Establishment of Permanent Renal Tubule Cell Lines by Infection With the Wild-Type and a Thermosensitive Mutant of the Simian Virus 40

1991 ◽  
Vol 17 (6) ◽  
pp. 619-621 ◽  
Author(s):  
Alain Vandewalle ◽  
Pierre Ronco ◽  
Roland Cassingena
Keyword(s):  
Cell Lines ◽  
Renal Tubule ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type ◽  
Tubule Cell ◽  
Renal Tubule Cell

scholarly journals Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells.

1985 ◽  
Vol 5 (5) ◽  
pp. 1043-1050 ◽  
Author(s):  
R E Lanford ◽  
C Wong ◽  
J S Butel
Keyword(s):  
Cell Lines ◽  
Mouse Embryo ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Mouse Embryo Fibroblasts ◽  
Established Cell Lines ◽  
Established Cell ◽  
Embryo Fibroblasts

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.

scholarly journals Overproduction of Rb protein after the G1/S boundary causes G2 arrest

1993 ◽  
Vol 13 (11) ◽  
pp. 6640-6652
Author(s):  
V Karantza ◽  
A Maroo ◽  
D Fay ◽  
J M Sedivy
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Single Amino Acid ◽  
Permissive Temperature ◽  
Wild Type ◽  
Facs Analysis ◽  
Rb Protein ◽  
G2 Phase

The Rb protein is known to exert its activity at decision points in the G1 phase of the cell cycle. To investigate whether it may also play some role(s) at later points in the cell cycle, we used a system of rapid inducible gene amplification to conditionally overexpress Rb protein during G2 phase. A cell line expressing a temperature-sensitive simian virus 40 large T antigen (T-Ag) was stably transfected with plasmids containing the Rb cDNA linked to the simian virus 40 origin of replication: pRB-wt, pRB-fs, and pRB-Dra, carrying wild-type murine Rb cDNA, a frameshift mutation close to the beginning of the Rb coding region, and a single-amino-acid deletion in the E1A/T-Ag binding pocket, respectively. Numerous independent cell lines were isolated at the nonpermissive temperature; cell lines displaying a high level of episomal amplification of an intact Rb expression cassette following shiftdown to the permissive temperature were chosen for further analysis. Plasmid pRB-fs did not express detectable Rb antigen, while pRB-Dra expressed full-length Rb protein. The Dra mutation has previously been shown to abrogate phosphorylation as well as T-Ag binding. Fluorescence-activated cell sorting (FACS) analysis revealed that cultures induced to overexpress either wild-type or Dra mutant Rb proteins were significantly enriched for cells with a G2 DNA content. Cultures that amplified pRB-fs or rearranged pRB-wt and did not express Rb protein had normal cell cycle profiles. Double-label FACS analysis showed that cells overexpressing Rb or Rb-Dra proteins were uniformly accumulating in G2, whereas cells expressing endogenous levels of Rb were found throughout the cell cycle. These results indicate that Rb protein is interacting with some component(s) of the cell cycle-regulatory machinery during G2 phase.

Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells

1985 ◽  
Vol 5 (5) ◽  
pp. 1043-1050
Author(s):  
R E Lanford ◽  
C Wong ◽  
J S Butel
Keyword(s):  
Cell Lines ◽  
Mouse Embryo ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Mouse Embryo Fibroblasts ◽  
Established Cell Lines ◽  
Established Cell ◽  
Embryo Fibroblasts

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.

scholarly journals Overproduction of Rb protein after the G1/S boundary causes G2 arrest.

1993 ◽  
Vol 13 (11) ◽  
pp. 6640-6652 ◽  
Author(s):  
V Karantza ◽  
A Maroo ◽  
D Fay ◽  
J M Sedivy
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Single Amino Acid ◽  
Permissive Temperature ◽  
Wild Type ◽  
Facs Analysis ◽  
Rb Protein ◽  
G2 Phase

The Rb protein is known to exert its activity at decision points in the G1 phase of the cell cycle. To investigate whether it may also play some role(s) at later points in the cell cycle, we used a system of rapid inducible gene amplification to conditionally overexpress Rb protein during G2 phase. A cell line expressing a temperature-sensitive simian virus 40 large T antigen (T-Ag) was stably transfected with plasmids containing the Rb cDNA linked to the simian virus 40 origin of replication: pRB-wt, pRB-fs, and pRB-Dra, carrying wild-type murine Rb cDNA, a frameshift mutation close to the beginning of the Rb coding region, and a single-amino-acid deletion in the E1A/T-Ag binding pocket, respectively. Numerous independent cell lines were isolated at the nonpermissive temperature; cell lines displaying a high level of episomal amplification of an intact Rb expression cassette following shiftdown to the permissive temperature were chosen for further analysis. Plasmid pRB-fs did not express detectable Rb antigen, while pRB-Dra expressed full-length Rb protein. The Dra mutation has previously been shown to abrogate phosphorylation as well as T-Ag binding. Fluorescence-activated cell sorting (FACS) analysis revealed that cultures induced to overexpress either wild-type or Dra mutant Rb proteins were significantly enriched for cells with a G2 DNA content. Cultures that amplified pRB-fs or rearranged pRB-wt and did not express Rb protein had normal cell cycle profiles. Double-label FACS analysis showed that cells overexpressing Rb or Rb-Dra proteins were uniformly accumulating in G2, whereas cells expressing endogenous levels of Rb were found throughout the cell cycle. These results indicate that Rb protein is interacting with some component(s) of the cell cycle-regulatory machinery during G2 phase.

scholarly journals Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

1985 ◽  
Vol 5 (4) ◽  
pp. 642-648 ◽  
Author(s):  
J A Small ◽  
D G Blair ◽  
S D Showalter ◽  
G A Scangos
Keyword(s):  
Cell Lines ◽  
Choroid Plexus Papilloma ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Primary Cell ◽  
Tissue Samples ◽  
Primary Cell Lines

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.

scholarly journals A splice junction deletion deficient in the transport of RNA does not polyadenylate nuclear RNA.

1983 ◽  
Vol 3 (8) ◽  
pp. 1381-1388 ◽  
Author(s):  
L P Villarreal ◽  
R T White
Keyword(s):  
Simian Virus 40 ◽  
Splice Junction ◽  
Simian Virus ◽  
The Body ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Wild Type ◽  
Normal Position ◽  
Nuclear Rna ◽  
Late Region

A late region deletion mutant of simian virus 40 (dl5) was previously shown to be deficient in the transport of nuclear RNA. This is a splice junction deletion that has lost the 3' end of an RNA leader, an intervening sequence, and the 5' end of the splice acceptor site on the body of the mRNA. In this report, we analyzed the steady-state structure of the untransported nuclear RNA. The 5' ends of this RNA are heterogeneous but contain a prominent 5' end at the normal position (nucleotide 325) in addition to several other prominent 5' ends not seen in wild-type RNA. The 3' end of this RNA does not occur at the usual position (nucleotide 2674) of polyadenylation; instead, this RNA is non-polyadenylated, with the 3' end occurring either downstream or upstream of the normal position.

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