scholarly journals Functional rescue of temperature-sensitive defects in the cell-cycle mutants of rat 3Y1 fibroblasts by the small t-antigen deletion mutant of simian virus 40.

1989 ◽  
Vol 14 (4) ◽  
pp. 375-382 ◽  
Author(s):  
Akinobu Matsuzaki ◽  
Kousaku Ohno ◽  
Masumi Ohtsu ◽  
Genki Kimura
Keyword(s):  
Cell Cycle ◽  
Deletion Mutant ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Small T Antigen

scholarly journals Cell Cycle Progression in Monkey Cells Expressing Simian Virus 40 Small t Antigen from Adenovirus Vectors

1998 ◽  
Vol 72 (12) ◽  
pp. 9637-9644 ◽  
Author(s):  
Alan K. Howe ◽  
Stéphanie Gaillard ◽  
John S. Bennett ◽  
Kathleen Rundell
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mapk Pathway ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Dependent Manner ◽  
Cycle Progression ◽  
Adenovirus Vectors ◽  
Small T Antigen

ABSTRACT The simian virus 40 small t antigen (small-t) is required for optimal viral replication and transformation, especially during the infection of nondividing cells, suggesting that the function of small-t is to promote cell cycle progression. The mechanism through which small-t promotes cell growth reflects, in part, its binding and inhibition of protein phosphatase 2A (PP2A). The use of recombinant adenoviruses allows small-t expression in a majority of cells in a population, thus providing a convenient source of cells for biochemical analyses. In monkey kidney CV1 cells, small-t expressed from these adenovirus vectors activated the mitogen-activated protein kinase (MAPK) pathway, induced JNK activity, and increased AP-1 DNA-binding activity, all in a PP2A-dependent manner. Expression of small-t also caused an increase in the phosphorylation of the Na+/H+ antiporter, a mitogen-activated ion exchanger whose activity correlates with its phosphorylation. At least part of the antiporter phosphorylation induced by small-t reflected activation of the MAPK pathway, as suggested by results of assays using a chemical inhibitor of the MAPK-activating kinase, MEK. Finally, small-t expression from adenovirus vectors promoted efficient cell cycle progression by growth-arrested cells. These vectors should facilitate further analysis of effects of small-t on cell cycle mediators.

scholarly journals Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence.

1996 ◽  
Vol 16 (3) ◽  
pp. 859-867 ◽  
Author(s):  
E Hara ◽  
R Smith ◽  
D Parry ◽  
H Tahara ◽  
S Stone ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Temperature Sensitive ◽  
The Status ◽  
Population Doublings

p16CDKN2 specifically binds to and inhibits the cyclin-dependent kinases CDK4 and CDK6, which function as regulators of cell cycle progression in G1 by contributing to the phosphorylation of the retinoblastoma protein (pRB). Human cell lines lacking functional pRB contain high levels of p16 RNA and protein, suggesting a negative feedback loop by which pRB might regulate p16 expression in late G1. By a combination of nuclear run-on assays and promoter analyses in human fibroblasts expressing a temperature-sensitive simian virus 40 T antigen, we show that p16 transcription is affected by the status of pRB and define a region in the p16 promoter that is required for this response. However, the effect is not sufficient to account for the differences in p16 RNA levels between pRB-positive and -negative cells. Moreover, p16 RNA is extremely stable, and the levels do not change appreciably during the cell cycle. Primary human fibroblasts express very low levels of p16, but the RNA and protein accumulate in late-passage, senescent cells. The apparent overexpression of p16 in pRB-negative cell lines is therefore caused by at least two factors: loss of repression by pRB and an increase in the number of population doublings.

scholarly journals Overexpression of Simian Virus 40 Small-T Antigen Blocks Centrosome Function and Mitotic Progression in Human Fibroblasts

2001 ◽  
Vol 75 (20) ◽  
pp. 9799-9807 ◽  
Author(s):  
Stéphanie Gaillard ◽  
Kelly M. Fahrbach ◽  
Rajini Parkati ◽  
Kathleen Rundell
Keyword(s):  
Cell Cycle ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Human Diploid Fibroblasts ◽  
Biochemical Status ◽  
Phosphatase 2A ◽  
Small T Antigen ◽  
Sv40 Small T

ABSTRACT Recombinant adenoviruses that express high levels of the simian virus 40 (SV40) small-t (ST) antigen have been used to study the requirement for ST to drive cell cycle proliferation of confluent human diploid fibroblasts. This occurs when either large-T (LT) antigen or serum is added to provide a second signal. While cells readily completed S phase in these experiments, they were found to accumulate with 4N DNA content. Cellular and nuclear morphology, as well as the biochemical status of cyclin B complexes, showed that these cells entered mitosis but were blocked prior to mitotic metaphase. The defect appears to reflect an inability of cells overexpressing ST to form organized centrosomes that duplicate and separate normally during the cell cycle and, therefore, the absence of a mitotic spindle. The ability of ST to bind protein phosphatase 2A was required for this pattern, suggesting that altered phosphorylation of key centrosomal components may occur when ST is overexpressed. Although the possible significance of ST effects on the centrosome cycle is not fully understood, these findings suggest that ST could influence chromosomal instability patterns that are a hallmark of SV40-transformed cells and LT expression.

scholarly journals The Simian Virus 40 Small-t and Large-T Antigens Jointly Regulate Cell Cycle Reentry in Human Fibroblasts

1999 ◽  
Vol 73 (4) ◽  
pp. 3102-3107 ◽  
Author(s):  
Analía Porrás ◽  
Stéphanie Gaillard ◽  
Kathleen Rundell
Keyword(s):  
Cell Cycle ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
T Antigens ◽  
Cell Cycle Reentry ◽  
Small T Antigen ◽  
Hdf Cells

ABSTRACT Focus formation in human diploid fibroblasts (HDF cells) is known to require both the simian virus 40 (SV40) large-T and small-t antigens. Similarly, both SV40 proteins were required to stimulate confluent, density-arrested HDF cells to reenter the cell cycle. This study used defective recombinant adenoviruses to examine the roles of the individual SV40 proteins in altering specific steps in the cell cycle. Small-t antigen and, to a lesser extent, large-T antigen increased the level of the S phase cyclin cyclin A but without increasing the activity of associated cyclin kinases unless the two SV40 proteins were coexpressed. The absence of kinase activity reflected the presence in density-arrested cells of high levels of the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1. We report here that expression of SV40 large-T antigen reduced levels of p21WAF1, while expression of small-t antigen was required to decrease p27KIP1. The separate effects of large-T and small-t antigens on these two inhibitors may explain the joint requirement for the two proteins to drive cell cycle reentry of HDF cells and ultimately transform these cells.

scholarly journals Hyperphosphorylation of the retinoblastoma gene product is determined by domains outside the simian virus 40 large-T-antigen-binding regions.

1990 ◽  
Vol 10 (12) ◽  
pp. 6586-6595 ◽  
Author(s):  
P A Hamel ◽  
B L Cohen ◽  
L M Sorce ◽  
B L Gallie ◽  
R A Phillips
Keyword(s):  
Cell Cycle ◽  
Complex Formation ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Large T Antigen ◽  
T Complex ◽  
Cycle Dependent

With the murine retinoblastoma (RB) cDNA, a series of RB mutants were expressed in COS-1 cells and the pRB products were assessed for their ability (i) to bind to large T antigen (large T), (ii) to become modified by phosphorylation, and (iii) to localize in the nucleus. All point mutations and deletions introduced into regions previously defined as contributing to binding to large T abolished pRB-large T complex formation and prevented hyperphosphorylation of the RB protein. In contrast, a series of deletions 5' to these sites did not interfere with binding to large T. While some of the 5' deletion mutants were clearly phosphorylated in a cell cycle-dependent manner, one, delta Pvu, failed to be phosphorylated depsite binding to large T. pRB with mutations created at three putative p34cdc2 phosphorylation sites in the N-terminal region behaved similarly to wild-type pRB, whereas the construct delta P5-6-7-8, mutated at four serine residues C terminal to the large T-binding site, failed to become hyperphosphorylated despite retaining the ability to bind large T. All of the mutants described were also found to localize in the nucleus. These results demonstrate that the domains in pRB responsible for binding to large T are distinct from those recognized by the relevant pRB-specific kinase(s) and/or those which contain cell cycle-dependent phosphorylation sites. Furthermore, these data are consistent with a model in which cell cycle-dependent phosphorylation of pRB requires complex formation with other cellular proteins.

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