scholarly journals Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos.

1988 ◽  
Vol 8 (10) ◽  
pp. 4212-4216 ◽  
Author(s):  
K Maly ◽  
W Doppler ◽  
H Oberhuber ◽  
H Meusburger ◽  
J Hofmann ◽  
...  
Keyword(s):  
Growth Factors ◽  
Intracellular Ph ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
3T3 Cells ◽  
Ras Gene ◽  
Phosphatidylinositol Turnover ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

Desensitization of the Ca2+-mobilizing system to serum growth factors by Ha-ras and v-mos

1988 ◽  
Vol 8 (10) ◽  
pp. 4212-4216
Author(s):  
K Maly ◽  
W Doppler ◽  
H Oberhuber ◽  
H Meusburger ◽  
J Hofmann ◽  
...  
Keyword(s):  
Growth Factors ◽  
Intracellular Ph ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
3T3 Cells ◽  
Ras Gene ◽  
Phosphatidylinositol Turnover ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

An elevation of the intracellular pH and a rise in the cytoplasmic Ca2+ concentration are considered important mitogenic signals which are observed after stimulation by various growth factors. In a preceding report it was demonstrated that the expression of Ha-ras or v-mos in cells transfected with Ha-ras or v-mos, respectively, leads to an activation of the Na+/H+ antiporter and a concomitant rise in intracellular pH (W. Doppler, R. Jaggi, and B. Groner, Gene 54:145-151, 1987). This report describes the effect of the Ha-ras and v-mos oncogenes on intracellular Ca2+ release. The expression of Ha-ras in NIH 3T3 cells carrying a glucocorticoid-inducible transforming Ha-ras gene caused a desensitization of the Ca2+-mobilizing system to serum growth factors. The induction of p21ras in cells carrying the corresponding glucocorticoid-inducible proto-oncogene did not affect the Ca2+ response to growth factors. Conditions leading to the expression of the transforming Ha-ras gene but not those causing the induction of the normal Ha-ras gene yielded an increase in phosphatidylinositol turnover and a concomitant rise in inositol phosphates. Results similar to those obtained with the transforming Ha-ras gene were seen after the expression of v-mos. The data are consistent with a mechanism in which expression of the transforming Ha-ras gene leads to a release of Ca2+ from intracellular stores via elevated levels of inositol trisphosphate.

scholarly journals Rapid and selective alterations in the expression of cellular genes accompany conditional transcription of Ha-v-ras in NIH 3T3 cells.

1987 ◽  
Vol 7 (7) ◽  
pp. 2512-2520 ◽  
Author(s):  
R D Owen ◽  
M C Ostrowski
Keyword(s):  
Dna Sequences ◽  
Regulation Of Gene Expression ◽  
Hormone Treatment ◽  
Northern Blotting ◽  
3T3 Cells ◽  
Ras Gene ◽  
Cellular Genes ◽  
Conditional Expression ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Hormone treatment of NIH 3T3 cells that contain recombinant fusions between the mouse mammary virus long terminal repeat and the v-ras gene of Harvey murine sarcoma virus results in conditional expression of the ras p21 gene product. Levels of ras mRNA and p21 are maximal after 2 to 4 h of hormone treatment. Analysis of cellular RNA by Northern blotting and nuclease S1 protection assays indicates that the expression of two cellular RNA species increases with kinetics similar to v-ras: v-sis-related RNA and retrovirus-related VL30 RNA. Run-on transcription in isolated nuclei shows that the increase in v-sis-related RNA is not dependent on transcription and therefore must arise by a post-transcriptional mechanism. The increase in VL30 expression is a transcriptional effect. Hormone treatment of normal NIH 3T3 cells has no effect on the expression of these DNA sequences. These results suggest that v-ras stimulation of autocrine factors may play a role in transformation of cells by this gene and also suggest a reverse genetic strategy to determine the nucleic acid sequences and cellular factors involved in the regulation of gene expression that is observed.

Rapid and selective alterations in the expression of cellular genes accompany conditional transcription of Ha-v-ras in NIH 3T3 cells

1987 ◽  
Vol 7 (7) ◽  
pp. 2512-2520
Author(s):  
R D Owen ◽  
M C Ostrowski
Keyword(s):  
Dna Sequences ◽  
Regulation Of Gene Expression ◽  
Hormone Treatment ◽  
Northern Blotting ◽  
3T3 Cells ◽  
Ras Gene ◽  
Cellular Genes ◽  
Conditional Expression ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Hormone treatment of NIH 3T3 cells that contain recombinant fusions between the mouse mammary virus long terminal repeat and the v-ras gene of Harvey murine sarcoma virus results in conditional expression of the ras p21 gene product. Levels of ras mRNA and p21 are maximal after 2 to 4 h of hormone treatment. Analysis of cellular RNA by Northern blotting and nuclease S1 protection assays indicates that the expression of two cellular RNA species increases with kinetics similar to v-ras: v-sis-related RNA and retrovirus-related VL30 RNA. Run-on transcription in isolated nuclei shows that the increase in v-sis-related RNA is not dependent on transcription and therefore must arise by a post-transcriptional mechanism. The increase in VL30 expression is a transcriptional effect. Hormone treatment of normal NIH 3T3 cells has no effect on the expression of these DNA sequences. These results suggest that v-ras stimulation of autocrine factors may play a role in transformation of cells by this gene and also suggest a reverse genetic strategy to determine the nucleic acid sequences and cellular factors involved in the regulation of gene expression that is observed.

scholarly journals Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor.

1993 ◽  
Vol 13 (12) ◽  
pp. 7718-7724 ◽  
Author(s):  
H Cen ◽  
A G Papageorge ◽  
W C Vass ◽  
K E Zhang ◽  
D R Lowy
Keyword(s):  
The Other ◽  
Guanine Nucleotide ◽  
3T3 Cells ◽  
Ras Protein ◽  
Exchange Factor ◽  
Type Iv ◽  
Serum Dependent ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
In Cells

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.

scholarly journals Microinjection of ras p21 induces a rapid rise in intracellular pH.

1987 ◽  
Vol 7 (5) ◽  
pp. 1984-1988 ◽  
Author(s):  
N Hagag ◽  
J C Lacal ◽  
M Graber ◽  
S Aaronson ◽  
M V Viola
Keyword(s):  
Intracellular Ph ◽  
Rapid Rise ◽  
3T3 Cells ◽  
Ph Change ◽  
Ras P21 ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Quiescent mouse NIH 3T3 cells responded to microinjection of activated ras p21 with a rapid and sustained rise in intracellular pH (approximately 0.17 pH units). The p21-induced pH change was inhibited by amiloride treatment or growth of cells in medium low in sodium, suggesting a role for the Na+/H+ antiporter. Amiloride was found to suppress p21-induced mitosis, also.

scholarly journals A human Ki-ras oncogene encodes two transforming p21 proteins.

1986 ◽  
Vol 6 (4) ◽  
pp. 1326-1328 ◽  
Author(s):  
M S McCoy ◽  
R A Weinberg
Keyword(s):  
Ras Oncogene ◽  
3T3 Cells ◽  
Ras Gene ◽  
P21 Protein ◽  
Nih 3T3 ◽  
P21 Proteins ◽  
Nih 3T3 Cells

The human Ki-ras gene was previously reported to contain two alternative fourth exons which encode two distinct p21 proteins differing only at their carboxy termini. The present study shows that either p21 protein is able on its own to transform NIH 3T3 cells to a tumorigenic state.

Regulation of Na+-H+ exchange in normal NIH-3T3 cells and in NIH-3T3 cells expressing the ras oncogene

1989 ◽  
Vol 256 (4) ◽  
pp. C756-C763 ◽  
Author(s):  
N. E. Owen ◽  
J. Knapik ◽  
F. Strebel ◽  
W. G. Tarpley ◽  
R. R. Gorman
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Inositol Trisphosphate ◽  
Biologically Active ◽  
Ras Oncogene ◽  
3T3 Cells ◽  
Calmodulin Antagonist ◽  
Kinase C ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Our laboratory and others have demonstrated that Na+-H+ exchange can be regulated by two different pathways; one that is mediated by an inositol trisphosphate-stimulated increase in intracellular calcium activity, and one that is mediated by an increase in protein kinase C activity. To determine whether one of these pathways is more important than the other, or whether one pathway is physiologically relevant, we employed normal NIH-3T3 cells (3T3 cells) and NIH-3T3 cells expressing the EJ human bladder ras oncogene (EJ cells). The EJ cells were chosen because they provide a genetic model that does not exhibit serum- or platelet-derived growth factor (PDGF)-stimulated inositol trisphosphate release or Ca2+ mobilization. It was found that serum- or PDGF-stimulated Na+-H+ exchange was more pronounced in EJ cells than in control 3T3 cells. As expected, serum- or PDGF-stimulated Na+-H+ exchange in 3T3 cells was inhibited by chelating intracellular Ca2+ with the intracellular Ca2+ chelator quin2, by the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and by the calmodulin antagonist trifluoperazine. In contrast, these agents did not inhibit serum- or PDGF-stimulated Na+-H+ exchange in EJ cells. Activators of protein kinase C (e.g., 1-oleoyl-2-acetylglycerol or biologically active phorbol esters) were found to stimulate Na+-H+ exchange in EJ cells to the same extent as serum. However, these agents were considerably less effective than serum in control 3T3 cells. Despite these findings, PDGF did not stimulate diacylglycerol levels in EJ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of c-Ha-ras gene expression by double-stranded RNA and interferon requirement

1990 ◽  
Vol 10 (8) ◽  
pp. 4424-4426
Author(s):  
A Maran ◽  
I D Goldberg ◽  
B M Steinberg
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Ras Gene ◽  
Double Stranded Rna ◽  
Beta Interferon ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
Rna Levels

The addition of double-stranded RNA (dsRNA) to NIH 3T3 cells led to an increase in the RNA levels of c-Ha-ras. The double-stranded configuration was required for the increase in c-Ha-ras mRNA levels, as heat-denatured dsRNA and single-stranded RNA did not have any effect. Nuclear run-on transcription experiments indicated that the increase in c-Ha-ras mRNA levels stimulated by dsRNA was due to transcriptional activation of the gene. The induction of c-Ha-ras gene expression by dsRNA was inhibited by anti-beta interferon antibodies, suggesting that interferon might mediate the induction.

Microinjection of ras p21 induces a rapid rise in intracellular pH

1987 ◽  
Vol 7 (5) ◽  
pp. 1984-1988
Author(s):  
N Hagag ◽  
J C Lacal ◽  
M Graber ◽  
S Aaronson ◽  
M V Viola
Keyword(s):  
Intracellular Ph ◽  
Rapid Rise ◽  
3T3 Cells ◽  
Ph Change ◽  
Ras P21 ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Quiescent mouse NIH 3T3 cells responded to microinjection of activated ras p21 with a rapid and sustained rise in intracellular pH (approximately 0.17 pH units). The p21-induced pH change was inhibited by amiloride treatment or growth of cells in medium low in sodium, suggesting a role for the Na+/H+ antiporter. Amiloride was found to suppress p21-induced mitosis, also.

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