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.     

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.     

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.     

Loss of collagenase gene expression in immortalized clones of SV40 T antigen-transformed human diploid fibroblasts.

We isolated a cDNA clone whose expression was lost during immortalization. The subtractive hybridization was performed between a genetically matched pair of mortal and immortal lines of SV40 T antigen-transformed MRC-5. The clone was found to code human interstitial collagenase. The expression of collagenase gene was almost completely shut off in seven out of eight independent immortalized clones. In addition, the levels of collagenase expression were dramatically increased toward crisis in the T antigen-transformed but mortal cells. These findings suggest the possibility that the regulatory mechanism of collagenase expression is related to both processes of in vitro aging and immortalization.     

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.     

Down-regulation of T-STAR, a growth inhibitory protein, after SV40-mediated immortalization.

Normal human cells can undergo a limited number of divisions, whereas transformed cells may have an extended life span and can give rise to immortal cells. To isolate genes involved in the immortalization process, gene expression in SV40-transformed preimmortal human fibroblasts was compared with expression in SV40-transformed immortalized fibroblasts using an mRNA differential display. We found that the growth-inhibitory protein testis-signal transduction and activation of RNA (T-STAR) a homologue of cell-cycle regulator Sam68, is strongly down-regulated in immortalized cells. Overexpression of T-STAR in the SV40-transformed immortalized cells resulted in a strong reduction of colony formation, whereas deletion of the RNA-binding domain of T-STAR abrogated this effect. Down-regulation of testis-signal transduction and activation of RNA (T-STAR) expression is found only in immortal cells isolated after a proliferative crisis accompanied with massive cell death. The strict correlation of down-regulation of T-STAR expression only in those immortal cells that arose after a clear proliferative crisis suggests that the loss of T-STAR might be necessary to bypass crisis.     

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.     

Transcriptional control of SV40 T-antigen expression allows a complete reversion of immortalization

Conditional proliferation of mouse embryo fibroblasts was achieved with a novel autoregulatory vector for Tet-dependent expression of the SV40 T-antigen. The majority of cell clones that were isolated under induced conditions showed strict regulation of cell growth. Status switches were found to be fully reversible and highly reproducible with respect to gene expression characteristics. A consequence of T-antigen expression is a significant deregulation of >400 genes. Deinduced cells turn to rest in G0/G1 phase and exhibit a senescent phenotype. The cells are not oncogenic and no evidence for transformation was found after several months of cultivation. Conditional immortalization allows diverse studies including those on cellular activities without the influence of the immortalizing gene(s), senescence as well as secondary effects from T-antigen expression.     

12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced gene sequences in human primary diploid fibroblasts and their expression in SV40-transformed fibroblasts

We have isolated cDNA sequences from TPA-treated primary human fibroblasts, which indicate RNA species that are coordinately regulated after treatment of these cells with either ultraviolet light, mitomycin C, the UV-induced factor EPIF, or TPA. The levels of RNA are elevated in Bloom syndrome (cells of two out of three patients). After transformation with SV40 one of the sequences is overexpressed while another one is reduced. Both genes maintain their inducibility by the agents mentioned.     

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.     

Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H2O2-mediated oxidative stress

Proteins are modified by reactive oxygen species, and oxidation of specific amino acid residues can impair their biological functions, leading to an alteration in cellular homeostasis. Oxidized proteins can be eliminated through either degradation or repair. Repair is limited to the reversion of a few modifications such as the reduction of methionine oxidation by the methionine sulfoxide reductase (Msr) system. However, accumulation of oxidized proteins occurs during aging, replicative senescence, or neurological disorders or after an oxidative stress, while Msr activity is impaired. In order to more precisely analyze the relationship between oxidative stress, protein oxidative damage, and MsrA, we stably overexpressed MsrA full-length cDNA in SV40 T antigen-immortalized WI-38 human fibroblasts. We report here that MsrA-overexpressing cells are more resistant than control cells to hydrogen peroxide-induced oxidative stress, but not to ultraviolet A irradiation. This MsrA-mediated resistance is accompanied by a decrease in intracellular reactive oxygen species and is partially abolished when cells are cultivated at suboptimal concentration of methionine. These results indicate that MsrA may play an important role in cellular defenses against oxidative stress, by catalytic removal of oxidant through the reduction of methionine sulfoxide, and in protection against death by limiting, at least in part, the accumulation of oxidative damage to proteins.     

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.     

High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts.

Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells.     

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.     

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.