Isolation and partial characterization of virus-transformed cell lines representing the A, G and variant complementation groups of xeroderma pigmentosum

We have established viral-transformed, apparently permanent (immortalized) cell lines from diploid fibroblasts representative of normal and xeroderma pigmentosum (XP) A, G and variant individuals. The XP-G and XP-variant cells represent complementation groups not previously available as permanent lines. All the new permanent cell lines exhibit SV40 T-antigen expression. They are also aneuploid and have growth characteristics typical of viral transformants. They have retained the phenotypes of UV sensitivity, reduced repair synthesis or defective 'postreplication repair' appropriate to the XP complementation group they represent. Additionally, the new cell lines are all transfectable with the selectable plasmid pRSVneo. The XP-G and XP-variant cell lines show enhanced transfection with UV-irradiated plasmid DNA; a phenomenon previously reported for normal immortalized cells and for immortalized cells from the A and F complementation groups of XP.     

A strategy for immortalizing lines committed to endoderm, neuroectoderm or mesoderm from mouse teratocarcinoma

With the aim of immortalizing embryonic cells fixed at early embryonic stages, various plasmids carrying the SV40 early region were introduced into the mouse embryonal carcinomas (EC) F9 and 1003. Only the construction PK4, in which the SV40 oncogenes are placed under the control of the adenovirus E1A promoter, led to the immortalization of the cells at the onset of differentiation. Clones corresponding to committed precursors of each embryonic lineage (neuroectoderm, mesoderm and endoderm) were then selected with high efficiency according to the following strategy: selection of immature cells which: have lost EC cell markers, keep a stable phenotype, are immortalized by the expression of the SV40 oncogenes and are still able to differentiate along a restricted lineage in vitro or in vivo. Examples of an endodermal precursor (H7) which differentiates into extraembryonic and embryonic endoderm, of a neuroectodermic clone (ICII) committed to a serotoninergic differentiation, and of a mesodermal osteogenic clone (CI) which gives rise to bone in vivo and in vitro, are given.     

Transformation of DNA repair-deficient human diploid fibroblasts with a simian virus 40 plasmid

Fibroblasts from patients with xeroderma pigmentosum (XP) complementation groups A, C, D, E, and G, as well as Bloom syndrome (BS) and Fanconi anemia (FA) have been transfected with a plasmid, pSV7, containing the early region of Simian virus 40 (SV40). All of the cultures exhibited cytologic changes characteristic of transformed cells and expressed T-antigen. They also contained integrated copies of DNA derived from the vector, and in several cases, extrachromosomally replicated DNA. Not all of the transfected cultures became immortalized. The transformed xeroderma pigmentosum (XP) cultures retained their UV-sensitive phenotype in all but one case. The BS and FA cell lines retained their characteristic phenotype. All of the cultures, except the BS cells, can be readily transfected with the plasmids, pSV2neo and pSV2gpt.     

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).     

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.     

Host-cell reactivation of ultraviolet-irradiated SV40 DNA in five complementation groups of xeroderma pigmentosum.

Host-cell reactivation of UV-irradiated double-stranded SV40 DNA was studied in BSC-1 monkey cells, normal human cells, heterozygous Xeroderma pigmentosum (XP) cells, representative cell strains of the five complemention groups of XP and in XP "variant" cells. The following percentages of survival of the plaque-forming ability of double-stranded SV40 DNA were found in XP cells compared with the value found in normal monkey and human cells: group A, 13%; group B, 30%; group C, 18%; group D, 14%; group E, 59%; and in the heterozygous XP cells almost 100%. The survival in XP "variant" cells was 66%. The survival of single-stranded SV40 DNA in BSC-1 cells was much lower than that of double-stranded SV40 DNA in XP cells of complementation group A, which possibly indicates that some repair of UV damage occurs even in XP cells of group A.     

Three complementation groups in Cockayne syndrome

After 16 Jm-2 of UV-irradiation non-dividing normal cells recover normal rates of RNA synthesis within 24 h, whereas in cells from donors with Cockayne syndrome (CS) the rate of RNA synthesis gradually declines. Cultures of a mixed population from 2 CS donors were fused with polyethylene glycol; subsequently they were UV-irradiated and RNA synthesis was measured autoradiographically in mono-, bi-, and multinuclear cells. Genetic complementation was indicated by high levels of RNA synthesis in bi- and multinuclear cells when compared with mononuclear cells. Using this assay, 11 CS strains have been assigned to three complementation groups: 2 into group A, 8 into group B and 1 into group C. The strain in group C is derived from an individual who also had xeroderma pigmentosum (XP), and was the sole known representative of XP-complementation group B.     

Enhanced induction of SV40 replication from transformed rat cells by fusion with UV-irradiated normal and repair-deficient human fibroblasts

Fusion of SV40-transformed rat (BRKSV) cells which do not spontaneously produce infectious virus, with permissive monkey cells resulted in a low level of production of infectious virus in the heterokaryons. UV-Irradiation of the BRKSV cells prior to fusion did not result in increased virus production, but irradiation of the monkey cells prior to fusion did result in enhanced induction (EI) of SV40, as compared to control experiments in which neither cell type was irradiated. This indicated that rat cells lack the ability to initiate replication of integrated SV40 upon UV-irradiation and do not contain "permissiveness" factors that are required to support SV40 replication. In contrast, monkey cells do contain such permissiveness factors which seem to be temporally enhanced by UV-irradiation, and thus may be responsible for the EI phenomenon. Expression of EI was dose-dependent and reached maximum values approximately 24 h after UV-irradiation. The kinetics of EI resembled that of EI previously established for SV40 induction in semi-permissive cells, and of enhanced reactivation (ER) and enhanced mutagenesis (EM) of SV40 in monkey cells. Similar kinetics of EI were obtained when human diploid fibroblasts were used for fusion with BRKSV cells. Similar levels of EI were found with normal human cells and repair-deficient xeroderma pigmentosum (XP) cells of complementation groups A and C, and XP variant cells. This suggests that expression of EI is not related to excision repair. Since EI is also normally expressed in XP cells which display an abnormal ER of HSV and in XP variant cells which show a delayed EM of HSV, we conclude that EI may occur independently of ER and EM. Finally it was shown that treatment of human cells with N-ethyl-N-nitrosourea results in similar induction of EI as irradiation with UV-light, and that addition of TPA in fusion experiments has no effect on EI.     

Assignment of SV40-Immortalized Cells to More Than One Complementation Group for Immortalization

Human cell lines have been assigned to four complementation groups for immortalization [O.M. Pereira-Smith and J. R. Smith, Proc. Natl. Acad. Sci. USA 85, 6042-6046, 1988]. Three SV40-immortalized epithelial cell lines were fused to cell lines representative of each of these four complementation groups. All three formed senescent hybrids with an SV40-immortalized cell line representative of group A, indicating that SV40 genes do not always cause immortalization via the same genetic mechanism. In contrast to the results of studies with other human cell lines, each of these three cell lines was assigned to more than one complementation group for immortalization. Thus these cell lines may have lost the function of two or more putative senescence genes.     

An immortalized xeroderma pigmentosum, group C, cell line which replicates SV40 shuttle vectors

We have established and characterized an immortalized xeroderma pigmentosum (XP), group C, cell line. Transformation of the human fibroblasts was carried out with a recombinant plasmid, pLAS-wt, containing SV40 DNA encompassing the entire early region with a defective origin of DNA replication. The transformed XP cell line, XP4PA-SVwt, and the normal transformed fibroblasts AS3-SVwt, both express SV40 T antigen together with enhanced levels of the transformation-associated cellular protein, p53. XP4PA-SVwt retains the XP UV-repair defective phenotype as demonstrated by low levels of unscheduled DNA synthesis and by the reduced survival of irradiated SV40 virus. Analysis of cellular DNA shows a single major, stable, integration site of pLAS-wt in the XP4PA-SVwt cells. The T antigen in these cells supports efficiently the replication of SV40 based shuttle vectors and should prove suitable for the introduction, expression and selection of genes related to DNA repair and to the study of mutagenesis using defined molecular probes.     

Untransformed xeroderma pigmentosum cells are not hypersensitive to sister-chromatid exchange production by ethyl methanesulphonate--implications for the use of transformed cell lines and for the mechanism by which SCE arise

A transformed cell line, XP12RO(SV40) has previously been found to be hypersensitive to several chemical mutagens including ethyl methanesulphonate, as judged by sister-chromatid exchange (SCE) formation. The hypersensitivity of this line has been confirmed for SCE formation and extended to cell survival. Measurements of SCE formation and survival show, however, that the hypersensitivity of the XP12RO(SV40) cell line is not typical of the primary strain (XP12RO) from which the transformed line was derived, nor it is typical of other primary strains also belonging to complementation group A (XP4LO, XP25RO). These results suggest that reports based on single cell lines must be viewed with caution and that the relationship between unexcised damage in DNA and SCE production is uncertain.     

Differential gene expression in SV40-mediated immortalization of human fibroblasts

Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A λcDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is redu ced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of J4-4 and J4-3 strongly suggest that they are involved in the process of immortalization and/or can serve as target genes for assessing regulators of gene expression in this process. J. Cell. Physiol. 171:325–335, 1997. © 1997 Wiley-Liss, Inc.     

Extension of lifespan of human T lymphocytes by transfection with SV40 large T antigen.

Lymphocytes have a finite and predictable proliferative life span in culture similar to that observed in fibroblasts. In general, the senescence of human fibroblasts is inevitable and irreversible, but their proliferative life span can be extended by certain DNA tumor virus oncogenes, such as the large T antigen of the SV40 virus. Here, we show that human T lymphocytes (HTL) can be stably transfected with SV40 large T and that expression of T antigen extended the life span of T cell cultures. PHA-stimulated HTL were transfected with pSV3neo, an expression vector containing the SV40 early region and the neomycin resistance gene. Transfectants were selected for neomycin (G418) resistance. Control HTL, either mock transfected or transfected with pSV2neo (containing the neomycin resistance gene only), ceased proliferation after about 17 population doublings. In contrast, HTL transfected with pSV3neo underwent more than 170 doublings. pSV3neo-transfected cells expressed SV40 large T RNA, detectable by in situ hybridization, and SV40 T antigen, detectable by immunofluorescence. Greater than 95% of the transfected cells were CD4 positive. These results clearly show that SV40 large T enables HTL to escape senescence. Transfection with SV40 large T may be a valuable method for obtaining long term human T cell lines for studies of both aging and immunology.     

Physical and Genetic Characterization of Deletion Mutants of Simian Virus 40 Constructed In Vitro

Mutants of simian virus 40 (SV40), with deletions ranging in size from fewer than 3 to 750 base pairs located throughout the SV40 genome, were obtained by infecting CV-1P cells with linear SV40 DNA and DNA of an appropriate helper virus. The linear DNA was obtained by complete cleavage of closed circular DNA with Hae II or Bam HI endonuclease or partial cleavage with either Hae III endonuclease or nuclease S1, followed, in some cases, by mild digestion with phage lambda 5' -exonuclease. The following mutants with deletions in the late region of the SV40 genome were obtained and characterized. Ten, containing deletions at the Hae II endonuclease site (map location 0.83), define a new genetic complementation group, E, grow extremely slowly without helper virus, and cause alterations only in VP2. Two mutants with deletions in the region 0.92 to 0.945 affect both VP2 and VP3, demonstrating that VP3 shares sequences with the C-terminal portion of VP2. The mutant with a deletion at 0.93 is the first deletion mutant in the D complementation group and is also temperature sensitive; the mutant with a deletion at 0.94 is viable and grows normally. Three mutants with deletions at the EcoRI endonuclease site (0/1.0) and eleven with deletions at the BamHI endonuclease site (0.15) fall into the B/C complementation group. Six additional mutants with deletions at the BamHI endonuclease site are viable, growing more slowly than wild type. VP1 is the only polypeptide affected by mutants in the B/C group. A mutant with a deletion of the region 0.72 to 0.80 has a polar effect, failing to express the E, D, and B/C genes. Mutants with deletions in the early region (0.67 counterclockwise to 0.17) at 0.66 to 0.59, 0.48, 0.47, 0.33, and 0.285 to 0.205 are all members of the A complementation group. Thus, the A gene is the only viral gene in the early region whose expression is necessary for productive infection of permissive cells. Since mutants with deletions in the region 0.59 to 0.54 are viable, two separate regions are essential for expression of the gene A function: 0.66 to 0.59 and 0.54 to 0.21. Mutants with deletions at 0.21 and 0.18 are viable. Approximate map locations of SV40 genes and possible models for their regulation are discussed.     

Retinoblastoma protein and simian virus 40-dependent immortalization of human fibroblasts.

Transformation and immortalization of human diploid fibroblasts by simian virus 40 (SV40) is at least a two-stage process, since transformants have a limited lifespan in culture. We have isolated immortalized derivatives (AR5 and HAL) from transformants generated with an origin-defective SV40 genome encoding a heat-labile large T protein (T antigen) and reported that both preimmortal and immortal transformants are continuously dependent on T antigen function for growth as determined by temperature shift experiments. In this study, we demonstrate complex formation between T antigen and the retinoblastoma susceptibility gene product (Rb) at 35 degrees C and observed a reduction in complexes under conditions of loss of T antigen function and growth inhibition at 39 degrees C. Viral oncogenes (polyomavirus large T protein and adenovirus E1A 12S protein) known to bind Rb were introduced into AR5 and HAL cells, both stably by gene transfer and transiently by virus vectors. Such double transformants are still unable to proliferate at 39 degrees C, although complex formation with the newly introduced oncogenes was demonstrated. We suggest that T antigen interacts with other cellular processes in addition to Rb to transform and immortalize human cells in culture. Our finding that p53-T antigen complexes are also temperature dependent in AR5 and HAL cells could provide such an additional mechanism.     

Localization of the xeroderma pigmentosum group B-correcting gene ERCC3 to human chromosome 2q21

The human excision-repair gene ERCC3 was cloned after DNA-mediated gene transfer to the uv-sensitive Chinese hamster ovary mutant cell line 27-1, a member of complementation group 3 of the excision-defective rodent cell lines. The ERCC3 gene specifically corrects the DNA repair defect of xeroderma pigmentosum (XP) complementation group B, which displays the clinical symptoms of XP as well as of another rare excision-repair disorder, Cockayne syndrome. The gene encodes a presumed DNA and chromatin binding helicase, involved in early steps of the excision-repair pathway. ERCC3 was previously assigned to human chromosome 2 (L.H. Thompson, A.V. Carrano, K. Sato, E.P. Salazar, B.F. White, S.A. Stewart, J.L. Minkler, and M.J. Siciliano (1987) Somat. Cell Genet. 13: 539-551). Here we report its subchromosomal localization in the q21 region of chromosome 2 via somatic cell hybrids containing a translocated chromosome 2 and in situ hybridization with fluorescently labeled ERCC3 probes.