Transformation of human cystinotic fibroblasts by SV40: characteristics of transformed cells with limited and unlimited growth potential.

Human skin fibroblasts derived from patients with nephropathic cystinosis were transformed with SV40 virions, cloned and permitted to enter the degenerative stage of growth termed "crisis," characteristic of SV40 transformed human cells. Nephropathic cystinosis is an autosomal recessively inherited metabolic disorder resulting in the intracellular accumulation of the amino acid cystine. A transformed cystinotic cell line which was recovered from the crisis stage was indistinguishable from its transformed precrisis parental cell strain in growth rate in media containing either 1% or 10% serum, cloning efficiency on plastic, in semisolid media, or upon confluent monolayers of normal skin fibroblasts, expression of SV40 T antigen, or production of virus. However, the modal DNA content of the recovered postcrisis transformed cystinotic cell line was different from that of the cloned parental precrisis transformed cell strain, suggesting that the postcrisis line was derived from a small subpopulation of the precrisis strain. The DNA content of the established cystinotic cell line continued to be unstable during subsequent subculturing and gave rise to subclones with both more and less DNA per cell. This line now has an apparently infinite growth potential and still has the hallmark of the cystinotic parental line, the storage of abnormally large amounts of intracellular nonprotein cystine.     

Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells

Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients (n = 9) and healthy controls (n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 +/- 0.95 nmol/mg versus 0.09 +/- 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 +/- 0.83 nmol/mg versus 0.29 +/- 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.     

Glutathione metabolism in normal and cystinotic fibroblasts

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.     

Impaired clearance of free cystine from lysosome-enriched granular fractions of I-cell-disease fibroblasts.

Cultured fibroblasts from patients with I-cell disease (mucolipidosis II) accumulate excessive amounts of free cystine, similarly to cells from patients with nephropathic cystinosis, a disorder of lysosomal cystine transport. To clarify whether the intralysosomal accumulation of cystine in I-cell-disease fibroblasts was due to a defective disposal mechanism, we measured the rates of clearance of free [35S]cystine from intact normal, cystinotic and I-cell-disease fibroblasts. Loss of radioactivity from the two mutant cell types occurred slowly (t 1/2 = 500 min) compared with the rapid loss from normal cells (t 1/2 = 40 min). Lysosome-rich granular fractions isolated from three different cystine-loaded normal, cystinotic and I-cell-disease fibroblast strains were similarly examined for non-radioactive cystine egress. Normal granular fractions lost cystine rapidly (mean t 1/2 = 43 min), whereas cystinotic granular fractions did not lose any cystine (mean t 1/2 = infinity). I-cell-disease granular fractions displayed prolonged half-times for cystine disposal (mean = 108 min), suggesting that I-cell-disease fibroblasts, like cystinotic cells, possess a defective carrier mechanism for cystine transport.     

Characterization of simian virus 40 transformed African green monkey cells (CV-1). I. Defective virion and viral genome.

Several clones of SV40 transformed CV-1 cells have been characterized for the production of T- and V-antigens and for the state of viral genome. The transformed CV-1 cells failed to produce infectious virions as assayed after sonication or cocultivation and fusion with normal CV-1 cells, and were resistant to super-infection by SV40. Some clones of the transformed cells contained V-antigens. The population of V-antigen positive cells varied from 0 to 100% depending on the passage number while the T-antigen positive cells were always 100%. The virions isolated from the transformed cells were similar in morphology to complete SV40, but lighter in density than complete SV40. In one clone, a small amount of SV40 DNA was detectable in a free state while a large proportion of the DNA hybridizable with SV40 3H cRNA was linearly integrated into the cell DNA. The free SV40 DNA was noninfectious, closed circular DNA with a size smaller than infectious SV40 DNA component I. Since the cell extracts of the transformed cells contained an agent(s) which induced T- and V-antigens in normal CV-1 cells, it was suggested that the SV40 transformed CV-1 cells contained free as well as integrated defective SV40 genomes responsible for the synthesis of T- and V-antigens.     

Cells transformed by a wide variety of agents express higher abundance levels of some cellular RNA species

A cDNA-cloned library was prepared from mRNA synthesized by SV40-transformed mouse cells. Eleven cDNA clones were selected based on their ability to hybridize higher levels of mRNA in SV40-transformed 3T3 cells than in 3T3 cells. These cDNA clones were employed to screen the steady-state levels of cytoplasmic RNAs in a wide variety of viral (SV40, polyoma, adenovirus, and Rous sarcoma virus) and nonviral (methylcholanthrene, embryonal carcinoma) transformed cell lines. Two of the cDNA clones—A17 and 104—detected greater than 40–100-fold higher levels of mRNA in all the transformed cell lines tested when compared to nontransformed cells (3T3, C3HEF). The levels of mRNA complementary to these two cDNAs were regulated in a temperature-sensitive fashion (87–100-fold) in both SV40tsA- and RSV ts-src-transformed murine cell lines. These two cDNA clones detected greater than 100-fold, higher levels of complementary RNA derived from SV40 tumor tissue than in normal mouse liver. RNA species complementary to cDNA clones A17 or 104 were not detected in either actively growing nontransformed cells or in serum-stimulated 3T3 cells. The abundance levels of mRNAs detected by these two cDNA clones appear to be regulated 100-fold or greater by the transformed state, independent of the transforming agent. The higher levels of these RNA species detected in transformed mouse cells appear not to be solely regulated by the state of growth of nontransformed cells.     

Quantification of expression of linked cloned genes in a simian virus 40-transformed xeroderma pigmentosum cell line.

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).     

Fine mapping two distinct antigenic sites on simian virus 40 (SV40) T antigen reactive with SV40-specific cytotoxic T-cell clones by using SV40 deletion mutants.

The existence of two distinct antigenic sites at the surface of simian virus 40 (SV40)-transformed H-2b cells has been previously demonstrated (A. E. Campbell, L. F. Foley, and S. S. Tevethia, J. Immunol. 130:490-492, 1983) by using two independently isolated SV40-specific cytotoxic T-lymphocyte (CTL) clones, K11 and K19. We identified amino acids in the amino-terminal half of SV40 T antigen that are essential for the recognition of antigenic sites by these CTL clones by using H-2b cells transformed by mutants that produce T antigen truncated from the amino-terminal or carboxy-terminal end or carrying overlapping internal deletions in the amino-terminal regions of SV40 T antigen. The results show that CTL clone K11 failed to recognize and lyse target cells missing SV40 T-antigen amino acids 189 to 211, whereas CTL clone K19 lysed these cells. The cell lines missing SV40 T-antigen amino acids 220 to 223 and 220 to 228 were not lysed by CTL clone K19 but were susceptible to lysis by CTL clone K11. Two other cell lines missing amino acids 189 to 223 and 189 to 228 of SV40 T antigen were not lysed by either of the CTL clones but were lysed by SV40-specific bulk-culture CTL if sufficient amounts of relevant restriction elements were expressed at the cell surface. The SV40 T-antigen amino acids critical for the recognition of an antigenic site by CTL clone K11 were identified to be 193 to 211; 220 to 223 were identified as critical for recognition by CTL clone K19. The deletion of these amino acids from the T antigen resulted in the loss of antigenic sites specific for CTL clones K11 and K19.     

Difference in O6-methylguanine methyltransferase activity among transformed NIH3T3 cell clones

We examined the sensitivity to the lethal effects of methylating agents and the O6-methylguanine methyltransferase (MTR) activities of in vitro transformed NIH3T3 cell clones. The sensitivities to the lethal effects of MNNG were not different among all 49 transformed cell clones examined and do not correlate with the MTR activities. All 8 spontaneously transformed cell clones showed the same sensitivities to ACNU as the parental cell line. 2 of 20 transformants induced by UV or MNNG showed higher sensitivities to the ACNU although the MTR activity was normal. One cell clone transformed by UV was sensitive to ACNU and showed about half MTR activity. 5 of 19 cell clones transformed by oncogenes (Ha-ras or SV40 ori-) were sensitive to the lethal effects of ACNU and showed the low MTR activities, but were not as much sensitive as a Ha-MuSV transformed cell clone, Ha821.     

Demonstration of multiple antigenic sites of the SV40 transplantation rejection antigen by using cytotoxic T lymphocyte clones

Multiple antigenic sites on the simian virus 40 (SV40) tumor-specific transplantation antigen (TSTA) were detected by the use of cytotoxic T lymphocyte (CTL) clones isolated from continuous cultures of SV40-specific CTL (H-2b). Two independently derived clones, K11 and K19, specific for the SV40 TSTA in association with H-2Db, each recognized a different antigenic determinant of the SV40 TSTA. This conclusion was based on the observation that a human papovavirus BK virus (BKV) transformed cell line, which possesses a T antigen serologically cross-reactive with that of SV40, was lysed by a heterogeneous population of SV40-immune lymphocytes and by clone K19 but not by K11. Therefore, these CTL clones must recognize two different antigenic determinants of the SV40 TSTA:K19 recognizes a cross-reactive determinant of the SV40 and BKV TSTA, whereas K11 is reactive against an SV40-specific determinant.     

Simian virus 40-permissive cell interactions: selection and characterization of spontaneously arising monkey cells that are resistant to simian virus 40 infection.

A fraction of permissive cells survive simian virus 40 (SV40) infection. The frequency of such surviving cells depends only upon the concentration of infecting virus, both parental and progeny, to which the cells are exposed during the course of selection. Surviving clones, which can be freed of virus by cloning in the presence of SV40 antiserum, are indistinguishable from parental cells in their growth of characteristics and display no SV40 antigen; thus they are not transformed. Most surviving clones are less than 10% as susceptible as parental cells to SV40 infection; 5 to 10% are less than 1% as susceptible. None of these SV40-resistant clones is absolutely resistant to SV40 infection. Analysis of 16 independently arising resistant clones indicates that they all block SV40 infection at an early stage after adsorption and eclipse but before full uncoating. Viral mutants have been isolated that partially overcome the block to infection in these cells; these host range viruses plaque on resistant lines fivefold more efficiently than wild-type SV40 and have a characteristic plaque morphology. Fluctuation analysis indicates that resistant cells arise spontaneously during the growth of normally susceptible permissive cells. Thus, SV40-resistant cells are selected for, not induced by, SV40 infection.     

Activation of endogenous retroviral transcription in SV40-transformed mouse cells.

We have previously isolated a number of cDNA clones that correspond to mRNAs present at higher levels in SV40-transformed cells than in the untransformed parental cells (Scott, M.R.D., Westphal, K.-H. and Rigby, P.W.J. (1983) Cell 34, 557-567). We have now determined the nucleotide sequence of the archetypal Set 2 clone, pAG59, and can thus identify it as corresponding to the env gene of the endogenous, ecotropic C-type retrovirus of Balb/c mice, Emv-1. We have shown that in the subset of SV40-transformed cells that express the provirus both of the proteins encoded by env, gp70 and p15E, are synthesised and that the former is displayed on the cell surface. We discuss the significance of these observations for the biology of SV40 transformation.     

Increased mutation rates in doubly viral transformed Chinese hamster cells.

An untransformed parental Chinese hamster cell line, one polyoma transformed clone, and two clones containing both SV40 and polyoma DNA were tested for the appearance of variants resistant to 6-thioguanine or oubain. The frequency of such variants was found to be highest in doubly transformed cells. The mutation rate at these loci was correlated with the level of transformation. The mutants observed were stable and had low reversion frequencies with no gross cytogenetic changes. These results establish a quantitative basis for the evaluation of phenotypic variability observed in transformed cell populations.     

Virogenic Properties of Bromodeoxyuridine-sensitive and Bromodeoxyuridine-resistant Simian Virus 40-transformed Mouse Kidney Cells

When simian virus 40 (SV40)-transformed mouse kidney cells (mKS) were grown in the presence of susceptible indicator cells, SV40 was readily recovered from: (i) 15 transformed cell lines, (ii) transformed cells subcultured 45 times over a 7-month period in medium containing antiviral serum and bromodeoxyuridine (dBU), (iii) 45 of 46 clonal lines isolated in the presence of antiviral serum, (iv) 19 of 19 secondary clones isolated from two clonal lines, and (v) dBU-resistant transformed cell lines. dBU-resistant SV40-transformed mouse kidney cell lines were selected and shown to contain the T antigen and to have normal levels of thymidylate kinase and deoxyribonucleic acid (DNA) polymerase, but to be deficient in thymidine (dT) kinase. Radioautographic and biochemical experiments demonstrated that very little (3)H-dT was incorporated into DNA of dBU-resistant cells during a 6-hr labeling period. After infection of dT kinase-deficient mKS cells with vaccinia virus, high levels of dT kinase were induced. The properties of SV40 recovered from dBU-sensitive and dBU-resistant cells were studied. SV40 recovered from transformed cells was shown to express in CV-1 cells at least six functions characteristic of parental virus: synthesis of capsid antigen, synthesis of T antigen, synthesis of viral DNA, induction of dT kinase, induction of DNA polymerase, and induction of host cell DNA synthesis. In addition, SV40 recovered from the transformed cells induced T antigen, dT kinase, deoxycytidylate deaminase, thymidylate kinase, and DNA polymerase in abortively infected mouse kidney cultures, and the virus was also capable of transforming primary cultures of mouse kidney cells.     

The ability of large T antigen to complex with p53 is necessary for the increased life span and partial transformation of human cells by simian virus 40.

Simian virus 40 (SV40) T antigen binds to the tumor suppressor p53 protein, and this association may contribute to oncogenic transformation by the virus. We investigated the importance of this binding on transformation by examining three replication-competent mutants of SV40 (402DE, 402DN, and 402DH). These mutants express T antigens defective in binding to human and monkey p53s but retain some binding with mouse p53. All showed significant reduction in their ability to induce transformed cell foci of two normal human cell lines as well as a slight reduction with mouse embryo cells. Other comparable mutants which express T antigens retaining the ability to complex with p53 were able to induce foci at wild-type levels in both human and mouse cells. Further studies were performed with five T-antigen-positive clones isolated from the few human cell foci that appeared after transfection with 402 mutant DNAs. All five clones reached senescence at about the same point as did the parental untransformed cells. However, six other human cell clones obtained after transfection with DNA from nondefective mutants or wild-type virus were still growing well at more than 10 passages beyond their expected life span. These results suggest that the ability of T antigen to form stable complexes with p53 is necessary for SV40 to extend the life span and partially transform human cells in culture.     

Effect of passage in culture on a clone of BALB/c 3T3 cells transformed by simian virus 40.

Most simian virus 40 (SV40)-transformed BALB/c 3T3 clones employed for biochemical studies have been used without regard to passage level. To determine whether virus-induced properties are stable as a function of passage, we have extensively characterized one transformed clone, FNE, which was isolated after SV40 infection BALB/c 3T3 cells in factor-free medium. From the initial testing at passage 5 and for at least 50 subsequent subcultures, the cells stably maintained many transformed growth properties, including high saturation density, morphology, colony formation on contact-inhibited monolayers, tumorigenicity, and synthesis of viral-specific RNA. However, other properties varied as a function of passage. There was a slight decrease in viral genome equivalents per cell from 1.1 copy/cell at passage 5 to 0.7 copies at passage 40. Initially, the cells were negative for all type C virus; however, cells carried at low density for 13 to 20 passages (65 to 100 generations) began to release an endogenous type C virus that then persisted in the culture. Spontaneous release of type C virus did not occur in control BALB/c 3T3 cells carried under identical culture conditions for 90 passages. When the cultures were releasing type C viruses they stained uniformly and brightly positive for SV40 tumor (T) antigen by immunofluorescence, whereas T antigen staining was variable at early passage. These experiments suggest that subtle but perhaps important differences in viral gene expression can occur as a function of passage; they also demonstrate the importance of evaluating the interactions between SV40 and endogenous type C viruses.     

Instability of extrachromosomal cosmid DNA in SV40-transformed human (ataxia-telangiectasia) cells

The ability of SV40-transformed human (ataxia-telangiectasia) fibroblasts to maintain Epstein-Barr virus (EBV)-based plasmids and cosmids extrachromosomally has been investigated. Transfection of a culture of cells with two different plasmids gave rise to cell clones which were able to maintain both plasmids extrachromosomally. When an EBV-based cosmid library was transfected into the cells and an individual cell clone was isolated, the extrachromosomal DNA derived from the cosmid contained numerous deletions and rearrangements. When individual cosmids were transfected into the culture, and several cell clones were isolated, the intracellular cosmid-derived DNA again showed the presence of multiple deletions and rearrangements. We conclude that although SV40-transformed cells are able to maintain more than one different EBV-based plasmid extrachromosomally, large EBV-derived molecules are extensively rearranged. SV40-transformed human fibroblasts cannot therefore be usefully used in attempting to clone genes from EBV-based cosmid libraries.     

SV40 T基因转化的山羊乳腺上皮细胞系及其生物学特性

目的建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系.方法根据已发表的SV40病毒T基因序列设计引物,以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板,用高保真PCR扩增SV40 T基因.将获得的SV40 T基因克隆入真核表达载体,并用获得的重组表达质粒转染山羊原代乳腺上皮细胞.经有限稀释和反复传代后获得转化细胞克隆,对其生物学特性进行研究.结果扩增出序列正确的SV40T基因,重组质粒转染获得的转化细胞的对数生长期为接种后第4天,细胞群体倍增时间为23.5*!h,克隆形成率为26.7%.DNA斑点杂交试验证明转化细胞的基因组中整合有SV40 T基因,染色体核型分析试验表明转化细胞的核型无明显异常,裸鼠接种试验证明转化细胞不能形成肿瘤,软琼脂集落形成试验表明转化细胞在软琼脂中不能生长.部分细胞克隆已在体外传30代以上,保持正常乳腺上皮细胞的形态特征,在胶原基质上能形成腺泡样结构.结论本研究获得的SV40 T基因转化的山羊乳腺上皮细胞具有转化细胞系的生物学特性.