A mouse fibroblast cell model to study human papillomavirus-associated tumorigenesis

Cervical cancer represents the second most common cancer in women worldwide. About 90% of cervical cancer contain high-risk human papillomavirus (HPV) DNA, most often HPV type 16. Animal models and mostly laboratory mice are excellent for carrying out diverse immunological studies. We transfected a fibroblast cell line, 3T3-A31, with human papillomavirus type 16 genome to develop an in vivo/in vitro malignant transformant model. Isolated clones inoculated to immunocompetent mice displayed a tumorigenic phenotype. Small clusters of metastatic cells were found in the liver of animal 45 days after receiving the inoculum. Integrated viral DNA and expression of E7 viral oncogene from the high-risk HPV-16 were demonstrated both in transfectans and tumor-derived cells. The observed high-grade neovascularization was correlated with the upregulation of vascular endothelial growth factor (VEGF) mRNA on HPV-16 transformed fibroblast cells. These observations emphasize the association between papillomavirus expression and progression to malignancy.     

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.     

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。     

Human papillomavirus type 16 (HPV-16) genomes integrated in head and neck cancers and in HPV-16-immortalized human keratinocyte clones express chimeric virus-cell mRNAs similar to those found in cervical cancers

Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.     

The human papillomavirus type 16 E7 oncogene is required for the productive stage of the viral life cycle

The production of the human papillomavirus type 16 (HPV-16) is intimately tied to the differentiation of the host epithelium that it infects. Infection occurs in the basal layer of the epithelium at a site of wounding, where the virus utilizes the host DNA replication machinery to establish itself as a low-copy-number episome. The productive stage of the HPV-16 life cycle occurs in the postmitotic suprabasal layers of the epithelium, where the virus amplifies its DNA to high copy number, synthesizes the capsid proteins (L1 and L2), encapsidates the HPV-16 genome, and releases virion particles as the upper layer of the epithelium is shed. Papillomaviruses are hypothesized to possess a mechanism to overcome the block in DNA synthesis that occurs in the differentiated epithelial cells, and the HPV-16 E7 oncoprotein has been suggested to play a role in this process. To determine whether E7 plays a role in the HPV-16 life cycle, an E7-deficient HPV-16 genome was created by inserting a translational termination linker (TTL) in the E7 gene of the full HPV-16 genome. This DNA was transfected into an immortalized human foreskin keratinocyte cell line shown previously to support the HPV-16 life cycle, and stable cell lines were obtained that harbored the E7-deficient HPV-16 genome episomally, the state of the genome found in normal infections. By culturing these cells under conditions which promote the differentiation of epithelial cells, we found E7 to be necessary for the productive stage of the HPV-16 life cycle. HPV-16 lacking E7 failed to amplify its DNA and expressed reduced amounts of the capsid protein L1, which is required for virus production. E7 appears to create a favorable environment for HPV-16 DNA synthesis by perturbing the keratinocyte differentiation program and inducing the host DNA replication machinery. These data demonstrate that E7 plays an essential role in the papillomavirus life cycle.     

Induction of cytotoxic T lymphocytes specific for a syngeneic tumor expressing the E6 oncoprotein of human papillomavirus type 16.

Human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical carcinomas, but it is unknown whether HPV-specific immunity can function in controlling the growth of HPV-associated carcinomas. We previously demonstrated that CD8+ T lymphocytes can inhibit the in vivo outgrowth of murine tumor cells transfected with the HPV-16 E7 gene and have now established a murine model to study the CTL responses to the E6 oncoprotein of HPV-16. Immunization of C3H/HeN mice with syngeneic fibroblasts expressing a transfected HPV-16 E6 gene induced regression of transplanted tumors expressing this gene. Populations of CTL isolated from the spleens of mice whose E6+ tumors had regressed were shown to specifically lyse E6+ target cells. The cytolytic activity was mediated by CD8+ CTL in a MHC restricted pattern. These data and our previous findings with transfected tumor cells expressing the E7 gene, support the conclusion that tumor cells associated with HPV-16 can be inhibited by CTL specific for molecules encoded by the HPV-16 E6 and E7 genes.     

Primary structure of rat renal dipeptidase and expression of its mRNA in rat tissues and COS-1 cells.

Three cDNA clones of rat renal dipeptidase (rrDP) were isolated from rat renal and pulmonary cDNA libraries using a DNA fragment of human renal DP cDNA clone, MDP4, as a probe. The complete amino acid sequence deduced from the cDNA contains 410 amino acid residues, beginning with a signal peptide of 16 amino acid residues. RNA blot hybridization analysis showed that 1.6 and 2.2 kb mRNAs were expressed in lung and kidney, however, only 1.6 kb mRNA was detected in small intestine. COS-1 cells transfected with the cDNA expressed enzymatically active rrDP.     

The Carboxyl-Terminal Region of the Human Papillomavirus Type 16 E1 Protein Determines E2 Protein Specificity during DNA Replication

The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.     

Expression of the human papillomavirus E7 oncogene during cell transformation is sufficient to induce susceptibility to lysis by activated macrophages.

Human papillomaviruses (HPV), and in particular HPV type 16, are etiologic agents in the development of cervical cancer, which is the second most common form of cancer in women worldwide. Mammalian cells are susceptible to transformation in vitro by the E6 and E7 oncogenes derived from the HPV-16 genome. NIH-3T3 cells transfected with the HPV-16 E7 oncogene were found to exhibit cytolytic susceptibility to murine-activated macrophages. In comparison, E6 oncogene-expressing cells were not susceptible to lysis by activated macrophages. The E7 oncoprotein is multifunctional, being capable of complexing with the retinoblastoma tumor suppressor gene (anti-oncogene) product, stimulating DNA synthesis, and causing cell transformation in vitro. Macrophage killing assays performed on cell lines expressing E7 mutants revealed that the ability to complex the retinoblastoma tumor suppressor gene product and stimulate DNA synthesis did not induce susceptibility to activated macrophages, whereas the ability of E7 to cause transformation was required to induce susceptibility to activated macrophages. These data suggest that cell transformation is a more important prerequisite for inducing susceptibility to activated macrophages than is the loss of tumor suppressor gene function. This study also provides an initial link between HPV-16 oncogene expression and the ability of activated macrophages to selectively recognize and destroy HPV-16-associated neoplastic cells.     

Analysis of integrated human papillomavirus type 16 DNA in cervical cancers: amplification of viral sequences together with cellular flanking sequences.

We have isolated four clones of integrated human papillomavirus type 16 (HPV-16) DNA from four different primary cervical cancer specimens. All clones were found to be monomeric or dimeric forms of HPV-16 DNA with cellular flanking sequences at both ends. Analysis of the viral sequences in these clones showed that E6/E7 open reading frames and the long control region were conserved and that no region specific for the integration was detected. Analysis of the cellular flanking sequences revealed no significant homology with any known human DNA sequences, except Alu sequences, and no homology among the clones, indicating no cellular sequence specific for the integration. By probing with single-copy cellular flanking sequences from the clones, it was demonstrated that the integrated HPV-16 DNAs, with different sizes in the same specimens, shared the same cellular flanking sequences at the ends. Furthermore, it was shown that the viral sequences together with cellular flanking sequences were amplified. The possible process of viral integration into cell chromosomes in cervical cancer is discussed.     

Thyrotropin receptor gene expression in oncogene-transfected rat thyroid cells: correlation between transformation, loss of thyrotropin-dependent growth, and loss of thyrotropin receptor gene expression.

Rat FRTL-5 and PC-Cl-3 thyroid cells are continuously cultured, clonal lines which require thyrotropin to grow and function. Both can be efficiently transformed when infected with RNA or DNA viruses carrying oncogenes or when directly transfected with activated oncogenes. Transformation, assayed by the appearance of cell growth in agar and by tumorigenicity in syngeneic rats or nude mice, is associated with the loss of thyrotropin-dependent cell division and thyrotropin-regulated functions such as thyroglobulin synthesis. In 16 clones of FRTL-5 or PC-Cl-3 cells transformed with different oncogenes, we show that loss of thyrotropin-dependent growth and function correlates with the loss of thyrotropin receptor gene expression, measured with a rat thyrotropin receptor cDNA probe.     

Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells.

The close association of human papillomavirus type 16 DNA with a majority of cervical carcinomas implies some role for the virus in this type of cancer. To define the transforming properties of HPV-16 DNA in vitro we have now performed transfection experiments on baby rat kidney cells using HPV-16 DNA in conjunction with an activated ras gene. We have demonstrated that a 6.6-kb DNA fragment, containing the early genes of HPV-16 under the control of Moloney murine leukaemia virus long terminal repeats (MoMuLV-LTRs), cooperates with EJ-ras in transforming these cells. Both DNAs are required and neither alone is effective. The cooperating activity appears to reside in a protein or proteins derived from the E6/E7 region of the HPV-16 genome.     

人MCP cDNA在NIH3T3细胞中表达及功能研究

通过构建表达人膜辅因子蛋白ＭＣＰ编码区全长ｃＤＮＡ的重级载体ｐｃＤＮＡ３ＭＣＰ,以磷酸钙沉淀法转染ＮＩＨ３Ｔ３细胞,用Ｇ４１８筛选阳性克隆,并鉴定ＭＣＰ ｃＤＮＡ在细胞中的表达。将血清梯度稀释并与细胞孵育,然后检测细胞活力：灭活的人血清不能引起对照细胞与ＭＣＰ转染细胞的胞毒作用,而新鲜人血清可使对照细胞发生补体依赖的细胞毒反应,ＭＣＰ转染细胞由于ＭＣＰ蛋白的合成,细胞受到保护不发生该反应。另外,Ｍ     

人肝细胞生长因子cDNA克隆在CHO细胞中的表达

肝细胞生长因子是一种由α、β链组成的杂合二聚体糖蛋白,能促进肝细胞、多种上皮细胞、内皮细胞和神经胶质细胞的有丝分裂,并对多种肿瘤细胞具有细胞毒性作用或者抑制其生长［１～４］,其作用无种属特异性,如人肝细胞生长因子能促进大鼠肝细胞增殖［５］。由于天然Ｈ...     

cDNA表达文库转染NIH3T3-从高转移人肺腺癌细胞系Anip973中克隆新肿瘤转移相关基因

培养人高转移肺腺癌细胞系Anip973,构建其cDNA表达文库并转染小鼠成纤维细胞NIH3T3,将经药物筛选后的转染细胞克隆消化为单细胞,接种到软琼脂中培养2周,根据细胞明显的形态学变化挑选出有意义的细胞克隆,扩增再培养,提取DNA,PCR扩增插入片段并进行测序分析。结果表明:软琼脂中挑选出克隆100多个,PCR测序后,得到3个已知基因包括人类核糖体蛋白L23、人类假定蛋白FLJ22104和人类丝氨酸蛋白酶抑制因子6亚型以及一些氨基末端截短的核酸序列。进一步的研究表明转染人类核糖体蛋白L23的细胞与转染空载体细胞相比具有较高的侵袭能力(P<0.02)。利用cDNA文库在NIH3T3细胞中的表达,随后筛查鉴定在软琼脂中发生形态学变化的细胞,是一种寻找恶性转化和癌转移相关基因的有效方法。人类核糖体蛋白L23基因在细胞的运动和转移中发挥重要作用。     

Functional expression of the bradykinin-B2 receptor cDNA in Chinese hamster lung CCL39 fibroblasts.

The bradykinin (BK) B2 receptor cDNA was synthesized by rt-PCR and transfected into the Chinese hamster lung fibroblasts, CCL39. The CCL39 do not contain the mRNA for this receptor and do not bind BK. Clones of transfected cells were screened for BK receptor mRNA, binding of BK, and for [Ca2+]i response to BK. The clones showed various levels of receptor mRNA. Scatchard analysis of three clones, B6, B5 and B1, each gave a Kd of approximately 1.0nM while the Bmax for each clone differed at 320, 38.7, and 5.39 fmoles per 10(6) cells respectively. The [Ca2+]i response of the three clones to BK decreased with the receptor number/cell. Thus, levels of mRNA, BK binding and [Ca2+]i response proved proportionally related in the transfected clones. The actions of BK and alpha-thrombin, which has an endogenous receptor in these cells, were assessed in clone B6. BK proved active but also distinct from thrombin. BK at 10nM and thrombin at 2units/ml both effectively increased cytosolic [Ca2+]i. BK at 10nM stimulated PGE2 production three fold over basal, while thrombin only marginally elevated PGE2 levels. Alone, BK stimulated a small increase in 3H-thymidine incorporation into DNA. However, in combination with insulin, BK stimulated DNA synthesis to 76% of thrombin, a potent mitogen in these cells. These results illustrate that the BK-B2 receptor cDNA can be stably transfected into a mammalian cell and can activate transmembrane signalling pathways.