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.     

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.     

The susceptibility of Werner's syndrome and other human skin fibroblasts to SV40-induced transformation and immortalization

In attempts to transform and immortalize human cell cultures, skin fibroblasts from normal donors of different ages, from patients with the premature ageing diseases Werner's syndrome (WS) and progeria (PR), and from donors with the cancer-prone diseases ataxia telangiectasia (AT), Bloom's syndrome (BS) and Fanconi's anaemia (FA), were infected with SV40 virus and their growth monitored thereafter. Lesch-Nyhan (LN) fibroblasts were also infected. SV40-infected cultures from two normal and from WS, AT and LN donors attained a spectrum of transformed properties, high mitotic activity at confluence, presence of T-antigen, anchorage independence and altered morphology. Most of these pretransformed cultures died in the crisis period. However, two cultures from the WS and LN patients survived the crisis period and have now been grown to more than 200 passages. For the LN culture the crisis period was at least 200 days. Both permanent lines retain the properties of pretransformed cells, but differ in their modal chromosome number and ability to grow in methionine-free medium. It can be concluded from these experiments that transformation by SV40 to permanent lines is a rare event in human skin fibroblasts, even when these cells were taken from patients predisposed to form cancers.     

p53 and transformation by SV40

The large T antigen of SV40 is able to immortalize and transform primary and established cells in culture, and can, at least in certain cases, confer a tumorigenic phenotype on the infected cell. T antigen has been shown to induce cellular DNA synthesis in the infected cell and this activity is likely to be instrumental in T antigen mediated oncogenesis. A property of T antigen which may be of paramount importance to its oncogenic and mitogenic activities is its ability to specifically bind and stabilize the cellular protein p53. p53 has been implicated in the control of the passage of the cell from G0 arrest to G1 and S phase. Furthermore, altered p53 expression is strongly associated with various phenotypes of the transformed state, and p53 has been identified as an immortalizing oncogene. Thus it is possible that p53-fixation by T antigen is responsible for its transforming potential. In this article, the transforming activities of T antigen and p53 are reviewed, and the possible relevance of p53-binding to T antigen-induced transformation is discussed.     

Heterogeneity in distribution and content of p53 in SV40-transformed mouse fibroblasts

The high level and intranuclear location of the cellular phosphoprotein p53 are usually regarded as invariant features of SV40-transformed fibroblast lines. During the development of improved methods for immunocytochemical detection of p53 using SVA31 E7 mouse fibroblasts, we have observed unexpected and marked variations in its distribution. Cells grown on plastic coverslips were fixed in acetone and the content and distribution of p53 and SV40 large T-antigen analysed by an indirect immunoperoxidase procedure using monoclonal antibodies PAb122 and PAb 248 for p53, and PAb416 for large T. First, we observed in all cultures an apparent reversal of intracellular compartmentalization, with strong cytoplasmic and absent nuclear/chromatin positivity in mitotic cells and young daughter cells. More importantly, for a short period, between 18-24 h after trypsinization and cell passage, we observed a marked overall reduction in detectable nuclear p53 content in all cells with both PAb122 and PAb248 antibodies. The first observation also held for SV40 large T-antigen, the second only for p53. These variations have important practical implications for the immunocytochemical analysis of cellular content and intracellular compartmentalization of p53. The biological implications of our findings are also discussed.     

Random integration of SV40 in SV40-transformed, immortalized human fibroblasts

We have studied the relationship between immortalization of SV40-transformed human embryonic fibroblasts and their SV40 integration sites. From several independently transformed cell pools, we have isolated clones which do not harbor unintegrated SV40 DNA. We have analysed whole-cell DNA from these clones, using the Southern blot method. Our results suggest that no specific integration sites in the cellular genome exist which are a prerequisite for the immortalization process. Although some integration sites were found to be predominant in pre-crisis clones, they could not be detected in the post-crisis clones. This suggests that none of these predominating sites is selected for during the crisis period.     

SV40-immortalized human fibroblasts as a source of SV40 infectious virions

Human fibroblasts immortalized by Simian Virus 40 (SV40) are widely employed for cell and molecular biology model of study. Indeed, SV40 transmission to humans was believed to occur only under exceptional situations. The oncogenic potential of SV40 in laboratory animals is well established, whereas its involvement in human carcinogenesis is still a matter of active investigations. A recent report links SV40 exposure with the development of a brain tumor in a laboratory researcher. In previous studies, episomal viral DNA was detected in SV40 stably transformed and immortalized fibroblast cell lines. In this study, we report molecular and biological characterizations of SV40 DNA in human fibroblast cells. Our results indicate that SV40 is able to establish a persistent infection in long-term immortalized human fibroblasts, resulting in the production of an infectious viral progeny, which is able to infect both monkey and human cells. These data indicate that SV40-immortalized human fibroblasts may represent a source of SV40 infection. To avoid the SV40 infection, careful attention should be given by operators to this SV40-cell model of study.     

The immortalization of thymic nurse cells by SV40 virus

Thymic nurse cells (TNCs) are stromal elements that contain between 20 and 200 T cells within their cytoplasm. Because of this unique feature they are believed to play a role in thymocyte development. Unfortunately, it has been difficult to obtain pure TNCs in quantities sufficient for extensive evaluation of their thymic function. As a result, only a limited amount of information is available that characterizes TNCs or the T cell population(s) found within their cytoplasm. We have now used SV40 to infect and immortalize TNCs from C57BL/6 mice. SV40-transformed TNCs were found to specifically bind and internalize cells from an immature thymocyte line isolated in our laboratory. These results describe a method of obtaining pure populations of TNCs for future studies of their thymic function, and suggest that binding to specific subpopulations of lymphoblasts may be necessary for internalization.     

Efficient immortalization and morphological transformation of human fibroblasts by transfection with SV40 DNA linked to a dominant marker

Immortal cell lines are essential for genetic and biochemical studies. Unlike rodent cells, which will form continuously growing cultures either spontaneously or after infection with an oncogenic virus (e.g., Simian Virus 40 (SV40)), human cells fail to form continuous cell lines spontaneously and in only rare cases from cell lines after oncogenic virus infection. We have used a plasmid (pSV3gpt) containing both the SV40 early region encoding T antigen and the bacterial gene xanthine-guanine phosphoribosyl transferase (gpt) to achieve high efficiency morphological transformation and immortalization of primary human skin fibroblasts. Transfection of this plasmid into primary human skin fibroblasts derived from a normal individual, two Cockayne's syndrome patients, and an immuno-deficient patient and selection for the gpt gene resulted in an altered cell morphology and growth properties characteristic of previously described SV40-transformed cells. Transfected cultures subsequently senesced, entered crisis and in each case formed a rapidly growing culture. The high efficiency of immunortalization described here (four out of four cell strains) is in contrast to previously described procedures utilizing focal overgrowth. We suggest that the use of a dominant selectable marker linked to the SV40 early region increases the probability of establishing an immortal human cell line.     

The introduction of dominant-negative p53 mutants suppresses temperature shift-induced senescence in immortal human fibroblasts expressing a thermolabile SV40 large T antigen

Immortal human fibroblasts, SVts8 cells, which express a heat-labile SV40 large T antigen, induces a senescence-like phenomenon in response to upward shift in temperature. Cells with arrested division show strong induction of senescence-associated beta-galactosidase. We examined how p53 and pRB are involved in this phenomenon since they are major targets of the T antigen. Transfection of cells with plasmids encoding the wild-type T antigen or human papilloma virus type 16 E6/E7 proteins completely abolished the arrest in cell division, a plasmid encoding the E6 protein suppressed it markedly, while a plasmid encoding E7 had no effect. Plasmids encoding dominant-negative p53 mutants also suppressed the arrest in cell division to various degrees. Upon temperature shift, p21 mRNA was upregulated 10-fold in SVts8 cells, but only slightly in clones expressing the wild-type T antigen or dominant-negative p53 mutants. These data demonstrate that p53 plays a major role in this senescence-like phenomenon.     

Primate's p53 inhibits SV40 DNA replication in vitro

Previous reports indicated that rodent p53 inhibits simian virus 40 (SV40) DNA replication in vitro as well as in vivo while that from primate cells does not (1-4). Here we report the evidence that p53 of primate origin also inhibits SV40 DNA replication in vitro. p53-SV40 large tumor antigen (T antigen) complex purified from SV40 infected COS-1 cells had little replication activity and inhibited SV40 DNA replication in vitro. These results suggest that inhibition of SV40 DNA replication by p53 should be regarded as general property of the protein and does not determine the mode of species specific replication of SV40 DNA.     

Changes in HL-A antigen profiles on SV40-transformed human fibroblasts

The histocompatibility antigen profile of human fibroblasts transformed with SV40 virus has been investigated by determining their ability to specifically absorb HL-A alloantisera. Four out of four human adult skin fibroblast lines acquired, after SV40 transformation, the ability to absorb the HL-A alloantiserum VICTOR directed against the specificities HL-A5, W5. On the contrary, none of six embryonic fibroblast lines showed any qualitative or quantitative change of their HL-A antigenic profile. Similarly, murine, monkey and hamster cells could not absorb the activity of the HL-A alloantiserum VICTOR after SV40 transformation. It is suggested that the ‘new HL-A antigen’ specificity on human fibroblasts after SV40 transformation may be due to either a cross-reaction between HL-A specificities and antigenic structures present on glycoprotein(s) coded by the viral genome and expressed on the cell or to a change in the regulating mechanism of the expression of histocompatibility antigens on the cell surface, should the genetic information for all the HL-A specificities be present in the cell genome.     

SV40 T-antigen is required for maintenance of immortal growth in SV40-transformed human fibroblasts.

Two lines of immortal human fibroblasts were isolated following transfection of TIG-3 cells with plasmid DNA, pMT-1ODtsA, that contained SV40 early gene with a deletion in replication origin and ts mutation in coding sequence for T-antigen. These cells continued proliferation at 34 degrees C, over 565 population doubling level (PDL) which is far over the limited division potential of untransformed normal TIG-3 of 70-80 PDL. When the culture temperature was shifted to 40 degrees C after 70 PDL, they ceased proliferation immediately. One of these immortal clones, SVts8, lost its ts phenotype after retransformation with wtT-antigen gene. These results indicated that the function of intact T-antigen is required for maintenance of immortal proliferation, at least in one of the SV40 transformed immortal clones.     

SV40-transformed human fibroblasts exhibit a characteristic chromosomal pattern

A karyotype study of seven SV40-transformed human fibroblast cell lines was performed using R-banding. Although large variations existed from line to line and, to a lesser degree, from cell to cell in a given line, many common features were found. The most characteristic were chromosome imbalances. Some of the chromosomes or chromosome segments present in excess were, in decreasing order of frequency, the early replicating X, 12q, 3q, 12p, 19, 1p, and 6p. Losses of other chromosomes and chromosome segments were also frequent and involved 11p, 2p, 6q, 10p, 18, 4q, 8p, 4p, 10q, and 16. These imbalances seemed to correlate with metabolic characteristics, previously described, such as low activities of catalase (11p), superoxide dismutase 2 (6q), and acid phosphatase (2p, 11p) and a high ratio of lactate dehydrogenase B (LDHB, 12p) activity to LDHA (11p) activity.     

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.     

Analysis of genomic integrity and p53-dependent G1 checkpoint in telomerase-induced extended-life-span human fibroblasts

Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279-282, 1998; A. G. Bodnar et al., Science 279:349-352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21(Waf1/Cip1/Sdi1). TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.     

Synthesis of collagen by human fibroblasts and their SV40 transformants

Synthesis of collagen was studied in human fibroblasts (WI26, WI38) and their SV40 transformants. Viral transformation decreased the amount of collagen synthesized by a factor of 8 during a 24 h pulse and affected the rate of conversion of procollagen to collagen. No change was observed in the proportions of type I and type III collagen, the degree of hydroxylation of α-chains of the newly synthesized collagen remained the same. The collagen of viral transformants contained substantial amounts of collagen molecules which were composed of α1(I)-chains only. Immunofluorescence analysis using specific antibodies for type I collagen and fibronectin showed less deposition of extracellular fibrils in the transformed cell layers than in the normal cells.