Enhanced phosphorylation of many endogenous protein substrates in human fibroblasts transformed by simian virus 40

This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.7.3093 ◽

1989 ◽

Vol 9 (7) ◽

pp. 3093-3096 ◽

Cited By ~ 61

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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Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum.

Journal of Virology ◽

10.1128/jvi.39.2.481-489.1981 ◽

1981 ◽

Vol 39 (2) ◽

pp. 481-489 ◽

Cited By ~ 12

Author(s):

H L Ozer ◽

M L Slater ◽

J J Dermody ◽

M Mandel

Keyword(s):

Xeroderma Pigmentosum ◽

Human Fibroblasts ◽

Simian Virus 40 ◽

Simian Virus ◽

Normal Human

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Transformation of ultraviolet-irradiated human fibroblasts by simian virus 40 is enhanced by cellular DNA repair functions

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis ◽

10.1016/0005-2787(81)90121-0 ◽

1981 ◽

Vol 652 (2) ◽

pp. 314-323 ◽

Cited By ~ 6

Author(s):

Jennifer D. Hall

Keyword(s):

Dna Repair ◽

Human Fibroblasts ◽

Simian Virus 40 ◽

Simian Virus ◽

Cellular Dna

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Altered chromosome 6 in immortal human fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2273-2281.1992 ◽

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.

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Molecular Regulation of the IGF-Binding Protein-4 Protease System in Human Fibroblasts: Identification of a Novel Inducible Inhibitor

Endocrinology ◽

10.1210/endo.143.4.8729 ◽

2002 ◽

Vol 143 (4) ◽

pp. 1199-1205 ◽

Cited By ~ 18

Author(s):

Bing-Kun Chen ◽

Michael T. Overgaard ◽

Laurie K. Bale ◽

Zachary T. Resch ◽

Michael Christiansen ◽

...

Keyword(s):

Cell Growth ◽

Conditioned Medium ◽

Phorbol Ester ◽

Protease Activity ◽

Actinomycin D ◽

Human Fibroblasts ◽

Simian Virus 40 ◽

Simian Virus ◽

Igf Binding ◽

Igf Binding Protein

Abstract The IGF-binding protein-4 (IGFBP-4) protease system is an important regulator of local IGF bioavailability and cell growth. Recently, the IGF-dependent IGFBP-4 protease secreted by cultured human fibroblasts was identified as pregnancy-associated plasma protein A (PAPP-A). In pregnancy serum, PAPP-A circulates as a disulfide-bound complex with the precursor form of major basic protein (pro-MBP), and in this complex PAPP-A’s proteolytic activity is not evident. In this study we analyzed the IGFBP-4 protease system in normal human fibroblasts to determine regulation outside of pregnancy. Treatment with the phorbol ester tumor promoter, β-phorbol 12,13-didecanoate (β-PDD), resulted in time-dependent inhibition of the IGF-dependent IGFBP-4 protease activity in cell-conditioned medium, which was evident at 6 h and complete by 24 h. PAPP-A mRNA was constitutively expressed in control cells, and levels were decreased only after 24 h of β-PDD treatment. Secretion of PAPP-A protein into conditioned medium did not change with β-PDD treatment. On the other hand, pro-MBP mRNA was undetectable in control human fibroblasts, and treatment with β-PDD induced pro-MBP mRNA and protein expression within 6 h. β-PDD-induced pro-MBP mRNA expression and protease inhibition were blocked with an inhibitor of RNA synthesis, actinomycin D. Actinomycin D had no effect on PAPP-A mRNA levels in the absence or presence of β-PDD. Similarly, transformation of human fibroblasts with simian virus 40 large T antigen resulted in the synthesis of pro-MBP mRNA and protein and inhibition of IGFBP-4 protease activity. Coculture of fibroblasts with cells transfected with pro-MBP cDNA resulted in inhibition of IGFBP-4 proteolytic activity without having any effect on PAPP-A synthesis. In summary, phorbol ester tumor promoters and simian virus 40 transformation regulate IGFBP-4 proteolysis in human fibroblasts through induction of a novel inhibitor of PAPP-A, pro-MBP. These findings expand our understanding of the IGFBP-4 protease system and suggest an additional level of local cell growth control.

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Abolition of Cyclin-Dependent Kinase Inhibitor p16Ink4a and p21Cip1/Waf1 Functions Permits Ras-Induced Anchorage-Independent Growth in Telomerase-Immortalized Human Fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.23.8.2859-2870.2003 ◽

2003 ◽

Vol 23 (8) ◽

pp. 2859-2870 ◽

Cited By ~ 55

Author(s):

Wenyi Wei ◽

Wendy A. Jobling ◽

Wen Chen ◽

William C. Hahn ◽

John M. Sedivy

Keyword(s):

Kinase Inhibitor ◽

Human Fibroblasts ◽

Ectopic Expression ◽

Simian Virus 40 ◽

Fibroblast Cell ◽

Simian Virus ◽

T Antigen ◽

Human Cells ◽

Large T Antigen ◽

Anchorage Independent Growth

ABSTRACT Human cells are more resistant to both immortalization and malignant transformation than rodent cells. Recent studies have established the basic genetic requirements for the transformation of human cells, but much of this work relied on the expression of transforming proteins derived from DNA tumor viruses. We constructed an isogenic panel of human fibroblast cell lines using a combination of gene targeting and ectopic expression of dominantly acting mutants of cellular genes. Abolition of p21 Cip1/Waf1 and p16 Ink4a functions prevented oncogenically activated Ras from inducing growth arrest and was sufficient for limited anchorage-independent growth but not tumorigenesis. Deletion of the tumor suppressor p53 combined with abolition of p16 Ink4a function failed to mimic the introduction of simian virus 40 large T antigen, indicating that large T antigen may target additional cellular functions. Ha-Ras and Myc cooperated only to a limited extent, but in the absence of Ras, Myc cooperated strongly with the simian virus 40 small t antigen to elicit aggressive anchorage-independent growth. The experiments reported here further define specific components of human transformation pathways.

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

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7151 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7151-7158 ◽

Cited By ~ 93

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.

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