Insulin-induced mitogenesis associated with transformation by the SV40 large T antigen.

Simian virus 40 (SV40) large T antigen-transformed cells typically show a markedly reduced serum requirement for growth and the inability to growth arrest and differentiate. An SV40 large T antigen-transformed 3T3 T cell line, CSV3-1, that can growth arrest and differentiate into adipocytes with high efficiency has, however, recently been described (Scott et al: Proc. Natl. Acad. Sci. U.S.A. 86:1652-1656, 1989; Estervig et al: J. Virol. 63:2718-2725, 1989; J. Cell. Physiol. 142:552-558, 1990). The results of the current studies using these cells show that whereas quiescent 3T3 T cells show no mitogenic response to insulin, quiescent CSV3-1 cells show a highly significant insulin-induced mitogenic responsiveness in the absence of other added growth factors. Maximum mitogenesis was observed at an insulin concentration of 1 microgram/ml, which induced 40-70% of the cells to undergo DNA synthesis within 48 hours. The half maximum response was achieved with 1-10 ng/ml of insulin. Insulin's mitogenic effect on CSV3-1 cells was evident under several different culture conditions that induce quiescence and was not mediated by any detectable autocrine growth factors that might make CSV3-1 cells competent to respond to insulin. In CSV3-1 cells insulin appears to act on its own receptor rather than on the IGF-1 receptor, because at comparable dosages IGF-1 is 10- to 100-fold less effective than insulin. Insulin also is shown to be a mitogen for another SV40-transformed cell line, CSV3-35, which can be growth arrested; in contrast insulin has no mitogenic effect on two control cell lines that are stably transfected with pSV2neo, a plasmid containing SV40 early promoter/enhancer but lacking large T antigen gene: These results suggest a significant relationship between SV40 T antigen-associated transformation and the expression of mitogenic responsiveness to insulin.     

Increased tyrosine phosphorylation of the insulin receptor,the insulin receptor substrate-1 and a 73 kDa protein associated with insulin-induced mitogenesis in SV40-transformed 3T3T cells

Insulin selectively induces mitogenesis in quiescent SV40 large T antigen-transformed murine 3T3T (CSV3-1) cells but not in quiescent nontransformed 3T3T cells. This mitogenic effect induced by insulin in CSV3-1 cells requires an induction of AP-1 activity associated with c-Jun and JunB. To further investigate the mechanisms that are involved in insulin-induced mitogenesis in CSV3-1 cells, the current experiments were performed. The results show that following insulin stimulation, the insulin receptor -subunit and the insulin receptor substrate-1 undergo a much more significant tyrosine phosphorylation in CSV3-1 cells than in 3T3T cells. Insulin also induces tyrosine phosphorylation of a 73 kDa protein that is coprecipitated with the tyrosine-phosphorylated insulin receptor in CSV3-1 cells but not in 3T3T cells. The increased tyrosine phosphorylation in response to insulin stimulation in CSV3-1 cells does not appear to be due to an increase in the level of expression of the insulin receptor and does not appear to result from a significant change in tyrosine phosphatase activity compared to nontransformed cells. The results also show that the insulin effect in CSV3-1 cells is not mediated by insulin-like growth factor 1 receptor because insulin at the concentrations that induce mitogenesis does not increase the tyrosine phosphorylation of the insulin-like growth factor 1 receptor and the expression level of the receptor is not significantly changed in CSV3-1 cells compared to nontransformed cells. These data together indicate that the selective mitogenic effect of insulin on CSV3-1 cells involves increased tyrosine phosphorylation of the insulin receptor, the insulin receptor substrate-1 and the 73 kDa protein, although the underlying mechanisms need to be further elucidated.     

Unique and selective mitogenic effects of vanadate on SV40-transformed cells

Vanadate and insulin both function as unique complete mitogens for SV40-transformed 3T3T cells, designated CSV3-1, but not for nontransformed 3T3T cells. The mitogenic effects induced by vanadate and insulin in CSV3-1 cells are mediated by different signaling mechanisms. For example, vanadate does not stimulate the tyrosine phosphorylation of the insulin receptor -subunit nor the 170 kDa insulin receptor substrate-1. Instead, vanadate induces a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins that is not observed in insulin-stimulated CSV3-1 cells. Perhaps most interestingly, vanadate-induced mitogenesis is associated with the selective induction ofc-jun andjunB expression without significantly inducingc-fos orc-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation andc-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation andc-jun andjunB expression may serve the critical roles in mediating vanadate-induced mitogenesis in SV40-transformed cells.     

Selective induction of c-jun and jun-B but not c-fos or c-myc during mitogenesis in SV40-transformed cells at the predifferentiation growth arrest state.

Insulin has recently been reported to function as a complete mitogen for SV40 large T antigen-transformed 3T3 T-cells, designated CSV3-1, but not for nontransformed 3T3 T-cells (H. Wang and R. E. Scott, J. Cell. Physiol., 147: 102-110, 1991). It is now reported that sodium orthovanadate mimics this effect of insulin. For example, when exposed to 1-5 microM vanadate, most predifferentiation growth-arrested CSV3-1 cells undergo DNA synthesis within 24 h, but neither vanadate nor insulin induces mitogenesis in nontransformed 3T3 T-cells. To investigate the possible mechanisms by which mitogenesis is induced in CSV3-1 cells, the effects of insulin and vanadate on the expression of growth-related genes were examined. Whereas insulin and vanadate had no effect on the expression of c-fos, c-myc, c-jun, jun-B, or ornithine decarboxylase activity in nontransformed 3T3 T-cells, insulin and vanadate showed different effects on the expression of these genes in CSV3-1 cells. Insulin induced a rapid and transient accumulation of c-fos mRNA followed by induction of c-myc, c-jun, jun-B, and ornithine decarboxylase. In contrast, vanadate induced the expression of c-jun, jun-B, and ornithine decarboxylase without inducing c-fos and c-myc. These observations suggest that SV40 large T antigen may play an important role in insulin- and vanadate-induced mitogenesis and that insulin and vanadate may mediate their mitogenic effects by different signal transduction pathways.     

Inhibition of simian virus 40 T-antigen expression by cellular differentiation.

Murine 3T3T stem cells transfected with pSV3neo DNA were employed to study the effects of somatic cell differentiation on simian virus 40 (SV40) T-antigen expression. This experimental approach was used because the 3T3T cell line is a well-characterized in vitro adipocyte differentiation system and the pSV3neo plasmid contains the early region of the SV40 genome and a selective marker, G418 resistance. Cell clones containing stably integrated pSV3neo which expressed T antigen were isolated in G418-containing medium. Most of these cell clones differentiated poorly. However, several clones retained the ability to efficiently differentiate into adipocytes, and with these cell clones, it was established that adipocyte differentiation markedly repressed T-antigen expression. The differentiation-specific repression of T-antigen expression did not result from a loss of proliferative potential associated with terminal differentiation, because it was observed in adipocytes that could be restimulated to proliferate. In such cells, restimulation of cell growth induced reactivation of T-antigen expression. Repression of T-antigen expression was also demonstrated during differentiation of SV40 T-antigen-immortalized human keratinocytes. These results establish that the process of cellular differentiation can repress T-antigen expression in at least two distinct biological systems.     

Induction of c-jun independent of PKC, pertussis toxin-sensitive G protein, and polyamines in quiescent SV40-transformed 3T3 T cells.

CSV3 clones of simian virus 40 large T antigen-transformed murine 3T3 T cells can be made quiescent as part of a differentiation process. In these quiescent cells, insulin- and vanadate-induced mitogenesis are both associated with the induction of the c-jun proto-oncogene (Wang and Scott 1991 J. Cell. Physiol. 147, 102-110; Wang et al. 1991 Cell Growth Differ. 2, 645-652). The current studies were therefore designed to compare the early signal transduction pathways employed by insulin and vanadate to regulate c-jun expression. In quiescent CSV3-1 cells, down-regulation of protein kinase C by prolonged exposure to 12-O-tetra-decanoylphorbol-13-acetate or inhibition of protein kinase C activity by treatment with the protein kinase C antagonist staurosporine is shown not to affect c-jun induction by insulin or vanadate. This suggests that both insulin and vanadate act in a protein kinase C-independent manner. Insulin's effect on c-jun induction does, however, involve a G protein because insulin's effect can be inhibited by pertussis toxin. In contrast, vanadate induction of c-jun is not affected by pertussis toxin. Genistein, a general tyrosine kinase inhibitor, can inhibit the ability of vanadate to induce c-jun but it does not inhibit insulin's effect. Finally, the depletion of polyamines, particularly spermidine, by DL-alpha-difluoromethylornithine treatment also prevents c-jun induction by insulin but DL-alpha-difluoromethylornithine treatment has no effect on c-jun induction by vanadate. These observations indicate that the c-jun induction by insulin and vanadate in CSV3-1 cells is mediated by different signal transduction mechanisms. Together with our previously published data, these results suggest that c-jun can be induced independent of protein kinase C activation, without involvement of pertussis toxin-sensitive G protein, independent of induction of c-fos, and without expression of high levels of intracellular polyamines.     

Distinct protein tyrosine phosphorylation during mitogenesis induced in quiescent sv40-transformed 3T3 T cells by insulin or vanadate

Insulin and vanadate selectively induce mitogenesis in quiescent SV40 large T antigen-transformed 3T3 T cells (CSV3–1) but not in quiescent nontransformed 3T3 T cells. Insulin and vanadate mediate this effect in CSV3–1 cells by distinct signal transduction mechanisms that involve protein tyrosine kinase activity. To further study these processes, changes in protein tyrosine phosphorylation induced by insulin and vanadate were investigated. Using immunoprecipitation and Western blotting techniques with antiphosphotyrosine antibodies, we report distinct protein phosphorylation characteristics in insulin- and vanadate-stimulated CSV3–1 cells. The insulin receptor β-subunit is phosphorylated within 2 min after insulin stimulation of transformed CSV3–1 cells. Insulin also stimulates a rapid increase in tyrosine phosphorylation of the 170 kDa insulin receptor substrate-1 and complex formation between the phosphorylated insulin receptor substrate-1 and the 85 kDa subunit of phosphatidylinositol 3'-kinase. In contrast, vanadate does not initially increase detectable phosphorylation of any proteins, including neither the insulin receptor nor the insulin receptor substrate-1. After 60 min, however, a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins is observed in vanadate-treated CSV3–1 cells. Furthermore, treatment of CSV3–1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation but only minimally inhibits the effects of insulin. Finally, insulin stimulates the phosphorytion of a 33 kDa protein, whereas vanadate does not. By comparison, in nontransformed 3T3 T cells, insulin induces a delayed and weaker tyrosine phosphorylation of the insulin receptor β-subunit and vanadate does not enhance the tyrosine phosphorylation of the 55 and 64 kDa proteins. These data together indicate that the mitogenic effects of insulin and vanadate are associated with distinct protein phosphorylation patterns that appear to be differentially regulated in SV40-transformed and nontransformed 3T3 T cells. © 1994 Wiley-Liss, Inc.     

Inhibition of distinct steps in the adipocyte differentiation pathway in 3T3 T mesenchymal stem cells by dimethyl sulphoxide (DMSO)

Abstract. The process of adipocyte differentiation in murine 3T3 T mesenchymal stem cells involves three well-defined steps: 1 predifferentiation growth arrest; 2 nonterminal (reversible) differentiation and 3 terminal differentiation associated with the irreversible loss of proliferative potential. To further investigate these processes, the effects of dimethyl sulphoxide (DMSO), an agent that affects differentiation in several other cell systems, was tested. The results show that DMSO modulates two distinct steps of adipocyte differentiation. The first effect is evident when growing 3T3 T cells are cultured in differentiation-inducing medium in the presence of DMSO. Therein the expression of adipocyte phenotype is inhibited because the cells fail to growtharrest at the predifferentiation growth arrest state. Instead in the presence of DMSO, cells growth-arrest at a biological state that does not support differentiation. The second effect is evident if nonterminally differentiated adipocytes are cultured in terminal differentiation-inducing medium containing DMSO. Therein the terminal step in differentiation is inhibited. These inhibitory effects occur in a dosage-dependent manner; maximum inhibition of differentiation requires 2% DMSO. Therefore, whereas DMSO typically promotes differentiation in other cell systems, DMSO inhibits multiple steps in the process of adipocyte differentiation. These observations support the conclusion that a single pharmacological agent can have markedly different effects on specific cell types. Even more important, the data establish that DMSO can now be used as a tool to study the molecular mechanisms involved in the multistep process of adipocyte differentiation.     

N18-RE-105 cells: differentiation and activation of p53 in response to glutamate and adriamycin is blocked by SV40 large T antigen tsA58

Process extension was induced in cells of the N18-RE-105 neuroblastoma-retinal hybrid line by toxic agents, including glutamate and the p53-inducing anticancer agents adriamycin and etoposide. Both adriamycin and glutamate activated p53 as measured by a plasmid transfection assay. It was therefore hypothesized that SV40 large T antigen, which binds p53, would interfere with cellular differentiation. To test this hypothesis, the temperature-sensitive form of SV40 large T was transduced into N18-RE-105 cells by retroviral infection. SV40 large T-infected cells became de-differentiated, grew in tightly-packed colonies, lost expression of neurofilament, and lost the ability to differentiate in response to glutamate and adriamycin. The de-differentiating effect of SV40 large T antigen may be due to binding and inactivation of cellular proteins, such as p53, p107, p130, p300, and retinoblastoma protein, which are important in cellular growth and differentiation. It is suggested that p53 may play a role in cellular differentiation, perhaps under unusual circumstances involving stress or cytotoxicity. Received: 29 April 1997 / Accepted: 18 June 1997     

Three distinct effects of SV40 T-antigen gene transfection on cellular differentiation

SV40 large T-antigen-induced transformation has been reported to block differentiation, but the mechanism(s) of this effect has not been established. The results presented here show that stable transfection of the SV40 T-antigen gene, via the pSV3neo plasmid, has at least three distinct effects on 3T3T adipocyte differentiation. Cells first show a decreased ability to undergo predifferentiation growth arrest, which is a prerequisite for in vitro 3T3T adipocyte differentiation. However, if predifferentiation growth arrest is accomplished by use of stringent differentiation-inducing culture conditions, adipocyte differentiation can occur with high frequency. The pSV3neo-transfected cell clones also show other modifications of the adipocyte differentiation process, including changes in nonterminal (reversible) and terminal (irreversible) steps of adipocyte differentiation. When compared to nontransfected 3T3T cells, the cell clones containing pSV3neo require markedly reduced growth factor concentrations to restimulate proliferation of nonterminally differentiated adipocytes and the terminal step of differentiation is also blocked. These results suggest that integration of the T-antigen gene, through pSV3neo transfection, has multiple effects on the cellular mechanisms of differentiation. It does not block the differentiation process per se; rather it appears to make cells highly sensitive to proliferation signals, thereby making differentiation more difficult.     

A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells

As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.     

Phenotypic reversion of SV40-transformed 3T3 cells by dimethylsulfoxide

With dimethylsulfoxide (DMSO) (0.5 to 1.5%) in the medium, SV40-transformed 3T3 cells (SV3T3) changed morphologically from a round to a flat fibroblastic shape. The saturation density of the treated SV3T3 cells decreased and the generation time increased. These cells showed an increased anchorage dependency in soft agar. Hexose uptake by SV3T3 cells was reduced to the level in the parent 3T3 cells and susceptibility of the SV3T3 cells to concanavalin A (con A) also decreased. These phenotypes of transformed cells appeared to change concomitantly from the transformed toward the normal state with the increase of DMSO concentration.     

In vivo expansion of the residual tumor antigen-specific CD8+ T lymphocytes that survive negative selection in simian virus 40 T-antigen-transgenic mice

Mice that express the viral oncoprotein simian virus 40 (SV40) large T antigen (T-Ag) as a transgene provide useful models for the assessment of the state of the host immune response in the face of spontaneous tumor progression. Line SV11 (H2(b)) mice develop rapidly progressing choroid plexus tumors due to expression of full-length T-Ag from the SV40 promoter. In addition, T-Ag expression in the thymus of SV11 mice results in the deletion of CD8(+) T cells specific for the three H2(b)-restricted immunodominant epitopes of T-Ag. Whether CD8(+) T cells specific for the immunorecessive H2-D(b)-restricted epitope V of T-Ag survive negative selection in SV11 mice has not been determined. Immunization of SV11 mice with rVV-ES-V, a recombinant vaccinia virus expressing epitope V as a minigene, resulted in the induction of weak, but reproducible, epitope V-specific cytotoxic T-lymphocyte (CTL) responses. This weak lytic response corresponded with a decreased frequency of epitope V-specific CTL that could be recruited in SV11 mice. In addition, CTL lines derived from rVV-ES-V-immunized SV11 mice had reduced avidities compared to that seen with CTL derived from healthy mice. Despite this initial weak response, significant numbers of epitope V-specific CD8(+) T cells were detected in SV11 mice ex vivo following a priming-boosting approach and these cells demonstrated high avidity for epitope V. The results suggest that low numbers of tumor-reactive CD8(+) T cells with high avidity for epitope V survive negative selection in SV11 mice but can be expanded by specific boosting approaches in the tumor bearing host.