Immortalization of human fibroblasts transformed by origin-defective simian virus 40

1987 ◽  
Vol 7 (8) ◽  
pp. 2794-2802
Author(s):  
D S Neufeld ◽  
S Ripley ◽  
A Henderson ◽  
H L Ozer
Keyword(s):  
Cell Lines ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Experimental System ◽  
Cell Interaction ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Origin ◽  
Karyotypic Analysis ◽  
Transformed Cell Lines

Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori- transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SV/HF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre- and postimmortalization, should be useful in defining molecular changes associated with immortalization.

scholarly journals Immortalization of human fibroblasts transformed by origin-defective simian virus 40.

1987 ◽  
Vol 7 (8) ◽  
pp. 2794-2802 ◽  
Author(s):  
D S Neufeld ◽  
S Ripley ◽  
A Henderson ◽  
H L Ozer
Keyword(s):  
Cell Lines ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Experimental System ◽  
Cell Interaction ◽  
Simian Virus ◽  
T Antigen ◽  
Viral Origin ◽  
Karyotypic Analysis ◽  
Transformed Cell Lines

Simian virus 40 (SV40)-mediated transformation of human diploid fibroblasts has provided an effective experimental system for studies of both "senescence" in cell culture and carcinogenesis. Previous interpretations may have been complicated, however, by the semipermissive virus-cell interaction. In earlier studies, we previously demonstrated that the human diploid fibroblast line HS74 can be efficiently transformed by DNA from replication-defective mutants of SV40 containing a deletion in the viral origin for DNA synthesis (SVori-). In the current study, we found that such SVori- transformants show a significantly increased life span in culture, as compared with either HS74 or an independent transformant containing an intact viral genome, but they nonetheless undergo senescence. We have clonally isolated six immortalized derivatives of one such transformant (SV/HF-5). Growth studies indicate that the immortalized cell lines do not invariably grow better than SV/HF-5 or HS74. Genetic studies involving karyotypic analysis and Southern analysis of integrated viral sequences demonstrated both random and nonrandom alterations. All immortalized derivatives conserved one of the two copies of SV40 sequences which expressed a truncated T antigen. These cloned SV40-transformed cell lines, pre- and postimmortalization, should be useful in defining molecular changes associated with immortalization.

Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants

1984 ◽  
Vol 4 (8) ◽  
pp. 1653-1656
Author(s):  
K Van Doren ◽  
Y Gluzman
Keyword(s):  
Cell Lines ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Origin Of Replication ◽  
Viral Dna ◽  
Early Region ◽  
Transformed Cell Lines ◽  
Sv40 Dna

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.

scholarly journals Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants.

1984 ◽  
Vol 4 (8) ◽  
pp. 1653-1656 ◽  
Author(s):  
K Van Doren ◽  
Y Gluzman
Keyword(s):  
Cell Lines ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Origin Of Replication ◽  
Viral Dna ◽  
Early Region ◽  
Transformed Cell Lines ◽  
Sv40 Dna

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.

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.

Altered chromosome 6 in immortal human fibroblasts

1992 ◽  
Vol 12 (5) ◽  
pp. 2273-2281
Author(s):  
K Hubbard-Smith ◽  
P Patsalis ◽  
J R Pardinas ◽  
K K Jha ◽  
A S Henderson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Fibroblasts ◽  
Rare Event ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chromosome 6 ◽  
Karyotypic Analysis ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Immortal Cells

Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.

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.

scholarly journals Altered chromosome 6 in immortal human fibroblasts.

1992 ◽  
Vol 12 (5) ◽  
pp. 2273-2281 ◽  
Author(s):  
K Hubbard-Smith ◽  
P Patsalis ◽  
J R Pardinas ◽  
K K Jha ◽  
A S Henderson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Fibroblasts ◽  
Rare Event ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Chromosome 6 ◽  
Karyotypic Analysis ◽  
Diploid Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Immortal Cells

Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.

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.

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.

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