scholarly journals Quantitative correlation between simian virus 40 T-antigen synthesis and late viral gene expression in permissive and nonpermissive cells

1978 ◽  
Vol 77 (1) ◽  
pp. R1 ◽  
Author(s):  
A Graessmann ◽  
M Graessmann ◽  
E Guhl ◽  
C Mueller
Keyword(s):  
Time Course ◽  
Viral Gene Expression ◽  
Simian Virus 40 ◽  
Threshold Value ◽  
Simian Virus ◽  
T Antigen ◽  
Threshold Concentration ◽  
Viral Gene ◽  
3T3 Cells ◽  
V Antigen

The time-course of intranuclear Simian virus 40 (SV40) tumor (T) antigen synthesis and accumulation in permissive CV1 monkey cells and nonpermissive 3T3 mouse cells has been studied by immunofluorescence and cytofluorometry. CV1 cells accumulate T antigen continuously over a period of 48 h after infection, whereas in 3T3 cells the T-antigen content remains about constant and at a comparatively low level. Only those CV1 cells which have attained a threshold concentration of intranuclear T antigen synthesize viral capsid proteins (V antigen). In nonpermissive 3T3 cells, the T-antigen threshold value for the onset of V-antigen synthesis is higher than in CV1 cells and is never reached by infected cells. However, 3T3 cells microinjected with sufficient amounts of SV40 DNA easily surpass this value and behave permissively.

scholarly journals Phosphate and the regulation of DNA replication in normal and virus-transformed 3T3 cells

1983 ◽  
Vol 214 (3) ◽  
pp. 695-702 ◽  
Author(s):  
W Engström ◽  
A Zetterberg
Keyword(s):  
Dna Synthesis ◽  
Time Course ◽  
Simian Virus 40 ◽  
Complete Removal ◽  
Simian Virus ◽  
Phosphate Concentration ◽  
Serum Starvation ◽  
Cell Multiplication ◽  
Inhibitory Effects ◽  
3T3 Cells

3T3 cells were cultured in media with different phosphate concentrations and the effects on DNA synthesis were examined. Even a modest phosphate depletion markedly inhibited DNA synthesis and cell multiplication in proliferating cultures. Furthermore, the decrease in the proportion of DNA-synthesizing cells observed after phosphate starvation followed the same time-course as the decrease seen after serum starvation. Cells starved to quiescence in a medium with a 100-fold decrease in phosphate concentration remained viable but non-proliferating for up to 3 weeks, i.e. they had entered a state of quiescence comparable with that seen after serum starvation. Addition of phosphate to phosphate-depleted cultures restored DNA synthesis within 24h. Furthermore, the kinetics of [3H]thymidine labelling after phosphate addition were nearly identical with the labelling kinetics following addition of serum to serum-depleted cultures. In contrast, phosphate deprivation had no inhibitory effects on DNA synthesis in simian-virus-40-transformed 3T3 cells. Furthermore, the inhibitory effects on DNA synthesis in such cells caused by a complete removal of serum could not be further enhanced by decreasing the phosphate concentration in the culture medium.

scholarly journals C/EBPα Inhibits Cell Growth via Direct Repression of E2F-DP-Mediated Transcription

2000 ◽  
Vol 20 (16) ◽  
pp. 5986-5997 ◽  
Author(s):  
Beatrix A. Slomiany ◽  
Kenneth L. D'Arigo ◽  
Margaret M. Kelly ◽  
David T. Kurtz
Keyword(s):  
Cell Growth ◽  
Ectopic Expression ◽  
Simian Virus 40 ◽  
Normal Liver ◽  
Simian Virus ◽  
Binding Activity ◽  
T Antigen ◽  
S Phase ◽  
3T3 Cells ◽  
L Cells

ABSTRACT Using an inducible transcription system which allows the regulated expression of C/EBP isoforms in tissue culture cells, we have found that the ectopic expression of C/EBPα, at a level comparable to that found in normal liver tissue, has a pronounced antimitogenic effect in mouse L cells and NIH 3T3 cells. The inhibition of cell division by C/EBPα in mouse cells cannot be reversed by simian virus 40 T antigen, by oncogenic ras, or by adenovirus E1a protein. When expressed in thymidine kinase-deficient L cells or 3T3 cells, C/EBPα is detected in a protein complex which binds to the E2F binding sites found in the promoters of the genes for E2F-1 and dihydrofolate reductase (DHFR). Bacterially expressed C/EBPα has no affinity for these E2F sites, but when recombinant C/EBPα is added to nuclear extracts from mouse fibroblasts, a new E2F binding activity appears, which contains the C/EBPα protein. Using an E2F-DP1-responsive promoter linked to a reporter gene, it can be shown that C/EBPα directly inhibits the induction of this promoter by E2F-DP1 in transient-transfection assays. Furthermore, C/EBPα can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines. These findings delineate a straightforward mechanism for C/EBPα-mediated cell growth arrest through repression of E2F-DP-mediated S-phase transcription.

scholarly journals Growth of immortal simian virus 40 tsA-transformed human fibroblasts is temperature dependent.

1989 ◽  
Vol 9 (7) ◽  
pp. 3093-3096 ◽  
Author(s):  
R L Radna ◽  
Y Caton ◽  
K K Jha ◽  
P Kaplan ◽  
G Li ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Cellular Aging ◽  
Cellular Growth ◽  
Viral Gene ◽  
Large T Antigen ◽  
Temperature Dependent

Simian virus 40 (SV40)-mediated transformation of human fibroblasts offers an experimental system for studying both carcinogenesis and cellular aging, since such transformants show the typical features of altered cellular growth but still have a limited life span in culture and undergo senescence. We have previously demonstrated (D. S. Neufeld, S. Ripley, A. Henderson, and H. L. Ozer, Mol. Cell. Biol. 7:2794-2802, 1987) that transformants generated with origin-defective mutants of SV40 show an increased frequency of overcoming senescence and becoming immortal. To clarify further the role of large T antigen, we have generated immortalized transformants by using origin-defective mutants of SV40 encoding a heat-labile large T antigen (tsA58 transformants). At a temperature permissive for large-T-antigen function (35 degrees C), the cell line AR5 had properties resembling those of cell lines transformed with wild-type SV40. However, the AR5 cells were unable to proliferate or form colonies at temperatures restrictive for large-T-antigen function (39 degrees C), demonstrating a continuous need for large T antigen even in immortalized human fibroblasts. Such immortal temperature-dependent transformants should be useful cell lines for the identification of other cellular or viral gene products that induce cell proliferation in human cells.

The growth-stimulatory effect of simian virus 40 T antigen requires the interaction of insulinlike growth factor 1 with its receptor

1992 ◽  
Vol 12 (11) ◽  
pp. 5069-5077
Author(s):  
P Porcu ◽  
A Ferber ◽  
Z Pietrzkowski ◽  
C T Roberts ◽  
M Adamo ◽  
...  
Keyword(s):  
Growth Factor ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Serum Free Medium ◽  
Free Medium ◽  
3T3 Cells ◽  
Sv40 T Antigen ◽  
Serum Free ◽  
Insulinlike Growth Factor

We have used a plasmid expressing a temperature-sensitive (ts) mutant of simian virus 40 (SV40) T antigen, stably transfected into 3T3 cells, to study the role of insulinlike growth factor 1 (IGF-1) and its receptor in T-antigen-mediated growth. While 3T3 cells do not grow in serum-free medium, in 1% serum, or with the sole addition of either platelet-derived growth factor (PDGF) or IGF-1, cells expressing the tsA T antigen (BALB 58 cells) grow at 34 degrees C in either PDGF or 1% serum but not in IGF-1. At the restrictive temperature (39.6 degrees C), these cells can only grow in 10% serum. We show that BALB 58 cells, at 34 degrees C, have a markedly increased expression of IGF-1 and IGF-1 mRNA and that their growth in 1% serum (at 34 degrees C) is inhibited by an antisense oligodeoxynucleotide to the IGF-1 receptor RNA. When this tsA plasmid is stably transfected into cells constitutively overexpressing the human IGF-1 receptor cDNA, the resulting cell lines show a constitutively phosphorylated IGF-1 receptor and grow in serum-free medium at 34 degrees C (but not at 39.6 degrees C). A functional SV40 T antigen also increases the expression of a plasmid in which the reporter luciferase gene is under the control of a rat IGF-1 promoter. We conclude (i) that the SV40 T antigen induces the expression of IGF-1 and IGF-1 mRNA, at least in part by a transcriptional mechanism, thus altering the growth factors requirements, and (ii) that, in BALB/c3t3 cells, the SV40 T antigen necessitates a functional IGF-1 receptor for its growth-stimulating effect in low serum (or PDGF).

scholarly journals Correlation of DNA content, p53, T antigen, and V antigen in simian virus 40-infected human diploid cells

Cytometry ◽  
1989 ◽  
Vol 10 (2) ◽  
pp. 205-213 ◽  
Author(s):  
Judith Laffin ◽  
David Fogleman ◽  
John M. Lehman
Keyword(s):  
Dna Content ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
V Antigen ◽  
Human Diploid Cells ◽  
Diploid Cells

Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the nucleus

1988 ◽  
Vol 8 (12) ◽  
pp. 5495-5503
Author(s):  
L Fischer-Fantuzzi ◽  
C Vesco
Keyword(s):  
Amino Acid ◽  
Nuclear Transport ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
3T3 Cells ◽  
Rat Embryo ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We investigated the requisites for, and functional consequences of, the relocation to the nucleus of a transforming nonkaryophilic mutant of the simian virus 40 large T antigen (a natural deletion mutant lacking an internal large-T-antigen domain that includes the signal for nuclear transport). Synthetic oligonucleotides were used to obtain gene variants with one or more copies of the signal-specifying sequence inserted near the gene 3' end, in a region dispensable for the main large-T-antigen functions. The analysis of stable transfectant populations showed that mouse NIH 3T3 cells, rat embryo fibroblasts, and simian CS cells (a subclone of CV1 cells) differed considerably in their ability to localize some variant molecules into the nucleus. CS cells were always the most efficient, and NIH 3T3 cells were the least efficient. The nuclear localization improved either with reiteration of the signal or with a left-flank modification of the signal amino acid context. Three signals appeared to be necessary and sufficient, even in NIH 3T3 cells, to obtain a nuclear accumulation comparable to that of wild-type simian virus 40 large T antigen; other signal-cell combinations caused a large variability in subcellular localization among cells of the same population, as if the nuclear uptake of some molecules depended on individual cell states. The effect of the modified location on the competence of the protein to alter cell growth was examined by comparing the activity of variants containing either the normal signal or a signal with a mutation (corresponding to large-T-antigen amino acid 128) that prevented nuclear transport. It was found that the nuclear variant was slightly more active than the cytoplasmic variants in rat embryo fibroblasts and NIH 3T3 cells and was notably less active in CS cells.

scholarly journals Wild-Type p53 Enhances Efficiency of Simian Virus 40 Large-T-Antigen-Induced Cellular Transformation

2009 ◽  
Vol 83 (19) ◽  
pp. 10106-10118 ◽  
Author(s):  
Andrea Hermannstädter ◽  
Christine Ziegler ◽  
Marion Kühl ◽  
Wolfgang Deppert ◽  
Genrich V. Tolstonog
Keyword(s):  
Transformation Efficiency ◽  
Simian Virus 40 ◽  
Transcriptional Regulators ◽  
Cellular Transformation ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Wild Type ◽  
3T3 Cells ◽  
Sv40 Transformation

ABSTRACT Abortive infection of BALB/c mouse embryo fibroblasts differing in p53 gene status (p53+/+ versus p53−/ −) with simian virus 40 (SV40) revealed a quantitatively and qualitatively decreased transformation efficiency in p53−/− cells compared to p53+/+ cells, suggesting a supportive effect of wild-type (wt) p53 in the SV40 transformation process. SV40 transformation efficiency also was low in immortalized p53−/− BALB/c 10-1 cells but could be restored to approximately the level in immortalized p53+/+ BALB/c 3T3 cells by reconstituting wt p53, but not mutant p53 (mutp53), expression. Stable expression of large T antigen (LT) in p53+/+ 3T3 cells resulted in full transformation, while LT expression in p53−/− 10-1 cells could not promote growth in suspension or in soft agar to a significant extent. The helper effect of wt p53 is mediated by its cooperation with LT and resides in the p53 N terminus, as an N-terminally truncated p53 (ΔNp53) could not rescue the p53-null phenotype. The p53 N terminus serves as a scaffold for recruiting transcriptional regulators like p300/CBP and Mdm2 into the LT-p53 complex. Consequently, LT affected global and specific gene expression in p53+/+ cells significantly more than in p53−/− cells. Our data suggest that recruitment of transcriptional regulators into the LT-p53 complex may help to modify cellular gene expression in response to the needs of cellular transformation.

scholarly journals Nonlytic simian virus 40-specific 100K phosphoprotein is associated with anchorage-independent growth in simian virus 40-transformed and revertant mouse cell lines.

1981 ◽  
Vol 1 (11) ◽  
pp. 994-1006 ◽  
Author(s):  
S Chen ◽  
M Verderame ◽  
A Lo ◽  
R Pollack
Keyword(s):  
Molecular Weight ◽  
Cell Lines ◽  
Rank Correlation ◽  
Simian Virus 40 ◽  
Apparent Molecular Weight ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Gene ◽  
Semisolid Medium ◽  
Cell Extracts

Normal fibroblasts display two distinct growth controls which can be assayed as requirements for serum or for anchorage. Interaction of mouse 3T3 fibroblasts with simian virus 40 (SV40) thus generates four classes of transformed cells. We have examined viral gene expression in these four classes of cell lines. Immunoprecipitation of [35S]methionine-labeled cell extracts with an antiserum obtained from tumor-bearing hamsters detected the SV40 large T and small t proteins (94,000 molecular weight [94K], 17K) and the nonviral host 54K protein in all cell lines tested. A tumor antigen with an apparent molecular weight of 100,000 was also found in some, but not all, lines. Similar "super T" molecules have been found by others in many rodent transformed lines. We carried out an analysis of the relation of phenotype to relative amounts of these proteins in cell lines of the four classes, using the Spearman rank correlation test. The amount of the 100K T antigen relative to the 94K T antigen or to total viral protein was well correlated with the ability to form colonies in semisolid medium. No significant correlation was found between quantities of labeled 94K T antigen, 54K host antigen, or 17K t antigen and either serum or anchorage independence. Mouse cells transformed with the small t SV40 deletion mutant 884 synthesized a 100K T antigen, suggesting that small t is not required for the production of this protein. The 100K T antigen migrated more slowly than lytic T. Since mixtures of extracts from cells expressing and lacking the 100K T antigen yielded the expected amount of this protein, it is unlikely that the 100K T derives from the 94K protein by a posttranslational modification.

Sign in / Sign up

Export Citation Format

Share Document