A hypodiploid karyotype found in immortal human cells selected from a wide spectrum of posttransformation chromosomal complements

A simian virus 40 (SV40) DNA fragment, encompassing the whole early region but having a defective origin of DNA replication, was previously used to transform human fibroblast cells derived from a patient suffering from xeroderma pigmentosum complementation group C (XP-C). Two independent SV40 transformants had acquired immortality in culture. Unlike most SV40-transformed human fibroblasts, the two established XP-C cell lines possessed an identical hypodiploid karyotype of 44,XX,-19,Xq+,-22,15p+. We now show that prior to immortalization the two SV40 transformants display a very wide spectrum of karyotypes with regard to chromosome number. A similar variety of chromosomal complements is present in four independent mortal SV40 transformants of the same parental XP-C cell line as well as in a mortal SV40-transformed xeroderma pigmentosum group D cell line. The rarity of the immortalization event, coupled with the coincident occurrence of identical karyotypes in the two immortal cell lines, suggests that the immortal lines arose through selection of a peculiar karyotype from among those of the parent SV40-transformed fibroblasts, and that this peculiar hypodiploid karyotype may be related to, and perhaps even necessary for, the establishment of immortality.     

Identification and biochemical analysis of DNA replication-defective large T antigens from SV40-transformed cells

Nine commonly studied Simian virus 40 (SV40)-transformed rodent cell lines were screened for tumor (T) antigens defective in SV40 DNA replication using a simple polyethylene glycol-mediated cell fusion assay. Each line contained a functional origin of SV40 DNA replication, as shown by fusion with Cos 1 cells. Fusion with uninfected monkey cells revealed that T antigens from two lines lacked detectable replicative activity, while T antigens from five other lines exhibited only very weak replicative activity. One line, and a tumor cell line derived from it, expressed T antigen with wild-type replication activity. Biochemical analysis of these proteins revealed defects in DNA binding activity and ATPase activity. One line expressed large T antigen defective in both activities. All of the lines contained complexes of T antigen with the cellular protein p53 and all of the T antigens exhibited nucleotide-binding activity. The results indicate that some of these lines may constitute a useful source of new replication-defective T antigens.     

Host cell DNA synthesis as a possible factor in the enhancement of replication of human adenoviruses in simian cells by SV40

Temperature-sensitive mutants of SV40, ts-701 and ts-702, have been isolated after treatment of wild-type virus with nitrous acid and nitrosoguanidine, respectively. Although neither mutant produces infectious virus at the nonpermissive temperature, both can induce SV40 T and V antigens, virus DNA synthesis, and the stimulation of host cell DNA synthesis. Both are late mutants and appear to be in the same SV40 complementation group. Both mutants can also complement the replication of human adenovirus type 7 at the elevated temperature, as can another late mutant, pm-301, and an early mutant, tsA7. Therefore, the adenovirus complementation step appears to be prior to SV40 virus DNA synthesis. Since a BSC-1 clone in which SV40 infection stimulates host cell DNA synthesis allows the complementation of adenovirus type 7 by SV40 while parental BSC-1 cells neither support complementation of adenovirus type 7 by SV40 nor are stimulated to replicate their DNA after SV40 infection, the stimulation of host cell DNA synthesis appears to be critical to adenovirus enhancement. This stimulation appears to be specific since stimulation of DNA synthesis after nutrient deprivation does not induce the replication of human adenoviruses in simian cells.     

Caffeine toxicity is inversely related to DNA repair in simian virus 40-transformed xeroderma pigmentosum cells irradiated with ultraviolet light

Human cells transformed by simian virus 40 (SV40) are more sensitive to killing by ultraviolet light when grown in caffeine after irradiation. The degree of sensitization at 2 mM caffeine (expressed as the ratio of the 37% survival dose for control cells divided by the 37% survival dose for cells grown in caffeine, i.e., the dose modification factor) was approximately 1.9 in transformed normal cells and 3.8-5.8 in excision-defective xeroderma pigmentosum (XP) groups A, C, and D cells. A large dose modification factor of 12 was observed in a transformed XP variant cell line. Chinese hamster ovary cells were not significantly different from transformed normal human cells, with a maximum dose modification factor of 1.5. Two radioresistant XP revertants that do not excise cyclobutane dimers gave different responses; one resembled its group A parent in being sensitized by caffeine, and one did not. These results can be interpreted on the basis of a single hypothesis that cells are killed as a result of attempts to replicate damaged DNA. Increased replication rates caused by transformation, increased numbers of replication forks in DNA caused by caffeine, and increased numbers of damaged sites ahead of replication forks in excision-defective cells are all processes that will consequently increase killing according to this hypothesis. A corollary is that the XP variant may be highly sensitized to caffeine because of excision defects at the DNA replication forks, an idea that may be important in designing cloning strategies for the XP variant gene.     

Genetic expression of human adenoviruses in simian cells. Evidence for interserotypic inhibition of viral DNA synthesis

Most simian cells are permissive for SV40 and adenovirus-SV40 hybrids but nonpermissive for human adenoviruses, and the defect has been shown to take place at the level of processing of late viral mRNAs (Klessig and Grodzicker, 1979). Viral DNA synthesis and virus progeny production were studied in simian cells infected with different adenovirus serotypes. Adenoviruses belonging to oncogenic subgroups A and B (Ad31 and Ad3) failed to replicate their DNA in CV1 cells, whereas DNA replication occurred for all the other serotypes. Co-infection of CV1 cells with SV40 and Ad3 (or Ad31) resulted in the inhibition of SV40 DNA synthesis, as well as cellular DNA synthesis. The inhibition was not related to adenovirus DNA replication, since SV40 did not complement the Ad3/Ad31 replication defective function. Similar results were obtained in coinfected BSC and MK2 simian cell lines. Inhibition of Ad2ND1 DNA synthesis and gene expression also occurred in co-infection of simian cells with nondefective Ad2ND1 hybrid and defective Ad3/Ad31. In permissive human cell lines (HeLa or KB) co-infected with Ad2 and Ad3 (or Ad31), a dominant, inhibitory effect of Ad3 (or Ad31) over Ad2 was also observed. The inhibition appeared to function stoichiometrically and not catalytically, and to involve early adenovirus gene products. In both simian and human cells a hierarchy of dominance appeared between serotypes belonging to different subgroups. The degree of inhibitory effect occurred in the following decreasing order: Ad3 and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad31 (A), Ad2 and Ad5 (C).     

中国人着色性干皮病遗传互补组分析

目的　建立中国人着色性干皮病（ｘｅｒｏｄｅｒｍａ ｐｉｇ ｍ ｅｎｔｏｓｕ ｍ ， Ｘ Ｐ） 患者成纤维细胞株，分析其遗传互补组及分布。方法　对着色性干皮病患者成纤维细胞进行传代培养、建株，采用放射自显影和细胞融合技术分析 Ｘ Ｐ 细胞互补组。结果　４ 株 Ｘ Ｐ 细胞中，３ 株属 Ｃ 组，１ 株属 Ｅ 组， Ｅ 组 Ｘ Ｐ 细胞在中国人群中是第１ 次发现。结论　综合已往报道的中国人 Ｘ Ｐ 细胞互补组７ 例和本研究４ 例，共计１１ 例，其中 Ｃ 组９例， Ｆ 组１ 例， Ｅ 组１ 例。说明中国人 Ｘ Ｐ Ｃ 组居多。而日本人 Ｘ Ｐ 细胞分布以 Ｘ Ｐ Ａ 组居多， Ｘ Ｐ Ｃ 组极少。     

Simian virus 40-Chinese hamster kidney cell interaction II. The semipermissivity of the cell system

Summary Throughoutin vitro passages, Chinese hamster kidney (CHK) cells progressively lost susceptibility to SV 40 virus infection while remaining continuously susceptible to viral DNA infection. Upon infection with SV 40 virus or viral DNA, the CHK cell line supported viral DNA and virus replication at a low level. SV 40-transformed CHK cell lines spontaneously produced small amounts of viral DNA and virions. The percentage of virus-producing cells was low. Various clones derived from each of these lines behaved as the parental cell population, leading to the conclusion that each CHK cell, whether transformed or not with SV 40, is potentially permissive for this virus.With 4 Figures     

Immortalization of four new fanconi anemia fibroblast cell lines by an improved procedure

Fanconi anemia (FA) is an autosomal recessive disease characterized by birth defects, progressive bone marrow failure and increased risk for leukemia. FA cells display chromosome breakage and increased cell killing in response to DNA crosslinking agents. At least 5 genes have been defined by cell complementation studies, but only one of these, FAC has been cloned to date. Efforts to map and isolate new FA genes by functional complementation have been hapered by the lack of immortalized FA fibroblast cell lines. Here we report the use of a novel immortalization strategy to create 4 new immortalized FA fibroblast lines, including one from the rare complementation group D.     

Genome maps of simian virus 40 defectives propagated in human glioblastoma cells.

The DNA sequences present in four defective SV40 genomes propagated on human glioblastoma cells (A172) have been characterized by analysis of restriction enzyme digests and by hybridization to the wild-type genome. Like defectives grown on monkey cells, these molecules are 10 to 20% shorter than wild-type DNA and contain reiterations of specific segments of the nondefective genome, including multiple copies of the viral replication origin. However, unlike the monkey cell defectives, those grown on A172 cells have also retained multiple copies of the region of viral DNA around the BamHl site at map position 0.15. These defectives consist only of sequences derived from SV40 and contain no detectable host cell sequences.     

Analysis of adenovirus 12 temperature-sensitive mutants defective in viral DNA replication

Ten temperature-sensitive mutants of adenovirus 12 defective in viral DNA replication at the nonpermissive temperature (40°) were selected and seven of them were classified into three groups (A, B, and C) by complementation tests in viral DNA synthesis. The mutants in Groups A, B, and C were defective in initiation of viral DNA synthesis at 40°, as revealed by viral DNA chain elongation or ligation and density label in temperature shift-up experiments. Analysis by the M band technique indicated that the mutants in Groups B and C were less defective than the mutant in Group A in formation of viral DNA replication complex at the nonpermissive temperature. Additional differences among mutants in Groups A, B, and C were also shown by temperature shift-down experiments. All these mutants were not defective in formation of T antigen in permissive cells and in induction of cellular DNA synthesis in nonpermissive cells at the nonpermissive temperature. These observations suggest that at least three viral genes are involved at different steps in initiation of viral DNA replication.     

中国人着色性干皮病跗互补组分析

目的 建立中国人着色性干皮病（ｘｅｒｏｄｅｒｍａ ｐｉｇｍｅｎｔｏｓｕｍ，ＸＰ）患者成纤维细胞株，分析其遗传互补组及分布。方法 对着色性干皮病患者成纤维细胞进行传代培养、建株，采用放射自显影和细胞融合技术分析ＸＰ细胞互补组。结果 ４株ＸＰ细胞中，３株属Ｃ组，１株属Ｅ组，Ｅ组ＸＰ细胞在中国人群中是第１次发现。结论 综合已往报道的中国人ＸＰ细胞互补组７例和本研究４例，共计１１例，其中Ｃ组９例，Ｆ组１例     

Events preceding stable integration of SV40 genomes in a human cell line

We have examined the organization of integrated SV40 sequences in an uncloned population of a transformed human fibroblast cell line. Somatic cell hybrids between mouse B82 cells and human GM847 cells were examined for SV40 T-antigen expression and individual human chromosome presence. This analysis revealed that a functional SV40 genome is located on human chromosome 7. Restriction endonuclease digestion followed by blot hybridization of the parental human cell line revealed that it contains mutliple normal and defective SV40 copies integrated into the host genome in tandem. A similar analysis of several T-ag⁺ hybrid cell lines indicated that the integrated viral sequences in different hybrid cell lines (thus in different cells of the original population) are very closely related but not always identical. Analysis of subclones of GM847 also revealed such differences. Based upon these results, we postulate that following the initial integration event, viral as well as the flanking host DNA sequences become unstable and are subject to deletions and rearrangements. This short-lived structural instability is followed by highly stable integration of SV40 which is maintained in these cells or their hybrid derivatives for at least hundreds of cell generations.     

Complementation group assignments in fanconi anemia fibroblast cell lines from North America

Fanconi anemia is a rare autosomal recessive disease characterized by developmental defects of the thumb and radius, childhood onset of pancytopenic anemia and increased risk of leukemia. At least five complementation groups (A-E) have been defined but only theFAC gene has been cloned. Cells can be assigned to complementation group C by direct mutation analysis. To facilitate the search for additional FA genes and to measure the frequency of complementation groups, we have established new genetically marked immortalized FA-A and FA-D fibroblast cell lines and show their usefulness as universal fusion donors. These reference FA cell lines facilitated somatic cell fusion analysis and enabeled us to assign the complementation group in 16 unrelated FA patients from North America. The majority of patients, belong to FA complementation group A (69%), followed by FA-C (18%), FA-D (4%) and FA-B or FA-E (9%).     

Characterization of SV40-transformed xeroderma pigmentosum cell lines for their usability in HPRT mutation studies

We have characterized six SV40-transformed xeroderma pigmentosumcell lines (XP2OS and XP12BE derived from female donors, XP12RO-SV,XP3BR/12SV, XP4PA-SV and XP8CAC-SV from male donors) for theirusability in HPRT mutation studies. All cell lines exhibit hypersensitivity,compared with MRC5CV1 cells, towards the cytotoxic action ofUV-irradiation. They were all shown to be heteronuclear andhyperdiploid with pronounced variability in chromosome number.Fluorescence in situ hybridization (FISH) with an X-chromosomallibrary (X-chromosome painting) and BrdUrd-labelling of late-replicatingX-chromosomes demonstrated the presence of variable numbersof X-chromosomes and additional X-chromosomal material and suggestedthe presence of more than one genetically active HPRT allelein the majority of cells of five cell lines. The cell line XP8CAC-SV(complementation group C) seemed to be most suitable for HPRTmutation studies due to its near-diploid karyotype with onlyone X-chromosome in the majority of cells. From this cell line,a clonal subline was established (XP8CAC-SV-C1) which revealedthe same UV-hypersensitivity as the parental cell line and aneardiploid karyotype with one X-chromosome in 94% of the metaphases.Molecular analysis of the HPRT gene gave a normal PCR amplificationpattern for all exons and the normal wild-type sequence of thecDNA. HPRT tests with (+)-anti-benzo[a]pyrene-7,8-diol-9,10-oxide[(+)-anti-BPDE] showed a reproducible, concentration relatedincrease in mutant frequencies. Compared with results with MRC5CV1cells, the obtained data indicate spontaneous and (+)-anti-BPDE-inducedhypermutability of the XP line. XP8CAC-SV-C1 thus representsa permanent XP cell line with characteristic cellular XP featureswhich is convenient for studying the influence of deficientexcision repair on HPRT mutant frequencies and mutation spectra. ¹To whom correspondence should be addressed     

Adeno-associated viruses inhibit SV40 DNA amplification and replication of herpes simplex virus in SV40-transformed hamster cells

Coinfection with HSV-1 and any of the three defective parvoviruses AAV-2, AAV-3, or AAV-5 reduces the HSV-1-induced amplification of SV40 DNA in the SV40-transformed hamster cell lines CO631 and Elona. Moreover, all three viruses significantly decrease HSV-1 replication. The effects are measurable to the same extent in both cell lines and are dependent on the quantity of AAV DNA molecules rather than on infectious AAV particles. It seems unlikely that the inhibitions are due to simple competition but result from complex interactions that involve the terminal sequences of AAV DNA.     

Effect of mitomycin C and 60Co gamma-irradiation on the replication of SV40 in cell lines of varying permissivity for SV40 replication.

The effects of mitomycin C and 60Co gamma-irradiation, which induce production of SV40 from SV40-transformed hamster cells, on the replication of superinfecting SV40 or virus DNA in cells varying in permissivity for SV40 replication have been examined. These agents enhance replication of SV40 in an uninducible line of SV40-transformed hamster kidney cells and in nonpermissive secondary hamster kidney cells. The same treatments do not affect SV40 replication in semipermissive hamster (BHK21) and human (HEL, HEK) cells and inhibit SV40 replication in permissive monkey (TC-7) cells. We conclude that forms of induction treatment, such as mitomycin C or 60Co gamma-irradiation, modify the expression of host cell factors which determine the level of permissivity for SV40 infection.     

Maintenance of episomal SV40 genomes in GM637 human fibroblasts.

Episomal SV40 (SV40: simian virus 40, Polyomavirus maccacae) has been reported in SV40-transformed human fibroblast cell lines the integrated SV40 sequences of which are unlikely to give rise to episomal copies by recombinational mechanisms. The levels of episomal viral DNA in these lines are high, being easily visualized by ethidium staining of agarose gels after electrophoresis. We find that the episomal mutant gmSV40 in GM637 cells represents a persistent lytic infection that can be cured by treatment with neutralizing antibody, leaving only the chromosomally integrated viral genomes. The finding that maintenance of the gmSV40 in GM637 cells is due to persistent infection raises a note of caution for SV40-transformed lines with episomal SV40 genomes because these lines often are used in studies of DNA replication and repair. An infective center assay that does not depend on plaque formation shows that gmSV40 is a host range mutant, with poor infectivity for CV-1 monkey kidney cells and greatly increased infectivity for human cells. Passage of gmSV40 through monkey kidney cells selects for variants with greatly increased infectivity for monkey cells and, independently, for cytopathic variants that produce plaques. Thus plaque assays can give very unreliable infective center values in studies of host range mutants.