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

1981 ◽  
Vol 39 (2) ◽  
pp. 481-489 ◽  
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

Quantification of expression of linked cloned genes in a simian virus 40-transformed xeroderma pigmentosum cell line

1985 ◽  
Vol 5 (7) ◽  
pp. 1685-1693
Author(s):  
M Protić-Sabljić ◽  
D Whyte ◽  
J Fagan ◽  
B H Howard ◽  
C M Gorman ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cell Lines ◽  
Xeroderma Pigmentosum ◽  
Phenotypic Expression ◽  
Simian Virus 40 ◽  
Fibroblast Cell ◽  
Simian Virus ◽  
Selective Medium ◽  
Normal Human ◽  
Human Line

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).

scholarly journals Quantification of expression of linked cloned genes in a simian virus 40-transformed xeroderma pigmentosum cell line.

1985 ◽  
Vol 5 (7) ◽  
pp. 1685-1693 ◽  
Author(s):  
M Protić-Sabljić ◽  
D Whyte ◽  
J Fagan ◽  
B H Howard ◽  
C M Gorman ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cell Lines ◽  
Xeroderma Pigmentosum ◽  
Phenotypic Expression ◽  
Simian Virus 40 ◽  
Fibroblast Cell ◽  
Simian Virus ◽  
Selective Medium ◽  
Normal Human ◽  
Human Line

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).

scholarly journals Transformed human cells release different fibronectin variants than do normal cells.

1986 ◽  
Vol 103 (5) ◽  
pp. 1671-1677 ◽  
Author(s):  
P Castellani ◽  
A Siri ◽  
C Rosellini ◽  
E Infusini ◽  
L Borsi ◽  
...  
Keyword(s):  
Human Plasma ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Human Cells ◽  
Heparin Binding ◽  
Heparin Binding Domain ◽  
Normal Human ◽  
Different Sources ◽  
Adult Fibroblasts

Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.

scholarly journals Calmodulin antagonists decrease the binding of epidermal growth factor to transformed, but not to normal, human fibroblasts

1984 ◽  
Vol 218 (2) ◽  
pp. 629-632 ◽  
Author(s):  
P V Bodine ◽  
J T Tupper
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dependent Manner ◽  
Calmodulin Antagonists ◽  
Normal Human ◽  
Epidermal Growth

Four psychoactive agents which inhibit calmodulin activity were used to study their effect on the binding of epidermal growth factor (EGF) to normal and simian-virus-40-transformed human fibroblasts (WI38). These calmodulin antagonists decreased the binding of 125I-labelled EGF to the transformed, but not to the normal, cell in a dose-dependent manner. The mechanism of this effect appears to be due to a decrease in the apparent affinity of the plasma-membrane EGF receptor for the EGF molecule.

scholarly journals Fibroblast surface antigen produced but not retained by virus-transformed human cells.

1975 ◽  
Vol 142 (2) ◽  
pp. 530-535 ◽  
Author(s):  
A Vaheri ◽  
E Ruoslahti
Keyword(s):  
Human Serum ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Malignant Cells ◽  
Cell Type ◽  
Glycoprotein Antigen ◽  
A Cell ◽  
Normal Human ◽  
Cell Type Specific

Normal human fibroblasts contain a cell type-specific glycoprotein antigen (SF) that is known to be slowly shed into the medium and to be present also in human serum. Immunofluorescence with anti-SF antibodies showed that SF antigen has a highly nonrandom fibrillar distribution in surface of normal fibroblasts. Simian virus 40-transformed fibroblasts also produced the SF antigen, as shown by radioimmunoassay or immunodiffusion tests, but it was not retained by the surface of these cells. This creates a major difference between the surfaces of normal and malignant cells.

scholarly journals Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells.

1987 ◽  
Vol 104 (3) ◽  
pp. 595-600 ◽  
Author(s):  
L Borsi ◽  
B Carnemolla ◽  
P Castellani ◽  
C Rosellini ◽  
D Vecchio ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Alternative Splicing ◽  
Culture Medium ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Human Cells ◽  
Normal Human ◽  
Human Fibronectin ◽  
Different Sources

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.

scholarly journals A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.

1983 ◽  
Vol 3 (2) ◽  
pp. 280-289 ◽  
Author(s):  
H Okayama ◽  
P Berg
Keyword(s):  
Cdna Cloning ◽  
Mammalian Cells ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Plasmid Vector ◽  
Simian Virus ◽  
Cdna Clones ◽  
Alpha Globin ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Cloned Cdna

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).

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.

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