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In order to contribute to the understanding of the activation of the oncoprotein p53 we determined the metabolic stability of p53 in a variety of non-transformed, immortalized and SV40- and non-SV40-transformed cell lines. In addition, we analyzed the metabolic stability of the SV40 large T antigen in SV40 transformed cell lines. Pulse-chase experiments revealed a low stability (t1/2 = 20 min) of p53 in non-transformed cells and in cells immortalized by the p53 construct pLTRp53cG9. In cells transformed by an activated ras oncogene and pLTRp53cG9 and in methylcholanthrene induced mouse sarcoma cells p53 proved to be progressively more stable with half-lives ranging from 5.5 h to 7 h. Sequential immunoprecipitation with p53- or T antigen specific monoclonal antibodies allowed us to separate T-p53 complexes, uncomplexed p53 and free T antigen in cell extracts from cells transformed by SV40 and pLTRp53cG9. In these transformed cells uncomplexed p53 showed an increased stability (t1/2 = 2.8 h) when compared to p53 from non-transformed cells. Complex formation with T antigen resulted in an additional stabilization of p53 (t1/2 = 13.3 h). Furthermore, T-p53 complex formation also seems to increase the stability of T antigen nearly sixfold. In transformed cells two immunological variants of p53, a PAb246 precipitable and a non-precipitable form showed distinctly different stabilities, indicating a correlation between the ability of p53 subclasses to bind hsc70 protein and their metabolic stability. Moreover, binding to hsc70 correlated with the stabilization of T antigen in CTM cells also where the mutant T antigen is localized exclusively in the cytoplasm. In abortively infected cells p53, even in complex with T antigen, exhibited a relatively low stability (t1/2 = 87 min) indicating that complex formation per se is not sufficient for fully stabilizing p53.  相似文献   

3.
An albumin-simian virus 40 (SV40) large T-antigen (T-Ag) transgenic model and a chemically induced model of multistage hepatocarcinogenesis were created in our laboratory to study the molecular mechanisms involved in the genesis and progression of neoplasia in the rat liver. In the study presented here, these two models of rat hepatocarcinogenesis were used to perform a comparative mutational analysis of three tumor suppressor genes involved in hepatic neoplastic growth. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing, exons 5-8 of the p53 tumor suppressor gene and a region between nt 4325 and 4479 of the rat mannose 6-phosphate/insulin-like growth factor 2 receptor (M6p/Igf2r) coding sequence were screened. The latter is homologous to the human M6P/IGF2r coding sequence which is mutated in human hepatocellular carcinoma. A complete single strand conformation polymorphism analysis of the entire coding region of the rat adenomatous polyposis coli (Apc) gene was also performed for the first time in rat tumorigenic samples. Twenty-six chemically induced rat hepatocellular carcinomas, 21 neoplasms from the livers of SV40 T-Ag animals, and five immortalized hepatic cell lines from the transgenic rats were evaluated. None of the hepatic tumors exhibited mutations in the regions analyzed. The albumin-SV40 T-Ag transgenic cell line L-60, derived from normal hepatic tissue, had two mutations in contiguous codons of exon 5 of the p53 gene: a GGT --> GTT missense transversion in codon 183 and a silent mutation in codon 184. The transversion, which may affect the DNA binding domain of the p53 protein, probably originated during cell culture and may have been positively selected because it gave a growth advantage to the mutated cells. The studied region of the M6p/Igf2r gene was not found to be mutated in these two models of rat hepatocarcinogenesis. Although M6p/Igf2r, Apc, and p53 have been shown to be mutated in a variety of human hepatic proliferative diseases, our results indicate that aberrations in these genes may not be necessary for liver carcinogenesis in the rat.  相似文献   

4.
J Garbe  M Wong  D Wigington  P Yaswen  M R Stampfer 《Oncogene》1999,18(13):2169-2180
Our recent studies on the process of immortalization of cultured human mammary epithelial cells (HMEC) have uncovered a previously undescribed, apparently epigenetic step, termed conversion. When first isolated, clonally derived HMEC lines of indefinite lifespan showed little or no telomerase activity or ability to maintain growth in the presence of TGFbeta. Cell populations whose mean terminal restriction fragment length had declined to <3 kb also exhibited slow heterogeneous growth, and contained many non-proliferative cells. With continued passage, these conditionally immortal cell populations very gradually converted to a fully immortal phenotype of good growth+/-TGFbeta, expression of high levels of telomerase activity, and stabilization of telomere length. We now show that exposure of the early passage conditionally immortal 184A1 HMEC line to the viral oncogenes human papillomavirus type 16 (HPV16)-E6, -E7, or SV40T, results in either immediate (E6) or rapid (E7; SV40T) conversion of these telomerase negative, TGFbeta sensitive conditionally immortal cells to the fully immortal phenotype. Unlike conditional immortal 184A1, the HPV16-E7 and SV40T exposed cells were able to maintain growth in TGFbeta prior to expression of high levels of telomerase activity. A mutated HPV16-E6 oncogene, unable to inactivate p53, was still capable of rapidly converting conditional immortal 184A1. Our studies provide further evidence that the transforming potential of these viral oncogenes may involve activities beyond their inactivation of p53 and pRB functions. These additional activities may greatly accelerate a step in HMEC immortal transformation, conversion, that would be rate-limiting in the absence of viral oncogene exposure.  相似文献   

5.
Evidence against a role for SV40 in human mesothelioma   总被引:4,自引:0,他引:4  
SV40 has been implicated in the etiology of 40% to 60% of human mesotheliomas. These studies could have important medical implications concerning possible sources of human infection and potential therapies if human tumors are induced by this agent. We did PCR-based analysis to detect SV40 large T antigen DNA in human mesotheliomas. None of 69 tumors in which a single copy gene was readily amplified contained detectable SV40 large T antigen sequences. Under these conditions, it was possible to detect one copy of integrated SV40 DNA per cell in a mixture containing a 5,000-fold excess of normal cells using formalin-fixed preparations. Kidney, a known reservoir of SV40 in monkeys, from some of these individuals were also negative for SV40 large T antigen sequences. A subset of mesotheliomas was analyzed for SV40 large T antigen expression by immunostaining with a highly specific SV40 antibody. These tumors as well as several human mesothelioma cell lines previously reported to contain SV40 large T antigen were negative for detection of the virally encoded oncoprotein. Moreover, mesothelioma cell lines with wild-type p53 showed normal p53 function in response to genotoxic stress, findings inconsistent with p53 inactivation by the putative presence of SV40 large T antigen. Taken together, these findings strongly argue against a role of SV40 by any known transformation mechanism in the etiology of the majority of human malignant mesotheliomas.  相似文献   

6.
Progress in prostate cancer research has been hindered by thelack of well characterized, immortalized, human prostatic epithelialcell lines that express markers of normal prostatic epithelialcells and mimic normal growth and differentiation responsesto androgens. The objectives of this study were to: (i) establishimmortalized cell lines from non-neoplastic, adult human prostaticepithelium using adenovirus-12/simian virus-40 (Ad12-SV40) hybridvirus; (ii) establish their prostatic epithelial origin; (iii)demonstrate androgen responsiveness; and (iv) examine responseto growth factors. Primary epithelial cell cultures derivedfrom a non-neoplastic, adult human prostate were infected withthe Ad12-SV40 virus. Several immportalized clones were isolated.Single cell cloning of one clone, free of cytopathic effects,gave rise to the PWR-1E cell line. An immortalized cell linePWR-1E, which expresses many characteristics of normal prostaticepithelial cells was established. Immunostaining showed thatcells express cytokeratins 8 and 18 normally expressed by differentiated,secretory prostatic epithelial cells. The most remarkable characteristicsof PWR-1E cells are growth stimulation, increased expressionof androgen receptor and induction of prostate specific antigen(PSA) expression in response to androgens, which indisputablyestablish their prostatic epithelial origin. They are positivefor SV40 large-T antigen and show strong nuclear staining forp53. Cells from passages 23 and 40 were not tumorigenic in nudemice even when co-injected with Matrigel. They grow in a serum-freedefined medium and respond to EGF, bFGF and TGF-ß.Passage 42-cells showed a human male (XY), hyperdiploid karyotype.The PWR-1E cell line is the only known AD12-SV40-immortalizedhuman prostatic epithelial cell line. PWR-1E cells can be usedto study (i) the etiology and the multistep process of carcinogenesisand tumor progression in the human prostate; (ii) normal prostatephysiology and differentiation; and (iii) potential prostatecancer chemopreventive agents.  相似文献   

7.
The synthesis of essential fatty acids (EFAs) that have been shown to inhibit breast cancer cell growth in vitro and tumor growth in animals requires desaturation at C-6 of linoleic or alpha-linolenic acid. This observation, combined with reports that many tumors and tumor cell lines are deficient in Delta(6) desaturation and/or contain low levels of 6-desaturated EFAs, has led to the suggestion that loss of Delta(6) desaturating ability is relevant to the process of malignant transformation. This study was undertaken in search of direct evidence that malignant transformation of mammary epithelial cells alters EFA metabolism. We used two cell lines derived from the spontaneously immortalized human mammary epithelial cell line MCF-10A and expressing either the c-Ha-ras protooncogene (MCF-10AneoN) or an activated c-Ha-ras oncogene (MCF-10AneoT), and a cell line immortalized by transfection of human mammary epithelial cells with SV40 T antigen. We compared these cell lines in terms of ability to convert EFAs (30 mu M) to other EFAs of the same family. MCF-10AneoT cells lose the ability to perform Delta(6) and Delta(4) desaturations, whereas MCF-10AneoN cells and the SV40 T antigen-transformed cell line do not. No significant changes in growth response to culture with 6-desaturated EFAs were noted for MCF-10AneoT cells compared with MCF-10AneoN and parental MCF-10A cells, suggesting that FA metabolism alone cannot account for the effects of EFAs on the growth of neoplastic and non-neoplastic mammary epithelial cells.  相似文献   

8.
S G Kuhar  J M Lehman 《Oncogene》1991,6(9):1499-1506
Infection of normal human diploid fibroblasts (HF) with the DNA tumor virus simian virus 40 (SV) leads to an extension of lifespan and concomitant increase in the levels of the viral large tumor antigen (T antigen) and the cellular protein p53. The intracellular localization of T antigen and p53 was mostly nuclear in both SVpre-crisis and SVpost-crisis cells, however certain population doubling (PD) of the SVpre-crisis cells exhibited some cytoplasmic staining. The DNA content of SVpre-crisis cells shifted to tetraploidy and the SVpost-crisis cells were near-tetraploid. Quantitation of T antigen and p53 in single cells by flow cytometry demonstrated that for all antibodies tested the levels of T antigen were higher in the SVpre-crisis HF than in the SVpost-crisis. The quantity of p53 increased with increasing age of SVpre-crisis HF, and the levels of p53 were higher in the SVpost-crisis HF populations. Immunoprecipitation of p53, T antigen and complexes demonstrated that all p53 was bound to T antigen in SVpre-crisis HF and SVpost-crisis HF. The SVpre-crisis HF cells showed that 33% of all T antigen was bound to p53, while 67% was free, and the SVpost-crisis HF exhibited 50% free T antigen and 50% bound to p53. The half-life of p53 was similar in all SVpre-crisis HF; however, the half-life was 2-3 times greater in SVpost-crisis HF than in SVpre-crisis HF. These results suggest that the interaction of DNA (ploidy), T antigen, p53 and complexes may be involved in formation of a stable SV40-transformed human cell line.  相似文献   

9.
Mammary epithelial cells (MECs) were isolated and cultured from mammary glands of healthy women undergoing reduction mammoplasty. Normal MECs were infected with the transforming hybrid virus adeno-5/SV40. Two transformed epithelial cell lines, M1 and M2, were obtained, characterised phenotypically and studied for the production of and the response to cytokines and growth regulators. In both cell lines, expression of the SV40 large T antigen was associated with loss of interleukin 6 (IL-6) production and responsiveness as well as with down-regulation of IL-8 and transforming growth factor (TGF)-alpha production. Both M1 and M2 cell lines were capable of forming colonies in semisolid media, but upon injection into severe combined immunodeficient (SCID) mice only M2 cells were tumorigenic. DNA synthesis in M1 cells was partially inhibited by serum or TNF-alpha and weakly stimulated by hydrocortisone (HC) and IL-8. In contrast, M2 cells were totally unresponsive to a variety of growth regulators. Both lines overexpressed the p53 protein at levels about 20-fold higher than those observed in primary MEC cultures, but no mutations of the p53 gene could be detected. The date confirm the view that the expression in human mammary cells of different oncogenes - including the SV40 T antigen - is frequently associated with alterations of cytokine production and responsiveness.  相似文献   

10.
The biological effects of exposures to high LET radiations haveparticular relevance to radiation protection and risk assessmentSince most cancers are of epithelial origin, it is importantto obtain a better understanding of radiationinduced oncogenictransformation in this cell type. Accordingly we have initiatedstudies to determine whether immortalized human epidermal keratinocytes(RHEK) can be transformed with high LET radiations. Exponentiallygrowing RHEK cells were treated with single doses (1, 10, 25,50 and 100 cGy) of 0.85 MeV fission neutrons from the Janusreactor. Neutron exposure led to the development of morphologicallyaltered cells and foci formation after 6 weeks at confluence.These transformed cultures grew with an increased saturationdensity, exhibited anchorageindependent growth and formed tumorshi athymlc mice. Single-strand conformatlonal polymorphism analysisand DNA sequencing demonstrated the absence of point mutationsin codons 12/13 and 61 in the Ha-ras, Ki-ras, or N-ras genesand exons 4–9 of the pS3 tumor suppressor gene. Thesestudies demonstrate that high LET radiations (fission neutrons)can transform immortalized human epithelial cells to a malignantphenotype that does notappear to involve mutations in eitherthe cellular p53 or ras genes.  相似文献   

11.
Ovarian cancer is developed from a single layer of thin epithelial cells covering the surface of ovary, named human ovarian surface epithelial cells. Like all primary human cells, human ovarian surface epithelial cells have a finite life span and will go into senescence and eventually die when cultured in vitro. Immortalized human ovarian surface epithelial cells will provide an important model system with which to study ovarian cancer initiation and progression. Here, we show that silencing p53 expression with retrovirus-mediated small interfering RNA can delay the senescence and extend cell passage number, but is not sufficient to immortalize normal ovarian surface epithelial cells. Introduction of the catalytic subunit of telomerase is similarly insufficient to achieve immortalization. However, concurrent disruption of p53 expression with small interfering RNA retroviral constructs and ectopic expression of the catalytic subunit of telomerase was sufficient to induce cellular immortalization in 3 of 3 human ovarian surface epithelial cell cultures tested. The immortalization is associated with increased telomerase activity and telomere length, and attenuated response of cell-cycle regulatory proteins to irradiation. The resultant immortal cells continued to express the same specific cytokeratins 8 and 18 as parental cells did, indicating that the epithelial characters are still maintained in the immortal cells. In addition, the immortalized cells are non-tumorigenic and nearly diploid, which is in constrast with one immortalized by SV40 T/t antigens and hTERT. As both p53 pathway dysfunction and activation of telomerase are commonly present in human ovarian cancer, these immortal cells provide an authetic cell model system for the study of the human ovarian cancer initiation, progression, differentiation and chemoprevention.  相似文献   

12.
13.
Maiorana A  Tu X  Cheng G  Baserga R 《Oncogene》2004,23(42):7116-7124
The murine and human homologs of the zebrafish pescadillo protein (Pes1 and PES1, respectively) play important roles in ribosome biogenesis and DNA replication. We investigated the effect of Pes1 on the growth of mouse embryo (3T3-like) fibroblasts and conditionally immortalized human fibroblasts expressing the SV40 T antigen (AR5 cells). Increased expression of Pes1 causes transformation of mouse and human fibroblasts in culture (colony formation in soft agar). Although Pes1 can replace the SV40 T antigen in inducing colony formation in soft agar, it cannot substitute the T antigen in the immortalization of human fibroblasts, indicating that it distinguishes between the two functions. As the biological effects of Pes1 are similar to those of the insulin receptor substrate-1 (IRS-1), we investigated the interactions of Pes1 with IRS-1 itself and with the SV40 T antigen. The Pes1 protein (which localizes to the nuclei and nucleoli of cells) interacts with both IRS-1 and the SV40 T antigen, and markedly decreases the interaction of T antigen with p53. Taken together, these results suggest mechanisms for the ability of Pes1 to transform cells, and its failure to immortalize them.  相似文献   

14.
Knowledge of the function of the cell cycle checkpoints in tumour cells may be important to develop treatment strategies for human cancers. The protein p53 is an important factor that regulates cell cycle progression and apoptosis in response to drugs. In human malignant mesothelioma, p53 is generally not mutated, but may be inactivated by SV40 early region T antigen (SV40 Tag). However, the function of p53 has not been investigated in mesothelioma cells. Here, we investigated the function of the cell cycle checkpoints in six human mesothelioma cell lines (HMCLs) by studying the cell distribution in the different phases of the cell cycle by flow cytometry, and expression of cell cycle proteins, p53, p21(WAF1/CIP1) and p27(KIP1). In addition, we studied p53 gene mutations and expression of SV40 Tag. After exposure to gamma-radiation, HMCLs were arrested either in one or both phases of the cell cycle, demonstrating a heterogeneity in cell cycle control. G1 arrest was p21(WAF1/CIP1)- and p53-dependent. Lack of arrest in G1 was not related to p53 mutation or binding to SV40 Tag, except in one HMCL presenting a missense mutation at codon 248. These results may help us to understand mesothelioma and develop new treatments.  相似文献   

15.
16.
SV40T介导的人胎儿永生化气管成纤维细胞系的建立   总被引:1,自引:0,他引:1  
建立永生化气管成纤维细胞系为研究细胞恶性转化提供实验模型。用pZIPSV40T转染原代人胎气管成纤维细胞,G418筛选,挑取阳性克隆传代培养,并进行细胞生物学特性鉴定。结果表明,细胞经pZIPSV40T转染后,形态较正常细胞略短,排列不规则,当传到50代时仍生长良好。Southern blot 证实此转化细胞整合了SV40T基因。永生化细胞接种的第4天细胞为指数生长期,染色体核型以非整倍体为主,不  相似文献   

17.
We established a human osteoblastic cell line immortalized by simian virus 40 (SV40) in vitro , and designated it SV-HFO. Immunocytochemically, the cells were positive for SV40 large T-antigen, vimentin and osteocalcin, but negative for keratin and epithelial membrane antigen. The cells had characteristic morphologic and ultrastructural features of osteoblasts, produced alkaline phosphatase, and synthesized osteocalcin, the levels of which were elevated by treatment of the cells with 1α,25-dihydroxyvitamin D3. The cells proliferated and showed such osteoblastic properties even under serum-free conditions. The cells grew in soft agar, but did not form tumors when transplanted into athymic nude mice. Karyotypic analysis by the Q-banding technique showed that these cells were of human origin. The SV-HFO cell line is expected to serve as a suitable model for studying metabolism and carcinogenesis in human bone.  相似文献   

18.
A methylation profile of in vitro immortalized human cell lines   总被引:2,自引:0,他引:2  
Normal human diploid cells have a limited life span and undergo replicative senescence after various limited population doublings. Cells must pass the senescence barrier to become immortal. The exact mechanisms of immortalization are not clear, although inactivation of the RB pathway, and/or the p53 pathway and activation of telomerase has been shown to be necessary for immortalization of certain cell types with DNA viruses or hTERT. Methylation-associated inactivation of tumor suppressor genes plays an important role in tumor progression. To test if gene-specific methylation contributes to the immortalized and transformed phenotype, we analyzed the methylation status of 17 genes in normal cells immortalized with SV40, hTERT, Ad5, Ad12-SV40 or HPV-18. Some of these immortalized lines were progressively transformed and tumorigenic in nude mice. We observed gene-specific methylation in the in vitro immortalized and transformed cells. SV40 and HPV18 immortalization resulted in different methylation spectra. In SV40- and h-TERT-immortalized prostate epithelial cells, the most frequently methylated gene was RASSF1A, while in HPV18-immortalized cell lines, the RAR-beta2 gene was universally methylated. Immortalization with SV40 resulted in methylation of a greater number of genes than immortalization with HPV. Furthermore, in SV40-immortalized cell lines, methylation affected different genes in fibroblasts compared with epithelial cells, suggesting that different mechanisms may be used by SV40 to immortalize cell lines of different origins. In HPV18-immortalized and subsequently transformed cell lines, the most commonly methylated genes were hormone responsive genes, such as AR, ER-beta and RAR-beta2. In general, more genes were methylated in neoplastically-transformed cell lines than in only immortalized cell lines, indicating that accumulation of epigenetic abnormalities may contribute to oncogenesis.  相似文献   

19.
Codon 72 of human p53 gene is polymorphic, encoding arginine or proline. Here we report construction of a human p53 knock-in (Hupki) mouse encoding the codon 72(pro) variant. The new strain was crossed with the original Hupki mice (codon 72(arg/arg)) to obtain primary embryonic fibroblasts polymorphic at codon 72 or homozygous for codon 72(pro). The fibroblasts, cultured under standard conditions, immortalized within 12 weeks and acquired p53 mutations similarly to Hupki codon 72(arg/arg) cells investigated previously. Sequencing of human p53 exons 4-9 in immortalized cultures revealed missense mutations found repeatedly in human tumours. In cell lines ensuing from benzo(a)pyrene-treated cultures the combined p53 mutation pattern from experiments with the 3 codon 72 genotypes showed a predominance of strand-biased G to T transversions (18 of 36 mutations), and mutations recurring at smokers' lung tumour hotspot codons 157 and 273, supporting involvement of tobacco carcinogens in shaping the mutation signature in lung cancers of smokers. Mutations in cell lines from unexposed cultures did not cluster at these codons and G to T transversions were uncommon (2 of 52 mutations) (Fisher's exact test P<0.0001). Most mutations (13/16) in cell lines derived from cells polymorphic at codon 72 were found on the proline allele, with loss of the arginine allele.  相似文献   

20.
J S Rhim  J B Park  G Jay 《Oncogene》1989,4(11):1403-1409
Polybrene, in conjunction with dimethyl sulfoxide (DMSO) shock has been shown to increase the frequency of DNA-mediated gene transfer to mammalian cells as compared with the frequency obtained with calcium phosphate transfection. We have successfully adapted this procedure for use with human epidermal keratinocytes. Non-tumorigenic human epidermal epithelial cells immortalized by SV40 tumor antigen were neoplastically transfected, using Polybrene at a concentration of 10 micrograms ml-1, followed by a 4 min shock, with 30% DMSO, with a plasmid carrying the activated H-ras gene from the EJ bladder carcinoma cell line. The transfected cells showed morphological alterations and induced carcinomas when transplanted into nude mice. They contained integrated copies of the transfected H-ras gene and expressed high levels of the p21 protein. Polybrene-induced DNA transfection, therefore, offers the opportunity to transfer genes effectively into human epidermal keratinocytes and should accelerate the study of the interaction between oncogenes and human epithelial cells. This study appears to represent the first neoplastic conversion of nontumorigenic, immortalized human epidermal keratinocytes by an activated human oncogene.  相似文献   

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