distribution of tetrasomic cells in these raft cultures may reflect the involvement of additional viral genes or possibly differences in the pattern of viral oncogene and host gene expression.     

E6/E7 genes of human papilloma virus type 18 induced immortalization of human fetal esophageal epithelium

To study the role played by human papilloma virus (HPV) in carcinogenesis, immortalized esophageal epithelial cells were induced by E6 and E7 genes of HPV type 18 and the biological behavior was studied. Human fetal esophageal epithelial cells were transfected with recombined HPV18E6E7AAV and were cultured and passaged in medium M199. In both the 10th passage (SHEE10) and the 31st passage (SHEE31), their proliferative rates by flow cytometry and their abilities to grow and form colonies in soft agar, or to form tumors in SCID mice were examined. The HPV18 genes of E6E7 and its expression were determined using PCR methods. Cellular telomerase activity was detected by TRAP and chromosomes were analyzed by standard method. Immortalized cell lines of esophageal epithelium induced by the HPV18E6E7 were successfully established and cultured for >100 passages over 4 years. The result of PCR showed that the E6E7 gene of HPV18 was detectable in both cell clones. Both of them were unable to grow in soft-agarose medium and failed to produce tumors in SCID mice. Flow cytometry demonstrated an average of 43% proliferation index in SHEE31, but 28% in SHEE10. Telomerase activity was clearly identified in SHEE31 but not in SHEE10. Cytogenetic analysis demonstrated progression of chromosomal abnormalities with increasing trisome. Our data indicated that genes E6/E7 of the HPV18 were capable of inducing immortalization in fetal esophageal epithelial cells. The immortal phenotype requires both activation of telomerase and genetic alterations that abrogate normal differentiation and promote cellular proliferation. This cell line can assist us to characterize the role played by HPV in carcinogenesis.     

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.     

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.     

In vitro transformation of cell lines from human salivary gland tumors.

Explanted cells from salivary gland tumors are particularly difficult to propagate in vitro and not efficiently immortalized by agents such as simian virus 40. Human papillomavirus 16 (HPV16) has been widely used to transform cells of epithelial origin, but its use for salivary gland cell transformation has not been described. In this study, we employed viral constructs containing the E6/E7 genes of HPV16 to infect and stably transform 9 salivary gland tumor cell cultures. Four of the tumor cell cultures were derived from benign tumors and 5 from malignant tumors. All of the original cell cultures were diploid; however, 6 contained subpopulations of cells with structural abnormalities. All 9 cell cultures were successfully transformed, and 8 were immortalized. The resulting cell lines have decreased serum requirements, exhibit a high proliferation rate, are E6/E7-positive and form colonies in soft agar. Immuno-histochemical and molecular studies confirmed that the transformed cells were indeed epithelial/myoepithelial in origin. All of the transformed cell lines had a diploid or near-diploid karyotype, and 2 contained the original translocated chromosomes in all cells. Our report represents a new application of the E6/E7 system in immortalizing salivary gland cell cultures, resulting in retention of the cellular features found in the native tissue without a general destabilization of the karyotype. These types of tissue culture resources should prove useful for positional cloning and functional studies of genes involved in salivary gland oncogenesis.     

宫颈癌细胞系中HPV16 E6、E7及E6/E7基因对STK31基因甲基化状态及表达的影响

背景与目的：丝氨酸/苏氨酸蛋白激酶31(serine/threonine kinases 31,STK31)基因在人类多种癌症中扮演重要角色,且STK31基因的表达受其启动子及第一外显子区甲基化状态的影响;病毒感染与肿瘤组织中某些抑癌基因启动子区高甲基化有关。本研究旨在探讨宫颈癌细胞系中HPV16 E6、E7及E6/E7癌基因对STK31基因甲基化状态及表达的影响,以及不同种类甲基转移酶(DNA methyltransferases,DNMTs)基因在STK31基因甲基化中的潜在作用。方法：构建外源性HPV16 E6、E7以及E6/E7基因共表达慢病毒,分别感染人乳头瘤病毒(human papillomavirus,HPV)阴性宫颈癌细胞系HT-3及C33A,获得稳定转染的细胞系;采用亚硫酸氢盐基因组测序法(bisulfite genomic sequencing,BGS)和甲基化特异性PCR (methylation-specific PCR,MSP)检测3种HPV阳性宫颈癌细胞系HeLa、SiHa和CasKi以及HPV阴性宫颈癌细胞系HT-3和C33A转染前后STK31基因的甲基化状态;RT-PCR及蛋白［质］印迹法(Western blot)检测上述宫颈癌细胞系中STK31基因的表达以及DNMT1、DNMT2、DNMT3a、DNMT3b和DNMT3L基因在HPV16转染前后宫颈癌细胞系及HPV阳性、HPV阴性宫颈癌组织中的表达情况。结果：外源性HPV16 E6、E7以及E6/E7基因可在HPV阴性宫颈癌细胞系中稳定表达。3种HPV阳性细胞系HeLa、SiHa和CasKi中STK31基因呈低甲基化状态,STK31 mRNA及蛋白质表达阳性;2种HPV阴性细胞系HT-3、C33A中STK31基因则表现为高甲基化状态,STK31 mRNA及蛋白质表达缺失;与未感染慢病毒HT-3和C33A细胞系比较,外源性HPV16 E7以及E6/E7表达的HT-3和C33A细胞系STK31基因甲基化程度降低,其mRNA及蛋白质重新表达。DNMT1、DNMT3a和DNMT3b基因在HT-3E6/E7和C33AE6/E7细胞系中mRNA水平分别高于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(P<0.001)。DNMT1、DNMT3a和DNMT3b基因的mRNA水平在HPV16阳性宫颈癌组织中的表达高于其在HPV阴性宫颈癌组织中的表达,差异有统计学意义(t=5.997,P<0.001;t=6.743,P<0.001;t=7.926,P<0.001)。DNMT2在HT-3E6/E7和C33AE6/E7细胞系中mRNA表达水平分别低于HT-3空载细胞系和C33A空载细胞系,差异有统计学意义(t=7.451,P<0.001;t=2.451,P<0.05);DNMT2基因转录水平在HPV16阳性宫颈癌组织中低于HPV阴性宫颈癌组织(t=9.134,P<0.001)。DNMT3LmRNA表达水平在宫颈癌细胞系转染前后及HPV阴阳性宫颈癌组织中的差异无统计学意义(P>0.05)。结论：HPV感染可导致STK31基因启动子及第1外显子区甲基化状态降低,低甲基化状态促进该基因表达。STK31基因的表达受其启动子及第1外显子区甲基化状态的调控。HPV16 E7、E6/E7基因可能通过影响DNMT2的表达参与调控癌基因STK31基因启动子及第1外显子区甲基化状态。     

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.     

Antibodies against high‐risk human papillomavirus proteins as markers for invasive cervical cancer

Different human papillomavirus (HPV) genes are expressed during the various phases of the HPV life cycle and may elicit immune responses in the process towards malignancy. To evaluate their association with cervical cancer, antibodies against proteins from HPV16 (L1, E1, E2, E4, E6 and E7) and HPV18/31/33/35/45/52/58 (L1, E6 and E7) were measured in serum of 307 invasive cervical cancer cases and 327 controls from Algeria and India. Antibody response was evaluated using a glutathione S‐transferase‐based multiplex serology assay and HPV DNA detected from exfoliated cervical cells using a GP5+/6+‐mediated PCR assay. Among HPV16 DNA‐positive cases, seroprevalence of HPV16 antibodies ranged from 16% for HPV16 E1 to 50% for HPV16 E6 and all were significantly higher than controls. Seroprevalence of E6, E7 and L1 antibodies for HPV18 and for at least one of HPV31/33/35/45/52/58 were also higher in cases positive for DNA of the corresponding type (50% and 30% for E6 of HPV18 and HPV31/33/35/45/52/58 combined, respectively). E6 and E7 antibodies were rarely found in controls, but cross‐reactivity was evident among cancer cases positive for DNA of closely phylogenetically‐related HPV types. E6 or E7 antibodies against any of the eight HPV types were detected in 66.1% of all cervical cancer cases, as compared to 10.1% of controls. E6, and to a lesser extent E7, antibodies appear to be specific markers of HPV‐related malignancy. However, even among cases positive for the same type of HPV DNA, approximately one‐third of cervical cancer cases show no detectable immune response to either E6 or E7.     

ras,p53 and hpv status in benign and malignant prostate tumors

To study the role of ras, p53 genes and HPV virus (16 and 18) in the development of prostate cancer, we analyzed tissue sections from 27 patients affected with carcinomas (stages A to D) and from 24 patients with adenomas. Mutations of H, K and N-ras and p53 (exons 2-9) were studied by SSCP and DNA sequencing. Accumulation of p53 protein was studied by immu-nohistochemistry on tissue sections. Tumors were also analyzed for the presence of HPV 16 and -18 sequences by PCR and DNA hybridization with sequence-specific oligonucleotides. No mutation was found in the three ras genes studied, either in carcinomas or adenomas. By SSCP analysis we identified p53 mutations in only 2 of 19 carcinomas studied, both in exon 7. Immunohistochemical results strongly correlate with the SSCP results: p53 protein was positive in tumors with p53 mutation but not in others; 32% of studied adenomas had detectable HPV16 DNA, while 53% of carcinomas were HPV16+. Among these I presented a p53 mutation. No HPV 18 E6 sequence could be detected. Our data show that in prostate tumors from France, mutations of p53 and ras are rare events but that these tumors display detectable HPV 16 DNA at a high frequency. The low incidence of p53 mutation, associated to a significant proportion of tumors showing HPV16 DNA, could suggest that in prostate cancer HPV 16 infection could participate in p53 inactivation by E6. © 1995 Wiley-Liss, Inc.     

Cytogenetic analysis of eight human papillomavirus immortalized human keratinocyte cell lines

Cytogenetic analysis was performed on human papillomavirus (HPV)-immortalized cell lines. Lines were established by co-transfection of primary human keratinocyte cells with HPV type 16 or 18 DNA and pSV2neo. The resulting clonal lines contained integrated HPV DNA and exhibited extended life spans in culture but were non-tumorigenic in nude mice. Two HPV16-immortalized lines (FEPE1L8 and FEPE1L9) and 4 HPV18-immortalized lines (FEA, FEH18L, FEP18-5 and FEP18-11) were established. Two additional lines were derived by subsequent treatment of the FEA line with TPA and by further transfection with HSVII DNA. Cytogenetic analysis revealed that all lines were abnormal, containing a variety of numerical and structural aberrations. Six of the 8 lines were hyper-triploid and 2 were near-diploid. Examination of lines FEA, FEH18L and FEP18-11 at multiple passages in culture revealed that the lines were clonal and chromosomally stable over extended passage in culture. Structural rearrangements were most common in chromosomes 1 and 3 but also occurred in chromosomes 5, 7, 8, 12, 16 and 22. Marker chromosomes were present in all cell lines. A small metacentric marker, possibly an isochromosome for the short arm of chromosome 5, was consistently present in the FEA line and its derivatives (FEAB10 and FEAT) as well as the FEH18L line. A loss or reduction in copy number of chromosome 13 was seen in 5 of the 8 cell lines.     

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.     

食管癌高发区HPV检测及与p53的关系

目的 探讨食管癌高发区中人乳头瘤病毒（HPV）、爱泼斯坦-巴尔病毒（EBV）与食管癌发病的关系。并初步研究HPV感染与p53过表达之间的关系。方法 设计了多对引物进行PCR、原位杂交及原位PCR、免疫组化,并对30例高发区食管癌进行检测,结果 用PCR检出HPV-L1阳性率10.0%,HPV-16-E6阳性率60.0%,HPV-16-E7阳性率63.3%,HPV-18-E6及EBV的检出率分别为6.7%和0,p53蛋白的检出率为73.3%,用原位杂交和原位PCR检出HPV-16-E6阳性率均为53.3%.HPV-L1的低检出率及HPV-16E6、HPV-16-E7基因的较高检出率可能提示,在肿瘤细胞中HPV常以部分丢失的形式整合,而E6、E7常在整合中保留。结论 HPV-16型可能与高发区食管癌发生密切相关,而HPV-18、EBV则关系不大,p53突变在食管癌的发病中起重要作用。但其与HPV感染之间的关系不大。     

Antibodies against oncoproteins E6 and E7 of human papillomavirus types 16 and 18 in cervical-carcinoma patients from Russia

Certain human papillomaviruses (HPV), mainly types 16 and 18, have been widely recognized as an essential etiologic factor for the development of carcinoma of the uterine cervix. The early HPV proteins E6 and E7 are consistently expressed in the tumor cells, and cervical-carcinoma patients can develop antibodies against these oncoproteins. For cervical-carcinoma patients from Eastern Europe and Russia, detailed information on HPV DNA prevalence and HPV-specific immune responses is limited. The presence of HPV DNA in 128 Russian cervical-carcinoma tissues was determined: HPV16 DNA was found in 78% of the cases, HPV18 DNA in 14%, and no HPV-DNA in 10%. Using 4 recently developed sensitive and highly specific second-generation enzyme-linked immunosorbent assays, we also analyzed the prevalence of antibodies against HPV16 and -18 E6 and E7 proteins in sera from 95 cervical-carcinoma patients, from 61 female patients with non-HPV-associated tumors and from 83 female healthy controls. The strong association of E6 and/or E7 antibodies with cervical carcinoma was confirmed, with 36% seropositives in this group against only 2% in the control groups. The detected antibodies are highly HPV-type-specific since all 26 HPV16-E6- or -E7-antibody-positive patients had HPV16 DNA in their tumor and 6 out of the 8 HPV18-antibody-positive patients had HPV18 DNA. Antibody responses to HPV16 E6 and E7 appear to be dependent on clinical stage of the disease, with 21% seropositives found in FIGO stage I, 42% in stage II and 53% in stage III. Antibody response to HPV16 E6 is more frequent than to E7, especially in early stages.