Finite life span of hybrids formed by fusion of different simian virus 40-immortalized human cell lines.

Simian virus 40 (SV40) genes are able to induce immortalization of normal human cells after a culture crisis during which unknown cellular genetic changes presumably occur. To determine whether these genetic changes are always identical, we performed somatic cell hybridization analysis of an SV40-immortalized human bronchial epithelial cell line, BET-1A. Fusion of BET-1A with an SV40-immortalized fibroblast cell line resulted in hybrids that senesced, indicating that these cell lines are in different complementation groups for immortalization.     

Mutation ofp53Is Not a Prerequisite for Immortalization of Human Fibroblasts by SV40 T Antigen

Thein vitrolife span of human cells is under genetic control and limited. Immortalized cells, however, can be obtained at a low frequency following expression of the SV40 T antigen gene though the steps that lead to immortality are not well understood. p53 has been implicated in cell cycle regulation and evidence suggests it may have a role in controlling life span in rodent and human cells. In this study, we investigated whether allelic loss or mutation ofp53was an essential step during SV40 immortalization leading to the appearance of immortal cell lines. The gross structure of thep53gene was examined in a primary fibroblast cell strain (1BR.3) and two SV40-immortalized derivatives, 1BRMT1 and 1BRgn2. There was no evidence for allelic loss of thep53gene during immortalization. The primary cells and the immortal derivatives all expressed authenticp53mRNAs, though the immortal cell lines had higher levels of expression. Sequence analysis of exons 5–8 did not detect mutations associated with the immortal phenotype. These data are consistent with SV40 immortalization being independent of genetic changes inp53.     

Transformation of human cystinotic fibroblasts by SV40: characteristics of transformed cells with limited and unlimited growth potential.

Human skin fibroblasts derived from patients with nephropathic cystinosis were transformed with SV40 virions, cloned and permitted to enter the degenerative stage of growth termed "crisis," characteristic of SV40 transformed human cells. Nephropathic cystinosis is an autosomal recessively inherited metabolic disorder resulting in the intracellular accumulation of the amino acid cystine. A transformed cystinotic cell line which was recovered from the crisis stage was indistinguishable from its transformed precrisis parental cell strain in growth rate in media containing either 1% or 10% serum, cloning efficiency on plastic, in semisolid media, or upon confluent monolayers of normal skin fibroblasts, expression of SV40 T antigen, or production of virus. However, the modal DNA content of the recovered postcrisis transformed cystinotic cell line was different from that of the cloned parental precrisis transformed cell strain, suggesting that the postcrisis line was derived from a small subpopulation of the precrisis strain. The DNA content of the established cystinotic cell line continued to be unstable during subsequent subculturing and gave rise to subclones with both more and less DNA per cell. This line now has an apparently infinite growth potential and still has the hallmark of the cystinotic parental line, the storage of abnormally large amounts of intracellular nonprotein cystine.     

SV40 Large T Antigen Disrupts Embryogenesis of Canine and Porcine Somatic Cell Nuclear Transfer Embryo

Background

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development.

Results

SV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses.

Conclusions

Although lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.

     

Transformation and immortalization of diploid xeroderma pigmentosum fibroblasts

Diploid xeroderma pigmentosum (XP) skin fibroblast strains from various XP-complementation groups (B, C, G, and H) were transformed with an origin-defective SV40 early region or with the pSV3 gpt plasmid. In the latter case, transfected cells were selected for their ability to express the dominant xgpt gene. Immortalized cell lines were obtained, from XP-complementation groups C (8CA, 3MA, and 20MA; XP3MA and XP20MA were formerly considered to belong to complementation group I), G (2BI and 3BR), and H (2CS). No immortalized cells could be isolated from complementation group B (11BE). The immortalization frequency of wild-type diploid fibroblasts and diploid cultures from XP patients was not significantly increased by cotransfection with the SV40 early region plus several selected viral and cellular oncogenes. In fact, co-transfection with some of the oncogenes caused a marked decrease of the transformation frequency. The observed immortalization occurred at a frequency of approximately 5 x 10(-8).     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

Expression of SV40 T-antigen in lipoma cells with a chromosomal translocation T(3;12) is not sufficient for direct immortalization.

12q13-15 changes are the most frequent cytogenetic abnormalities in human tumor cells. To test their biological significance we used an assay based on lipoma cells with a limited in vitro lifetime and this type of chromosomal aberration. Lipoma cells with a reciprocal translocation t(3;12)(q28;q14) were transformed by transfection with a plasmid containing the SV40 "early region". The transformed cells showed an altered morphology with loss of contact inhibition, formation of foci, and T-antigen expression. They were immortalized after a growth crisis. The karyotypic patterns before and after the crisis show that the translocation together with expression of SV40 T-antigen is not sufficient for direct immortalization.     

Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.     

Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line

A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori-, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori--transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori--transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori- in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells.     

MDM2 is a target of simian virus 40 in cellular transformation and during lytic infection.

Phosphopeptide analyses of the simian virus 40 (SV40) large tumor antigen (LT) in SV40-transformed rat cells, as well as in SV40 lytically infected monkey cells, showed that gel-purified LT that was not complexed to p53 (free LT) and p53-complexed LT differed substantially in their phosphorylation patterns. Most significantly, p53-complexed LT contained phosphopeptides not found in free LT. We show that these additional phosphopeptides were derived from MDM2, a cellular antagonist of p53, which coprecipitated with the p53-LT complexes, probably in a trimeric LT-p53-MDM2 complex. MDM2 also quantitatively bound the free p53 in SV40-transformed cells. Free LT, in contrast, was not found in complex with MDM2, indicating a specific targeting of the MDM2 protein by SV40. This specificity is underscored by significantly different phosphorylation patterns of the MDM2 proteins in normal and SV40-transformed cells. Furthermore, the MDM2 protein, like p53, becomes metabolically stabilized in SV40-transformed cells. This suggests the possibility that the specific targeting of MDM2 by SV40 is aimed at preventing MDM2-directed proteasomal degradation of p53 in SV40-infected and -transformed cells, thereby leading to metabolic stabilization of p53 in these cells.     

Phenotypic conversion of SV40-immortalized human diploid fibroblasts to senescing cells by introduction of an antisense gene for SV40-T antigen.

Normal human lung fibroblast diploid cells, WI-38, become senescent after a definite number of divisions. VA-13 is a line of immortalized cells established by transformation of WI-38 cells by SV40 virus. To determine whether SV40 large T (SV40-T) antigen is essential for this immortalization of WI-38 cells we introduced an antisense gene for T antigen into VA-13. Two morphologically different types of antisense transformant (VA-AS5-8 and VA-AS37-8) were obtained. In both antisense transformants the expression of T antigen was reduced by more than 70% as compared to that in the parent cells. The morphology of the antisense transformants indicated a partial conversion to the senescent phenotype of WI-38. The relative number of cells in the S phase of the antisense transformants was decreased as compared to that in cultures of VA-13 and about 50% of cells were at G1/0. The doubling time of the transformants was prolonged to close to the doubling time of WI-38. The level of expression of retinoblastoma protein (pRB) complexed with SV40-T antigen of the antisense transformants was significantly decreased although the level of total pRB was much higher than that in VA-13. The pRB was present exclusively in the underphosphorylated form. Thus, the decreased level of formation of the complex between SV40-T and pRB or the underphosphorylation of pRB may explain the suppression of growth of antisense transformants. Together, these results show that an antisense gene for SV40-T antigen can efficiently block the cell proliferation and the cell immortalization of VA-13 cells.     

Enumeration of the simian virus 40 early region elements necessary for human cell transformation

While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.     

Evidence that a target-derived soluble factor is necessary for the selective lysis of SV40-transformed fibroblasts by activated mouse macrophages

Our laboratory is investigating the basis for the selective recognition of transformed cells by activated mouse macrophages. As targets we are using a panel of SV40-transformed, C3H.OL fibroblast cell lines (SV-COL) that display widely different levels of sensitivity to lysis, from highly sensitive to completely resistant. Our results show that adding conditioned medium from the macrophage-sensitive target SV-COL-E8 (CM(E8] to a cytolysis assay with the macrophage-resistant target SV-COL-F5f causes the macrophages to kill the resistant targets in a contact dependent fashion. We have termed this activity "macrophage cell lysis factor" (MCLF). MCLF activity was not detected in conditioned media from cells not killed by activated macrophages (i.e., 3T3-like cell lines or embryo fibroblasts) but was present in conditioned media from six other SV-COL cell lines at levels that were directly proportional to the sensitivity of those targets (r = 0.98). These data suggest that MCLF plays a key role in determining the lytic sensitivity of SV40-transformed fibroblasts. Finally, to ask whether the production of MCLF is sufficient to explain the selective recognition of SV40-transformed fibroblasts by activated macrophages we have tested whether CM(E8) will cause macrophages to kill normal cells. Our results show that in the presence of CM(E8) macrophages will kill immortalized, 3T3-like fibroblasts but will not kill normal embryo fibroblasts. These results suggest that the production of MCLF, or a similar activity, is necessary but not sufficient for macrophage cytolysis to occur and that a change in target cell phenotype that occurs during the process of immortalization is also required.     

Termination of lifespan of SV40-transformed human fibroblasts in crisis is due to apoptosis

Normal human fibroblasts in culture have a limited lifespan, ending in replicative senescence. Introduction of SV40 sequences encoding large T antigen and small t antigen into pre-senescent cells results in an extension of lifespan for an additional 20-30 population doublings. Rare clones of SV40-transformed cells are capable of indefinite growth and are described as immortal; however, the great majority of SV40-transformed cells terminate this extended lifespan in cell death, termed "crisis." We have examined the properties of cells in crisis to obtain further insights into mechanism of cell death and immortalization. Populations at the terminal cell passage show a balance between cell replication and cell death over a period of several weeks, with a progressive increase in cells undergoing cell death. During this period, there is less than a 3-fold increase in attached cell number, with two stages being identifiable on the basis of the focal pattern of cell survival. We also demonstrate that cells in crisis are undergoing apoptosis based on TUNEL assay, subG1 DNA content, annexin V reactivity, and activation of caspases 3 and 8. We suggest a model whereby SV40-transformed cells acquire increased sensitivity to apoptosis based on changes in properties which activate caspase 8 in addition to changes previously described involving shortening of telomeric sequences. While only telomere stabilization could be clearly shown to be essential for survival of cells through crisis, the extended period of cell replication and altered gene expression observed in SV40-transformed cells during crisis are compatible with other genetic alterations in immortal cells.     

Chromosomal rearrangements and their effects in spontaneous immortalization and transformation of rat embryo cells in vitro

G-banding analysis of LRec-1 and LRec-3, spontaneously immortalized cell lines from rat embryo fibroblast, revealed diploid karyotypes with specific clonal structural rearrangements of chromosomes 7 and 19 - del(7)(q11.2q22.1), t(7;19)(q11.1;q12) in malignant stage. Both clonal abnormalities of chromosomes 7 and 19 were also revealed in LRec-1k clone and LRec-1 sf cell line. Previous study of LRec-1 and LRec-3 cells showed the presence of karyotypes with pseudodiploid modal chromosome number, partial trisomy of chromosome 7 and same clonal structural rearrangements of chromosomes 7 and 19 in immortalized stage. In malignant stage, the trisomy 6 and new clonal structural rearrangements of chromosomes 1, 2, 11, 15, 18, 19 and of chromosomes 10, 20 were also found in LRec-1 sf and LRec-1 cells, accordingly. There were no new clonal structural chromosome rearrangements in LRec-1 k and LRec-3 cells. We compared locies of chromosomes involved in rearrangements with mapped genes on these chromosomes according to RATMAP. Supposedly these genes are involved in spontaneous immortalization of rat embryo fibroblast and malignant transformation of LRec-1 and LRec-3 cells and rearrangements of chromosomes 1, 2, 11, 15 and 18 facilitate expression of growth factors of LRec-1 sf cells.     

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.     

Generation of cytotoxic lymphocytes by SV40-induced antigens

In order to study the correlation of in vivo tumor transplantation immunity and in vitro immunologic assays, cell-mediated cytotoxicity against SV40-transformed cells was studied in AL/N strain mice by using 51Cr-release assay. Killing of SV40-transformed AL/N fibroblast cells was observed by spleen cells of AL/N mice immunized with syngeneic SV40-transformed cells. Immunization with the solubilized SV40 tumor-specific transplantation antigen (TSTA) that induced transplantation immunity in vivo did not elicit cytotoxic spleen cells in vitro. However, the spleen cells from mice immunized with solubilized TSTA and then sensitized in vitro with SV40-transformed cells became cytotoxic against SV40-transformed fibroblasts. Similarly, SV40 TSTA (T antigen) purified by immunoprecipitation was able to prime the lymphocytes in AL/N mice: the primed lymphocytes could differentiate into cytotoxic lymphocytes upon in vitro stimulation by SV40-transformed cells. These data indicate that SV40 TSTA (T antigen) plays a role in the induction of cytotoxic lymphocytes.     

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.