Enhanced induction of SV40 replication from transformed mammalian cells by fusion with UV-irradiated untransformed cells

DNA-damaging agents such as ultraviolet (UV) light are known to cause stimulation of virus replication in SV40-transformed hamster and human cells. The dose-response curves of UV-induced SV40 replication in transformed hamster cells resemble that obtained for UV-enhanced reactivation (ER) and UV-enhanced mutagenesis (EM) of SV40 or herpes viruses in mammalian cells. We have investigated whether UV-enhanced production of SV40 from transformed hamster (THK) and human (NB-E) cells belongs to the same category of conditional responses as ER and EM. To answer this question we have made use of the phenomenon that fusion of the SV40-transformed cells with monkey cells that are permissive to SV40 results in a considerable increase in the production of SV40 virus. When THK or NB-E cells were fused with UV-irradiated CV-1 cells at various times after irradiation, induction of SV40 was further increased and reached a maximum value of 2--3-fold when fusion was delayed for 24-48 h after irradiation. The kinetics of enhanced SV40 induction resembled that of ER and EM, suggesting that the UV-stimulated part of the induction represents one of the pleiotropic responses that are transiently induced in mammalian cells by DNA-damaging agents. Evidence is presented, showing that this transient effect can be induced only in cells that are permissive to SV40 replication. This suggests that the enhanced induction observed after fusion with irradiated monkey cells may be attributed to a transient increase in the activity of "permissiveness' factors. No enhanced induction was found when the THK or NB-E cells were fused with irradiated rodent cells, that are not or only slightly permissive to SV40 replication.     

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.     

Inhibition of postreplication repair and the enhancement of induction of SV40 virus from transformed hamster kidney cells.

The induction by ultraviolet light of simian virus 40 (SV40) from two SV40--transformed hamster kidney cell lines is enhanced by caffeine. In order to investigate the mechanism responsible for this enhancement, the effect of caffeine on postreplication repair of DNA damaged by UV light was studied utilizing alkaline sucrose-gradient sedimentation. Caffeine at concentrations of 0.5, 1.0 or 2.0 mM inhibited the filling of gaps during postreplication repair. In addition, caffeine was found to potentiate cell killing by mitomycin C, an alkylating agent, and to enhance SV40 induction by mitomycin C. We postulate that the persistence of gaps in DNA, caused by the presence of caffeine, results in the enhancement of SV40 virus induction.     

Beta-galactosidase overexpression in SV40-transformed Chinese hamster fibroblasts exposed to mutagens as a result of amplification of transfected bacterial lacZ DNA sequences

Genetic constructions in which the bacterial lacZ gene, encoding the enzyme beta-galactosidase, is fused to a viral (SV40) origin of replication have been introduced in an SV40-transformed hamster cell line (C1102). We have studied in detail 3 clones in which beta-galactosidase-specific activity increases after treatment with genotoxic agents. We show that this increase is dependent on the activity of the viral T protein and correlates with an amplification of lac sequences. This system provides a basis for the study of the induction of gene amplification by genotoxic agents in mammalian cells.     

Characterization and biological activity of cloned simian virus 40 DNA fragments

The biological activity of fragments of the SV40 genome was determined by manual microinjection of the fragments into the nuclei of mammalian cells. Fragments of the SV40 A gene (that codes for the T antigens) were obtained either directly by digestion with restriction endonucleases or after cloning into plasmid pBR322. Three different biological activities were studied: expression of T antigen, induction of cell DNA synthesis, and, in a few cases, reactivation of repressed ribosomal RNA genes. By using a number of fragments with deletions in the various portions of the SV40 A gene, we have been able to conclude that: 1) the sequences from 0.65 to 0.51 map units are not needed for the induction of cell DNA synthesis; 2) the sequences from 0.42 to 0.17 map units are not needed for the induction of cell DNA synthesis; and 3) the critical sequences for the induction of cell DNA synthesis, 0.51 to 0.42 map units, are different from those necessary for the reactivation of repressed ribosomal RNA genes (0.39-0.33 map units). These results indicate that the information for these two fundamental processes of cell proliferation resides in two separate and distinct domains of the SV40 A gene.     

Isolation of Defective Lysogens from Simian Virus 40-transformed Mouse Kidney Cultures

Rescue of simian virus 40 (SV40) from hamster and murine cell lines transformed by nonirradiated or by ultraviolet (UV)-irradiated SV40 (10(-3) to 10(-5) survival) was studied. A combination of tests was employed to detect induction of SV40 synthesis: (i) co-cultivation with susceptible monkey kidney (CV-1) cells; (ii) treating mixtures of transformed and CV-1 cells with UV-irradiated Sendai virus (UV-Sendai) prior to co-cultivation; and (iii) plating untreated or UV-Sendai-treated mixtures of transformed and CV-1 cells with freshly trypsinized CV-1 cells. The first and second tests provided a measure of the total infectious SV40 yield per culture, and the third test provided a measure of the frequency of induction (fraction of transformed cells giving rise to infectious centers). With the combination of tests, SV40 was rescued in all trials from TSV-5 hamster cells, mKS-BU100 mouse cells, and from several lines of mouse kidney cells transformed by UV-irradiated SV40 (mKS-U lines). The frequency of induction was about 7 x 10(-2) for TSV-5 cells, about 3 x 10(-3) for mKS-BU100 cells, greater than 10(-4) for the mKS-U lines which were "good" yielders, and about 10(-5) to 10(-4) for the mKS-U lines which were "average" yielders. SV40 of a plaque type different from parental virus was rescued from four of the mKS-U cell lines. Virus was also easily rescued from: (i) tumor cells produced from the mKS-A line of transformed mouse kidney cells; (ii) mouse kidney cells transformed by SV40 which had been rescued from mKS-BU100 cells; and (iii) tumor cells (HATS) which had been produced by inoculating newborn hamsters with SV40 rescued from mKS-BU100 cells. The frequency of induction of HATS cells was of the same order of magnitude as the frequency of induction of TSV-5 cells. In a study of the kinetics of virus induction, it was shown that SV40 could be detected 28, 40, and 48.5 hr after UV-Sendai treatment of mixtures of CV-1 and TSV-5, HATS, or mKS-BU100 cells, respectively. Although all of the mKS-U lines contained the SV40-specific tumor antigen, some were poor virus yielders (SV40 was recovered in less than 50% of the trials) and five lines were rare virus yielders (SV40 recovered only once in four or more trials). Forty-eight mKS-U lines were nonyielders; SV40 was never recovered by any test used thus far. UV-Sendai-treated mixtures of pairs of nonyielder mKS-U lines with CV-1 cells also did not yield infectious virus. Various factors affecting rescue have been discussed. The mKS-U lines which were poor virus yielders, rare yielders, or which never yielded virus have been classified tentatively as "defective lysogens" which contain mutational lesions at loci essential for detachment of SV40 from integration sites or for SV40 replication, or for both.     

Test of models for replication of simian virus 40 DNA following ultraviolet irradiation.

When mammalian cells are irradiated with ultraviolet light, semiconservative DNA replication is inhibited and the length of newly synthesized daughter strands is reduced. We have used the simian virus 40 (SV40) viral system to examine the molecular mechanism by which this inhibition of DNA replication occurs immediately following ultraviolet irradiation. We tested two models for DNA replication-inhibition by using a procedure first developed by Danna, K. J., and D. Nathans (1972, Proc. Natl. Acad. Sci. USA, 69:3097-3100) in which the distribution of 3H-label in segments of newly completed SV40 form-I molecules is measured after short pulse labeling with 3H-thymidine. Our experimental results were compared with those predicted by mathematical models that describe two possible molecular mechanisms of replication inhibition. Our data are best fit by a "blockage" model in which any pyrimidine dimer encountered by the replication fork prevents complete replication of the SV40 genome. An alternative model called "slowdown" in which DNA damage causes a generalized slowdown of replication fork movement on all genomes has more adjustable parameters but does not fit the data as well as the blockage model.     

The wavelength dependence of 8-methoxypsoralen photosensitization of radiation-enchanced reactivation in a mammalian cell-virus system.

The combined effect of 8-methoxypsoralen (8-MOP) and ultraviolet (UV) radiation on the ability of an irradiated mammalian cell (CV-1) to reactivate UV-irradiated mammalian virus (Herpes simplex) was tested. Prior treatment of cells with 8-MOP was found to increase Radiation-Enhanced Reactivation (RER) at one wavelength (297 nm) in the far ultraviolet but not at others (240-289 nm). This same treatment induced RER in the near UV (302-370 nm) and the visible region (380-400 nm). An action spectrum for the photo-sensitized induction of this cellular parameter was obtained. This action spectrum is consistent with the absorption spectrum for 8-MOP and the theory that damage to DNA is, at least in part, responsible for Radiation-Enhanced Reactivation.     

An SV40-transformed xeroderma pigmentosum group D cell line: establishment, ultraviolet sensitivity, transfection efficiency and plasmid mutation induction

Fibroblasts from a patient with xeroderma pigmentosum complementation group D were treated with Simian virus 40 to establish a transformed cell line suitable for studies of DNA-mediated gene transfer. After progressing through 2 crises, a stable line, XP6Be(SV40), was established and cultured for more than 1 year. This line retains the characteristic xeroderma pigmentosum ultraviolet hypersensitivity and is able to complement a SV40-transformed group A line when fused and assayed for ultraviolet radiation inhibition of colony-forming ability. XP6Be(SV40) expressed high levels of transfected chloramphenicol acetyltransferase activity (0.1 nmole X mg-1 X min-1) in a transient expression assay, showed stable expression of transfected gpt or neo genes (frequency 1-20 X 10(-5)), and permitted replication of the mutagenesis shuttle vector plasmid, pZ189. Ultraviolet treatment (500 J X m-2) of pZ189 prior to replication in XP6Be(SV40) resulted in a large reduction in plasmid yield (5% survival) and a 60-fold increase in the mutation frequency, reflecting the reduced ability of these cells to repair ultraviolet-damaged transfecting DNA. This cell line provides the opportunity to utilize transfection studies in cells with the xeroderma pigmentosum group D defect in excision repair.     

Rescue of Simian Virus 40 from Cell Lines Transformed at High and at Low Input Multiplicities by Unirradiated or Ultraviolet-irradiated Virus

The relation between simian virus 40 (SV40) input multiplicity during transformation of primary mouse kidney cultures and the subsequent rescue of SV40 from clonal lines of transformed cells has been studied. Primary mouse kidney cultures were transformed with unirradiated SV40 at input multiplicities varying from 0.06 to 200 plaque-forming units (PFU) /cell or with SV40 irradiated with ultraviolet (UV) light to a survival of 0.04 to 0.01. All of the transformed lines contained the intranuclear SV40 T antigen, but cell-free extracts prepared from the transformed cell lines failed to yield infectious virus when assayed on monkey kidney cell (CV-1) monolayers. After fusion with susceptible CV-1 cells induced by UV-inactivated Sendai, all of the lines transformed by unirradiated virus yielded infectious SV40. The frequency of induction and the incidence of successful trials did not depend on the multiplicity of infection. “Good” yielders were obtained from mouse kidney cells transformed at the low input multiplicity of 0.06 PFU /cell. In contrast, only 4 of 12 clonal lines transformed at moderately low input multiplicity, and none of the lines transformed at very low input multiplicity with UV-irradiated virus yielded infectious SV40. The four positive lines have been classified as “poor” or “rare” yielders.     

Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication

The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.     

Reactivation of herpes simplex virus in a cell line inducible for simian virus 40 synthesis

The reactivation of UV-irradiated herpes simplex virus (HSV) was investigated in irradiated and unirradiated transformed hamster cells in which infectious simian virus 40 (SV40) can be induced. Reactivation was enhanced when the cells were treated with UV light or mitomycin C prior to infection with HSV. The IV dose-response curve of this enhanced reactivation was strikingly similar to that found for induction of SV40 virus synthesis in cells treated under identical condictions. This is the first time that two SOS functions described in bacteria have been demonstrated in a single mammalian cell line.     

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.     

DNase I sensitivity of integrated simian virus 40 DNA.

We undertook an analysis of integrated simian virus 40 (SV40) DNA to learn whether the DNase I-sensitive region is retained in the integrated array of mouse transformants. Our results indicate that full-length integrated SV40 chromatin retains a DNase I-hypersensitive region at the same point as in nonintegrated SV40 chromatin. Thus, the lack of a DNase I-hypersensitive region is not likely to be the reason for nonpermissivity of SV40 in mouse cells. In addition, results reported here indicate that a deletion of about 200 base pairs of DNA in the region of the DNase I-hypersensitive site severely reduces the sensitivity of integrated SV40 chromatin. This result is similar to a previously reported result obtained with deletion mutants of SV40 analyzed in the lytic cycle. It is the first report of a DNA lesion affecting DNase I hypersensitivity of a mammalian chromosome.     

Analysis of single-stranded DNA stability and damage-induced strand loss in mammalian cells using SV40-based shuttle vectors

The fate and stability of fully or partially single-stranded DNA molecules transfected into mammalian cells have been analysed. For this, we constructed a simian virus 40 (SV40)-based shuttle vector containing the f1 bacteriophage replication origin in the two possible orientations (pi SVF1-A and pi SVF1-B). This vector contains the SV40 origin of replication, the late viral genes and DNA sequences for replication and selection in Escherichia coli. It also carries the lacO sequence, which permits the analysis of plasmid stability. Single-stranded DNA from pi SVF1-A and pi SVF1-B were produced in bacteria and annealed in vitro to form a heteroduplex molecule. We showed that, in monkey kidney COS7 cells, single-stranded vectors replicate to form duplex molecules. After transfection of the three forms of molecules (single-stranded, heteroduplex or double-stranded), replicated DNA was rescued in E. coli. Vector stability was analysed by checking for plasmid rearrangements and screening for lacO mutants. The single-stranded pi SVF1 has a lower rearrangement level, while the spontaneous mutation frequency (on the lacO target) is in the same range as for the double-stranded vector. In contrast, the level of spontaneous mutagenesis is higher for the heteroduplex than for the single- and double-stranded forms. In addition, we found that replication of heteroduplex with one strand containing ultraviolet light-induced lesions yields progeny molecules in which the irradiated strand is mostly lost. This result indicates for the first time the specific loss of the damaged strand in mammalian cells.     

Regulation of cellular and SV40 virus origins of replication by Chk1-dependent intrinsic and UVC radiation-induced checkpoints

DNA replication is inhibited by DNA damage through cis effects on replication fork progression and trans effects associated with checkpoints. In this study, we employed a combined pulse labeling and neutral-neutral two-dimensional gel-based approach to compare the effects of a DNA damaging agent frequently employed to invoke checkpoints, UVC radiation, on the replication of cellular and simian virus 40 (SV40) chromosomes in intact cells. UVC radiation induced similar inhibitory effects on the initiation and elongation phases of cellular and SV40 DNA replication. The initiation-inhibitory effects occurred independently of p53 and were abrogated by the ATM and ATR kinase inhibitor caffeine, or the Chk1 kinase inhibitor UCN-01. Inhibition of cellular origins was also abrogated by the expression of a dominant-negative Chk1 mutant. These results indicate that UVC induces a Chk1- and ATR or ATM-dependent checkpoint that targets both cellular and SV40 viral replication origins. Loss of Chk1 and ATR or ATM function also stimulated initiation of cellular and viral DNA replication in the absence of UVC radiation, revealing the existence of a novel intrinsic checkpoint that targets both cellular and SV40 viral origins of replication in the absence of DNA damage or stalled DNA replication forks. This checkpoint inhibits the replication in early S phase cells of a region of the repetitive rDNA locus that replicates in late S phase. The ability to detect these checkpoints using the well characterized SV40 model system should facilitate analysis of the molecular basis for these effects.     

Properties of Simian Virus 40 Rescued from Cell Lines Transformed by Ultraviolet-Irradiated Simian Virus 40

Simian virus 40 (SV40) strains have been rescued from various clonal lines of mouse kidney cells that had been transformed by ultraviolet (UV)-irradiated SV40. To learn whether some of the rescued SV40 strains were mutants, monkey kidney (CV-1) cells were infected with the rescued virus strains at 37 C and at 41 C. The SV40 strains studied included strains rescued from transformed cell lines classified as "good," "average," "poor," and "rare" yielders on the basis of total virus yield, frequency of induction, and incidence of successful rescue trials. Four small plaque mutants isolated from "poor" yielder lines and fuzzy and small plaque strains isolated from an "average" and a "good" yielder line, respectively, were among the SV40 strains tested. Virus strains rescued from all classes of transformed cells were capable of inducing the transplantation antigen, and they induced the intranuclear SV40-T-antigen, thymidine kinase, deoxyribonucleic acid (DNA) polymerase, and cellular DNA synthesis at 37 C and at 41 C. With the exception of four small plaque strains rescued from "poor" yielders, the rescued SV40 strains replicated their DNA and formed infectious virus with kinetics similar to parental SV40 at either 37 or 41 C. The four exceptional strains did replicate at 37 C, but replication was very poor at 41 C. Thus, only a few of the rescued virus strains exhibited defective SV40 functions in CV-1 cells. All of the virus strains rescued from the "rare" yielder lines were similar to parental SV40. Several hypotheses consistent with the properties of the rescued virus strains are discussed, which may account for the significant variations in virus yield and frequency of induction of the transformed cell lines.     

Selective gene amplification in mammalian cells after exposure to 60Co gamma rays, 241Am alpha particles, or uv light

Simian Virus 40 wild type (SV40)-transformed Chinese hamster embryo cells (Co631) contain about five viral copies integrated per cell genome. These SV40 sequences were used as endogenous indicator genes to study the response of mammalian cells to radiation at the gene level. An increase in copy number was detected by dispersed cell blotting and Southern analysis in combination with specific DNA hybridization. All types of radiation tested induce a 15- to 25-fold amplification of SV40 sequences without producing intact virus. The amplification is dose dependent and increases with time after irradiation: a maximum effect is observed at Day 3 after alpha particle or uv exposure and at Day 6 after gamma-ray exposure. A RBE of 6 can be calculated for alpha particles if amplification rates at Day 3 are compared. However, when the maximum effect is considered independent of time, no difference between different types of radiation is observed. Southern blots of genomic DNA show that not all integrated SV40 sequences are amplified upon radiation. Amplified sequences are found either in restriction fragments of relatively high molecular weight or in unit size fragments. SV40 amplification is selective in that the amplification of other genes, e.g., of alpha-actin, dhfr (dihydrofolate reductase), and of two oncogenes of the ras family (Kirsten ras and Harvey ras), was below detection level.