H-ras activation in benign and self-regressing skin tumors (keratoacanthomas) in both humans and an animal model system

1988 ◽  
Vol 8 (2) ◽  
pp. 786-793
Author(s):  
J Leon ◽  
H Kamino ◽  
J J Steinberg ◽  
A Pellicer
Keyword(s):  
Animal Model ◽  
Model System ◽  
Skin Tumors ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Genes ◽  
Ras Activation ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
Codon 61

The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, which are benign and self-regressing skin tumors, both in humans and in a corresponding animal model system. Keratoacanthomas were induced on rabbit ears by repeated applications of 7,12-dimethylbenz(a)anthracene. About 60% of the tumor DNAs produced transformed foci after transfection into NIH 3T3 cells, and in all of them the transforming gene was identified as H-ras by Southern and Northern (RNA) hybridization. Immunoprecipitation experiments suggested that the transforming rabbit H-ras protein carried a mutation in codon 61. In addition, an activated H-ras gene was detected in a human keratoacanthoma by using a nude mouse tumorigenesis assay after transfection of tumor DNA into NIH 3T3 cells. This is the first report of ras activation in a benign human tumor. The transforming human H-ras gene showed a point mutation in codon 61 that would result in leucine instead of the glutamine present in the normal gene product. The finding of ras activation in tumors that are not only benign but also self-regressing indicates that activated ras genes are not sufficient to maintain a neoplastic phenotype, although they likely play a role in early stages of tumorigenesis.

scholarly journals H-ras activation in benign and self-regressing skin tumors (keratoacanthomas) in both humans and an animal model system.

1988 ◽  
Vol 8 (2) ◽  
pp. 786-793 ◽  
Author(s):  
J Leon ◽  
H Kamino ◽  
J J Steinberg ◽  
A Pellicer
Keyword(s):  
Animal Model ◽  
Model System ◽  
Skin Tumors ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Genes ◽  
Ras Activation ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
Codon 61

The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, which are benign and self-regressing skin tumors, both in humans and in a corresponding animal model system. Keratoacanthomas were induced on rabbit ears by repeated applications of 7,12-dimethylbenz(a)anthracene. About 60% of the tumor DNAs produced transformed foci after transfection into NIH 3T3 cells, and in all of them the transforming gene was identified as H-ras by Southern and Northern (RNA) hybridization. Immunoprecipitation experiments suggested that the transforming rabbit H-ras protein carried a mutation in codon 61. In addition, an activated H-ras gene was detected in a human keratoacanthoma by using a nude mouse tumorigenesis assay after transfection of tumor DNA into NIH 3T3 cells. This is the first report of ras activation in a benign human tumor. The transforming human H-ras gene showed a point mutation in codon 61 that would result in leucine instead of the glutamine present in the normal gene product. The finding of ras activation in tumors that are not only benign but also self-regressing indicates that activated ras genes are not sufficient to maintain a neoplastic phenotype, although they likely play a role in early stages of tumorigenesis.

scholarly journals A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.

1986 ◽  
Vol 6 (5) ◽  
pp. 1843-1846 ◽  
Author(s):  
A G Papageorge ◽  
B M Willumsen ◽  
M Johnsen ◽  
H F Kung ◽  
D W Stacey ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
S Phase ◽  
3T3 Cells ◽  
Ras Gene ◽  
Mutant Proteins ◽  
Ras Protein ◽  
Ras Genes ◽  
Essential Function ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

scholarly journals Desensitization of prostaglandin F2α-stimulated inositol phosphate generation in NIH-3T3 fibroblasts transformed by overexpression of normal c-Ha-ras-1, c-Ki-ras-2 and c-N-ras genes

1990 ◽  
Vol 267 (3) ◽  
pp. 809-813 ◽  
Author(s):  
F M Black ◽  
M J O Wakelam
Keyword(s):  
Inositol Phosphate ◽  
Prostaglandin F2α ◽  
Exponential Growth Phase ◽  
Maximal Effect ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Genes ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The stimulation of inositol phosphate generation in control and ras-gene-transformed NIH-3T3 cells by prostaglandin F2 alpha (PGF2 alpha) was investigated. Compared with the control cells, a desensitization of the response was observed in cells transformed by the overexpression of N-, Ha-, or Ki-ras genes. This desensitization was without effect upon the concentration causing half-maximal effect (EC50), dissociation constant (Kd) or number of PGF2 alpha receptors. Inhibition of PG synthesis was without effect upon desensitization, demonstrating that the effect was not agonist-induced. Desensitization could be induced in NIH-3T3 cells by culturing under conditions where the cells were all in the exponential growth phase, or by a 12 h exposure to a C-kinase-activating phorbol ester. These results suggest that desensitization of certain agonist-induced inositol phospholipid responses in ras-transformed cells is a consequence of increased cell proliferation and associated amplification in C-kinase activity and is an indirect consequence of transformation by ras.

A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene

1986 ◽  
Vol 6 (5) ◽  
pp. 1843-1846
Author(s):  
A G Papageorge ◽  
B M Willumsen ◽  
M Johnsen ◽  
H F Kung ◽  
D W Stacey ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
S Phase ◽  
3T3 Cells ◽  
Ras Gene ◽  
Mutant Proteins ◽  
Ras Protein ◽  
Ras Genes ◽  
Essential Function ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

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 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.

scholarly journals Experimental metastasis in nude mice of NIH 3T3 cells containing various ras genes.

1986 ◽  
Vol 83 (14) ◽  
pp. 5277-5281 ◽  
Author(s):  
M. O. Bradley ◽  
A. R. Kraynak ◽  
R. D. Storer ◽  
J. B. Gibbs
Keyword(s):  
Nude Mice ◽  
Experimental Metastasis ◽  
3T3 Cells ◽  
Ras Genes ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

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.

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.

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