Accumulation of cells with 4N DNA content at nonpermissive temperature in rat embryo diploid cells transformed bytsA mutant of simian virus 40

1986 ◽  
Vol 127 (2) ◽  
pp. 303-310 ◽  
Author(s):  
Atsuyuki Okuda ◽  
Hideaki Tamura ◽  
Hideo Shimura ◽  
Genki Kimura
Keyword(s):  
Dna Content ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Nonpermissive Temperature ◽  
Rat Embryo ◽  
Diploid Cells

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

Efficient transfer of cloned DNA into human diploid cells: protoplast fusion in suspension

1984 ◽  
Vol 4 (11) ◽  
pp. 2549-2552
Author(s):  
P Litzkas ◽  
K K Jha ◽  
H L Ozer
Keyword(s):  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
Selective Medium ◽  
T Antigen ◽  
Human Diploid Fibroblasts ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Human Diploid Cells ◽  
Diploid Cells

A method for fusion of protoplasts bearing amplified plasmids and human diploid fibroblasts or other cell types in suspension is described. Transient expression of plasmid-encoded proteins occurs in up to 50% of the human cells, as demonstrated for simian virus 40 T antigen by immunofluorescence and the Escherichia coli xanthine-guanine phosphoribosyl transferase by autoradiography. In contrast, frequencies of stable transformants were similar to those obtained by the CaPO4 coprecipitation technique. However, experiments with both methods involving the recombinant pRSVneo (in which the Rous sarcoma virus long terminal repeat regulates expression of the antibiotic-inactivating aminoglycoside phosphotransferase) revealed a much higher frequency of colonies in G418 selective medium with constructions in which the early region of simian virus 40 DNA was present as well. We propose a role for the simian virus 40 T antigen in enhancing stable transformation in this system.

Maintenance of cellular proliferation by adenovirus early region 1A in fibroblasts conditionally immortalized by using simian virus 40 large T antigen requires conserved region 1

1990 ◽  
Vol 10 (12) ◽  
pp. 6664-6673
Author(s):  
T E Riley ◽  
A Follin ◽  
N C Jones ◽  
P S Jat
Keyword(s):  
Cell Line ◽  
Growth Arrest ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Growth Defect ◽  
Primary Cells ◽  
Nonpermissive Temperature ◽  
Large T Antigen ◽  
Adenovirus E1a

Various mutants of adenovirus E1A were assayed for their ability to complement the growth defect at the nonpermissive temperature for the cell line tsa14 which was isolated by immortalizing rat embryo fibroblasts with the thermolabile large T antigen of tsA58. This cell line grows indefinitely at the permissive temperature but undergoes rapid growth arrest upon shift up to the nonpermissive temperature. Since this growth arrest can be overcome by introduction of wild-type simian virus 40 large T antigen, human papillomavirus 16 E7, and adenovirus E1A, the tsa14 cells provided an excellent system for defining regions of E1A necessary for complementation of the growth defect. We demonstrate that conserved region 1 (CR1) is the region of E1A required for complementation. While CR2 of E1A has been shown to be required for the immortalization of primary cells and is also necessary for the binding of the 105-kDa retinoblastoma protein, mutations within this region did not abrogate complementation of the growth defect. However, since both CR1 and CR2 have previously been shown to be absolutely required for immortalization of primary cells by adenovirus E1A, this evidence suggests that the tsa14 system assays for the maintenance of proliferation and that this requires CR1.

scholarly journals Maintenance of cellular proliferation by adenovirus early region 1A in fibroblasts conditionally immortalized by using simian virus 40 large T antigen requires conserved region 1.

1990 ◽  
Vol 10 (12) ◽  
pp. 6664-6673 ◽  
Author(s):  
T E Riley ◽  
A Follin ◽  
N C Jones ◽  
P S Jat
Keyword(s):  
Cell Line ◽  
Growth Arrest ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Growth Defect ◽  
Primary Cells ◽  
Nonpermissive Temperature ◽  
Large T Antigen ◽  
Adenovirus E1a

Various mutants of adenovirus E1A were assayed for their ability to complement the growth defect at the nonpermissive temperature for the cell line tsa14 which was isolated by immortalizing rat embryo fibroblasts with the thermolabile large T antigen of tsA58. This cell line grows indefinitely at the permissive temperature but undergoes rapid growth arrest upon shift up to the nonpermissive temperature. Since this growth arrest can be overcome by introduction of wild-type simian virus 40 large T antigen, human papillomavirus 16 E7, and adenovirus E1A, the tsa14 cells provided an excellent system for defining regions of E1A necessary for complementation of the growth defect. We demonstrate that conserved region 1 (CR1) is the region of E1A required for complementation. While CR2 of E1A has been shown to be required for the immortalization of primary cells and is also necessary for the binding of the 105-kDa retinoblastoma protein, mutations within this region did not abrogate complementation of the growth defect. However, since both CR1 and CR2 have previously been shown to be absolutely required for immortalization of primary cells by adenovirus E1A, this evidence suggests that the tsa14 system assays for the maintenance of proliferation and that this requires CR1.

scholarly journals Regions and Activities of Simian Virus 40 T Antigen That Cooperate with an Activated ras Oncogene in Transforming Primary Rat Embryo Fibroblasts

2002 ◽  
Vol 76 (7) ◽  
pp. 3145-3157 ◽  
Author(s):  
Tina M. Beachy ◽  
Sara L. Cole ◽  
Jane F. Cavender ◽  
Mary J. Tevethia
Keyword(s):  
Amino Acids ◽  
Simian Virus 40 ◽  
Suppressive Effect ◽  
Simian Virus ◽  
T Antigen ◽  
Wild Type ◽  
Rat Embryo ◽  
Specific Amino Acid ◽  
Antigen Fragment ◽  
Transcriptional Transactivator

ABSTRACT Prolonged expression of a ras oncogene in primary cells accelerates the natural process of senescence. This ras-induced permanent growth arrest is bypassed in cells expressing the simian virus 40 large T antigen. Previously we showed that two regions of T antigen, a region consisting of the N-terminal 147 amino acids and a region consisting of amino acids 251 to 708 (T251-708), independently overcome ras-induced senescence. Coexpression of either T-antigen fragment and Ras results in the appearance of dense foci of transformed cells. Using a series of mutants that produce shorter T-antigen fragments, we show that the C-terminal limit of the N-terminal T-antigen fragment that cooperates with Ras lies between amino acids 83 and 121. The N-terminal limit of the C-terminal T-antigen fragment lies between amino acids 252 and 271. In addition, we present evidence that cooperation between the N-terminal fragment and Ras depends upon an intact T-antigen J domain and the ability of the T antigen to bind and inactivate the growth-suppressive effect of the tumor suppressor Rb. Introduction of specific amino acid substitutions surrounding residue 400 into T251-708 prevented the fragment from cooperating with Ras. T251-708 proteins with these same substitutions inhibited the transcriptional transactivating potential of p53 as effectively as did the wild-type protein. Thus, at least one activity contained within T251-708, other than inactivating p53 as a transcriptional transactivator, is likely to be required to bypass Ras-induced senescence.

scholarly journals L-myc cooperates with ras to transform primary rat embryo fibroblasts.

1988 ◽  
Vol 8 (6) ◽  
pp. 2668-2673 ◽  
Author(s):  
M J Birrer ◽  
S Segal ◽  
J S DeGreve ◽  
F Kaye ◽  
E A Sausville ◽  
...  
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Rat Embryo ◽  
Ras Gene ◽  
Reading Frame ◽  
Continuous Cell Lines ◽  
Embryo Fibroblasts ◽  
Soft Agar Cloning

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.

scholarly journals Elevated recombination in immortal human cells is mediated by HsRAD51 recombinase.

1997 ◽  
Vol 17 (12) ◽  
pp. 7151-7158 ◽  
Author(s):  
S J Xia ◽  
M A Shammas ◽  
R J Shmookler Reis
Keyword(s):  
Cell Lines ◽  
Cell Transformation ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Cell Strains ◽  
Immortal Cells ◽  
Diploid Cells ◽  
Untransformed Cell

Normal diploid cells have a limited replicative potential in culture, with progressively increasing interdivision time. Rarely, cell lines arise which can divide indefinitely; like tumor cells, such "immortal" lines display frequent chromosomal aberrations which may reflect high rates of recombination. Recombination frequencies within a plasmid substrate were 3.5-fold higher in nine immortal human cell lines than in six untransformed cell strains. Expression of HsRAD51, a human homolog of the yeast RAD51 and Escherichia coli recA recombinase genes, was 4.5-fold higher in immortal cell lines than in mortal cells. Stable transformation of human fibroblasts with simian virus 40 large T antigen prior to cell immortalization increased both chromosomal recombination and the level of HsRAD51 transcripts by two- to fivefold. T-antigen induction of recombination was efficiently blocked by introduction of HsRAD51 antisense (but not control) oligonucleotides spanning the initiation codon, implying that HsRAD51 expression mediates augmented recombination. Since p53 binds and inactivates HsRAD51, T-antigen-p53 association may block such inactivation and liberate HsRAD51. Upregulation of HsRAD51 transcripts in T-antigen-transformed and other immortal cells suggests that recombinase activation can also occur at the RNA level and may facilitate cell transformation to immortality.

The simian virus 40 sequences between 0.169 and 0.423 map units are not essential to immortalize early-passage rat embryo cells

1985 ◽  
Vol 5 (5) ◽  
pp. 1191-1194
Author(s):  
L Sompayrac ◽  
K J Danna
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rat Embryo ◽  
Early Passage ◽  
Early Region ◽  
Embryo Cells ◽  
Small T Antigen ◽  
Late Region ◽  
Control Sequences

F8dl is a simian virus 40 early-region deletion mutant that lacks the sequences between 0.169 and 0.423 map units. We show that cloned F8dl DNA immortalized early-passage Fisher rat embryo cells with an efficiency that was about 20% of that of cloned wild-type simian virus 40 DNA. In contrast, we detected no immortalized colonies when we transfected the cells with DNA of five other early-region deletion mutants that do not make stable truncated forms of T antigen. Since all five of these mutants have intact early- and late-region control sequences, we conclude that these control sequences are not sufficient for immortalization. Three of the mutants that did not immortalize did make a normal small t antigen, suggesting that the expression of this protein alone is not sufficient for immortalization of early-passage Fisher rat embryo cells.

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