Association of SV40 DNA sequences with nuclear matrix in SV40 transformed hamster fibroblasts

Nuclear matrices were isolated by the high-salt, non-ionic detergent method from SV40-transformed hamster fibroblasts (TSV5 cell line), and from hamster tumours derived from these cells. DNA isolated from matrices and total nuclei was hybridized with nick-translated SV40 DNA. The enrichment of matrix DNA with SV40 DNA sequences was observed in all five experiments with matrix DNA of TSV5 cells but only in five out of nine matrix DNA isolated from tumour cells.     

Comparison of gamma-radiation-induced accumulation of ataxia telangiectasia and control cells in G2 phase

Recent reports from a number of laboratories have linked radiosensitivity in ataxia telangiectasia (A-T) to a large and prolonged block of some cells in G2 phase. Previous results from this laboratory, largely with one Epstein-Barr virus-transformed A-T lymphoblastoid cell line, presented evidence for a dramatic increase in the number of cells in G2 phase over controls during a 24-h period post irradiation. We describe here a study of the effect of gamma-radiation on G2 phase delay in several A-T cell lines. Based on previous results with several cell lines 24 h post irradiation was selected as the optimum time to discriminate between G2 phase delay in control and A-T cells. All A-T homozygotes showed a significantly greater number of cells in G2 phase, 24 h post irradiation, than observed in controls. A more prolonged delay in G2 phase after irradiation was seen in different A-T cell types that included lymphoblastoid cells, fibroblasts and SV40-transformed fibroblasts. At the radiation dose used it was not possible to distinguish A-T heterozygotes from controls.     

Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line

A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori-, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori--transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori--transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori- in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells.     

Some aspects of glutathione metabolism in ataxia-telangiectasia fibroblasts

Levels of glutathione (GSH) and two enzymes involved in GSH metabolism, glutathione reductase (GR) and glutathione-S-transferase(s) (GST), were measured in four SV40-transformed human fibroblast cell lines. MRC5-V1 and GM0637, derived from normal individuals, had mean GSH levels of 4.2 and 6.5 nmoles/10(6) cells, respectively. TAT2SF and AT5BIVA, both from ataxia-telangiectasia (A-T) patients, respectively had 6.5 and 4.2 nmol/10(6) cells, indicating that basal GSH levels were similar in A-T and normal cells. There was some variation in GST activity among the four cell lines but deficiency in this enzyme cannot be associated with radiosensitivity in A-T. When GR activity was measured, A-T cells had approximately 82 per cent of the mean normal activity. Though statistically significant, (P = 0.05), this small deficiency could be due to chance and is unlikely to be responsible for the radiosensitive phenotype of A-T.     

The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses.     

Selective excision of gamma ray damaged thymine from the DNA of cultured mammalian cells.

Three mammalian cell lines (WI-38, SV40-transformed WI-38 and Chinese hamster ovary) were exposed to high doses of 137-Cs gamma rays and their DNA analysed, following various periods of postirradiation incubation, for products of the 5,6-dihydroxy-dihydrothymine type. Within fifteen minutes of incubation at 37 degrees C 70 to 90 percent of these radiation products were removed from acid-precipitable material in all three cell lines. The amount of DNA degradation induced by radiation varied from approximately one percent in WI-38 cells to 15 percent in SV40-transformed WI-38 cells. Comparison of DNA degradation with the amount of thymine radiation product removed indicates that a selective gamma ray-induced excision repair capability exists in mammalian cells. Because of its more rapid kinetics, gamma ray excision repair is probably a distinct process as compared with ultraviolet-induced pyrimidine dimer excision.     

Establishment and characterization of two human cell lines with amplified dihydrofolate reductase genes

Two SV40-transformed human cell lines, GM637, derived from a normal human subject, and GM5849, derived from a patient with ataxia-telangiectasia (A-T), were grown in increasing concentrations of the cytotoxic agent methotrexate (MTX). The GM637 line was naturally more resistant to methotrexate than was GM5849 and, over a 5-month period, became resistant even to very high concentrations (up to 100 microM). The GM5849 line became resistant to 500 nM methotrexate during the same period. However, dot blot and Southern blot analyses showed that both cell lines had amplified their dihydrofolate reductase (dhfr) genes to about the same extent, approx. 50-fold. Using the GM5849 line with amplified dhfr, we attempted to determine if interruption of DNA synthesis by hydroxyurea would cause DNA to be replicated twice within a single cell cycle, as has been reported for Chinese hamster ovary cells. No evidence for such a phenomenon was obtained.     

Effect of X-radiation on DNA and histone synthesis in ataxia telangiectasia and normal lymphoblastoid cells

The possibility that the radiosensitivity of lymphoblastoid cell lines from patients with ataxia telangiectasia (A-T) is due to an aberrant content of histones has been examined. The histone pattern of lymphoblastoid cell lines derived from A-T patients was found to be indistinguishable from that obtained from normal individuals. X-ray irradiation led to a greater decrease in cell growth rate in the A-T cells than in the normal cells but was accompanied by a greater decrease of DNA synthesis rate in the normal cells. This difference in radiosensitivity was not reflected in differences in the content or rates of synthesis of histones or of major non-histone proteins in these cells. Reduction in the rate of DNA synthesis was not associated with the appearance of the lysine-rich histone variant H1. We conclude that the hypersensitivity to ionizing radiation in A-T cells is not due to fundamental differences in the composition or synthesis of the major chromosomal proteins.     

Sensitivity of fibroblasts derived from ataxia-telangiectasia patients to calicheamicin gamma 1I.

We report the hypersensitivity of SV40-transformed fibroblasts derived from ataxia telangiectasia (AT) patients to calicheamicin gamma 1I. In common with other free-radical generating agents such as bleomycin and ionizing radiation, treatment with calicheamicin gamma 1I reveals AT derived lines to be 6-fold more sensitive to this drug when compared to controls. Furthermore, in common with ionizing radiation, AT cells did not show dose-dependent inhibition of DNA synthesis after treatment with calicheamicin gamma 1I.     

The effect of recombinant human tumour necrosis factor on growth and macromolecular synthesis of human epithelial cells

We have studied the effect of recombinant human tumour necrosis factor (rHuTNF) on growth and macromolecular synthesis in a range of normal and transformed epithelial cell types. Tumour necrosis factor did not affect the growth of normal human mammary epithelial cells, but its growth-inhibitory action on the SV40-transformed human mammary epithelial cell line HBL-100 increased with passage number in association with a progression of malignant phenotype. However, of two lines derived from nude mouse tumours of HBL-100 lines, one, HBLT-12, did not respond to rHuTNF, and the other, HBLT-11 showed some growth stimulation by high dose rHuTNF. Macromolecular synthesis in HBLT-11 was not affected by rHuTNF. The breast cancer cell lines MCF-7 and BT20 were sensitive to the cytotoxic effects of rHuTNF. In MCF-7 a gradual decrease in RNA and DNA synthesis occurred over 48 h, ending with an accumulation of cells in S and G2 phase of the cell cycle and cell death. The addition of alpha- or gamma-interferon increased, but did not accelerate the cytotoxicity of rHuTNF.     

Isolation of novel cDNA transformation markers from SV40-transformed human keratinocytes

Differential screening of a cDNA library was used to isolate probes for mRNAs that are induced in simian virus 40 (SV40)-transformed human keratinocytes. Several of these cDNAs hybrid select mRNAs which encode transformation-induced proteins found in the cytoskeletal component of SV40-transformed keratinocytes. One of these cDNAs was used to study the phenotype of normal and transformed cell lines derived from various tissues. We found that mRNA encoding the novel transformation-induced proteins is expressed in two squamous carcinoma cell lines derived from the oral epithelium, four SV40-transformed keratinocyte cell lines, and two SV40-transformed fibroblasts. Normal or transformed lymphoid cells or cell lines derived from carcinoma of the cervix do not express mRNAs which hybridize to these probes. The results from this study suggest that these probes may be used to detect markers of transformation in certain cell types.     

Response of Simian Virus 40-Transformed Cell Lines and Cell Hybrids to Superinfection with Simian Virus 40 and Its Deoxyribonucleic Acid

Whereas normal human and monkey cells were susceptible both to intact simian virus 40 (SV40) and to SV40 deoxyribonucleic acid (DNA), human and monkey cells transformed by SV40 were incapable of producing infectious virus after exposure to SV40, but displayed susceptibility to SV40 DNA. On the other hand, mouse and hamster cells, either normal or SV40-transformed, were resistant both to the virus and to SV40 DNA. Hybrids between permissive and nonpermissive parental cells revealed a complex response: whereas most hybrids tested were resistant, three of them produced a small amount of infectious virus upon challenge with SV40 DNA. All were resistant to whole virus challenge. The persistence of infectious SV40 DNA in permissive and nonpermissive cells up to 96 hr after infection was ascertained by cell fusion. The decay kinetics proved to be quite different in permissive and nonpermissive cells. Adsorption of SV40 varied widely among the different cell lines. Very low adsorption of SV40 was detected in nonsusceptible cells with the exception of the mKS-BU100 cell line. A strong increase in SV40 adsorption was produced by pretreating cells with polyoma virus. In spite of this increased adsorption, the resistance displayed by SV40-transformed cells to superinfection with the virus was maintained.     

Spontaneous chromosomal aberrations in Fanconi anaemia,ataxia telangiectasia fibroblast and Bloom's syndrome lymphoblastoid cell lines as detected by conventional cytogenetic analysis and fluorescence in situ hybridisation (FISH) technique

Several primary and transformed human cell lines derived from cancer prone patients are employed routinely for biochemical and DNA repair studies. Since transformation leads to some chromosomal instability a cytogenetic analysis of spontaneous chromosome aberrations in fibroblast cell lines derived from patients with Fanconi anaemia (FA), ataxia telangiectasia (AT), and in lymphoblastoid cell lines derived from patients with Bloom's syndrome (BS), was undertaken. Unstable aberrations were analysed in Giemsa stained preparations and the chromosome painting technique was used for evaluating the frequencies of stable aberrations (translocations). In addition, the frequency of sister-chromatid exchanges (SCEs) was determined in differentially stained metaphases. The SV40-transformed fibroblasts from these cell lines have higher frequencies of unstable aberrations than the primary fibroblasts. In the four lymphoblastoid cell lines derived from BS patients higher frequencies of spontaneously occurring chromosomal aberrations in comparison to normal TK6wt cells were also evident. The frequency of spontaneously occurring chromosome translocations was determined with fluorescence in situ hybridisation (FISH) and using DNA libraries specific for chromosomes 1, 2, 3, 4, 7, 8, 11, 14, 19, 20 and X. The translocation levels were found to be elevated for primary FA fibroblasts and lymphoblastoid cells derived from BS patients in comparison with control cell lines, hetero- and homozygote BS cell lines not differing in this respect. The SV40-transformed cell lines showed very high frequencies of translocations independent of their origin and almost every cell contained at least one translocation. In addition, clonal translocations were found in transformed control TK6wt and AT cell lines for chromosomes 20 and 14, respectively. The spontaneous frequencies of SCEs were similar in transformed fibroblasts derived from normal individuals and AT patients, whereas in SV40-transformed FA cells these were higher (4-fold). Among cell lines derived from BS patients, heterozygote lines behaved like control, whereas in homozygote cell lines very high frequencies of SCEs (about 12-fold) were evident.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

A derivative of an ataxia-telangiectasia (A-T) cell line with normal radiosensitivity but A-T-like inhibition of DNA synthesis

Ataxia-telangiectasia (A-T) cells are hypersensitive to the lethal effects of ionizing radiation and fail to inhibit DNA synthesis following radiation exposure. A cell line derived from an A-T line following DNA-mediated gene transfer has normal radiation sensitivity, but the kinetics of DNA synthesis after gamma-irradiation are similar to those of A-T cells.     

Radioimmunoassay of the cellular protein p53 in mouse and human cell lines

We have developed quantitative radioimmunological solid phase assays for the host protein p53 from mouse cells and from human cells. The first assay, for mouse p53, depends on having two monoclonal antibodies reacting with different determinants on the p53 molecule. With this assay we have shown that SV40-transformed cells have approximately 100-fold more p53 than untransformed mouse cells and that other transformed cells have intermediate levels. Embryonal carcinoma cell lines have approximately 50-fold less p53 than SV40-transformed cells. This is in contrast to the high levels of incorporation of [35S]methionine into p53 in these cells and indicates that metabolic labelling is not a valid approach for measuring p53 levels. The second assay, for human p53, required a different approach and made use of the anti-p53 antibodies detected in the sera of some breast cancer patients. Human tumour cell lines contained amounts of p53 varying from the high level seen in SV40-transformed human fibroblasts down to less than one hundredth of this amount. Normal human cells showed low levels of p53. The data confirm that many, but not all, human tumour cell lines contain more p53 than normal cells.     

Characterization of a simian virus 40-transformed Fanconi anemia fibroblast cell line

We have characterized a SV40-transformed human fibroblast cell line (GM6914) derived from a patient with Fanconi anemia (FA) in order to establish its usefulness for biochemical and genetic experiments, including DNA-mediated gene transfer. GM6914 cells have a growth rate similar to that of SV40-transformed normal human fibroblasts and an indefinite lifespan in culture. As has been established for other FA cell types, GM6914 cells have an increased sensitivity to DNA-crosslinking agents such as mitomycin C (MMC). The D10 for GM6914 cells is 8 times lower than for equivalent controls. GM6914 cells also have an elevated frequency of spontaneous chromosome aberrations and this frequency can be increased by MMC concentrations which show no effect on control cells. Genetic complementation studies with lymphoblasts derived from two affected sibs of the donor of GM6914 cells show that GM6914 belongs to FA complementation group A. In DNA-transfection studies using plasmid pRSVneo, colonies of GM6914 cells resistant to the drug G-418 were observed at frequencies ranging from 1.7 to 16 X 10(-4), values similar to those observed with several other SV40-transformed human cell lines. GM6914 should be a useful recipient cell line in experiments using DNA-mediated gene transfer to clone the normal allele of the gene which is defective in FA complementation group A. GM6914 would also be an excellent cell line for studies on mutagenesis, recombination and repair using plasmid vectors.     

DNA ligase III is the major high molecular weight DNA joining activity in SV40-transformed human fibroblasts: normal levels of DNA ligase III activity in Bloom syndrome cells.

The phenotypes of cultured cell lines established from individuals with Bloom syndrome (BLM), including an elevated spontaneous frequency of sister chromatid exchanges (SCEs), are consistent with a defect in DNA joining. We have investigated the levels of DNA ligase I and DNA ligase III in an SV40-transformed control and BLM fibroblast cell line, as well as clonal derivatives of the BLM cell line complemented or not for the elevated SCE phenotype. No differences in either DNA ligase I or DNA ligase III were detected in extracts from these cell lines. Furthermore, the data indicate that in dividing cultures of SV40-transformed fibroblasts, DNA ligase III contributes > 85% of high molecular weight DNA joining activity. This observation contrasts with previous studies in which DNA ligase I was reported to be the major DNA joining activity in extracts from proliferating mammalian cells.     

Tumor formation by SV40-transformed human cells in nude mice: the role of SV40 T antigens

Human cells transformed in vitro by SV40 rarely form tumors in nude mice. We examined whether these cells as a group are inherently nontumorigenic or whether they are potentially tumorigenic but rejected by the athymic host, possibly by nonspecific immune mechanisms. SV80 and NG8 are SV40-transformed human cell lines that express all of the transformed properties, including anchorage-independent growth, but do not form tumors in adult nude mice after injection of as many as 10(8) cells. Both the SV80 and NG8 cell lines have SV40-specific transplantation antigens which crossreact with those present on SV40-transformed (but tumorigenic) rodent cells. We found that SV80 cells, though not NG8 cells, induced progressively growing lethal tumors if the cells are injected repeatedly into neonatal nude mice. Somatic cell hybrids between SV80 or NG8 cells and a highly tumorigenic cell line derived from a human tumor continue to express the virus-induced antigens and fail to form tumors in adult nude mice. These results strongly suggest that at least for some SV40-transformed human cells, the failure to form tumors in nude mice may be due to their expression of virus-induced transplantation antigens rather than the absence of tumorigenic potential.     

Rearrangement of integrated viral DNA sequences in mouse cells transformed by simian virus 40.

The organization of viral DNA sequences in several cell lines derived from a primary colony of simian virus 40 (SV40)-transformed mouse cells was analyzed to examine the origin of the various distinctive patterns of SV40 sequence arrangement present in transformed cells. This analysis revealed a complex arrangement of viral sequences in the uncloned transformed cells but simplified arrangements in cloned derivatives of the primary transformant. The cell lines studied had certain SV40 sequence arrangements in common, but the cloned lines had lost some parental arrangements and acquired new arrangements. These results indicate that the arrangement of viral sequences in some SV40-transformed cells is not fixed but that alterations occur after integration, creating a heterogeneous population of transformants. In the process, expression of viral genes may be altered. Possible causes for and implications of this genetic instability are discussed.