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 NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

1985 ◽  
Vol 5 (1) ◽  
pp. 259-262 ◽  
Author(s):  
U P Thorgeirsson ◽  
T Turpeenniemi-Hujanen ◽  
J E Williams ◽  
E H Westin ◽  
C A Heilman ◽  
...  
Keyword(s):  
Nude Mice ◽  
Dna Sequences ◽  
Immune Cell ◽  
Human Tumor ◽  
3T3 Cells ◽  
Metastatic Phenotype ◽  
Ras Oncogenes ◽  
Nih 3T3 ◽  
Tumor Dna ◽  
Nih 3T3 Cells

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.

scholarly journals Identification of ErbB3-stimulated genes using modified representational difference analysis

1997 ◽  
Vol 323 (1) ◽  
pp. 113-118 ◽  
Author(s):  
Carl F. EDMAN ◽  
Sally A. PRIGENT ◽  
Andrea SCHIPPER ◽  
James R. FERAMISCO
Keyword(s):  
Dna Sequences ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Representational Difference Analysis ◽  
3T3 Cells ◽  
Difference Analysis ◽  
Total Rna ◽  
Egfr Family ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The epidermal growth factor receptor (EGFR) family of tyrosine kinases is involved in the growth of normal and tumour cells. The specific contribution of each of the four family members to these processes remains unclear. In the present study we have used a PCR-based subtractive approach to identify differences in messages induced in response to activation of ErbB3 and EGFR. The approach described is a modification of the representational difference analysis technique adapted for analysis of cDNA, which we have modified to permit identification of differential gene expression using as little as 20 μg of total RNA as the starting material. The mRNA obtained from EGF-stimulated NIH-3T3 cells expressing chimaeric EGFR-ErbB3 receptors provided the tester amplicons (small PCR-amplified fragments) which were subtracted against driver amplicons derived from unstimulated NIH-3T3 cells expressing the EGFR-ErbB3 chimaera or EGF-stimulated NIH-3T3 cells overexpressing the EGFR. A total of 22 different clones were isolated, 90% of which showed increased expression in the tester amplicons. Six of these, corresponding to known DNA sequences, were selected for further Northern blot analysis against total RNA prepared from the starting cell lines. Of these, the gene encoding the protein dlk (or a closely related protein, Pref-1) was identified as being regulated by ErbB3 but not by the EGFR. Other genes appeared to be elevated by both ErbB3 and EGFR, including those encoding c-jun, Ret finger protein (RFP), neuroleukin and amyloid protein precursor. One gene product, TIS11, was identified as being regulated by EGFR but not by ErbB3.

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice

1985 ◽  
Vol 5 (1) ◽  
pp. 259-262
Author(s):  
U P Thorgeirsson ◽  
T Turpeenniemi-Hujanen ◽  
J E Williams ◽  
E H Westin ◽  
C A Heilman ◽  
...  
Keyword(s):  
Nude Mice ◽  
Dna Sequences ◽  
Immune Cell ◽  
Human Tumor ◽  
3T3 Cells ◽  
Metastatic Phenotype ◽  
Ras Oncogenes ◽  
Nih 3T3 ◽  
Tumor Dna ◽  
Nih 3T3 Cells

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.

Induction of a mitogen-responsive gene after expression of the Ha-ras oncogene in NIH 3T3 fibroblasts

1989 ◽  
Vol 9 (11) ◽  
pp. 5207-5214
Author(s):  
A K Werenskiold ◽  
S Hoffmann ◽  
R Klemenz
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Transcriptional Level ◽  
3T3 Cells ◽  
Serum Factors ◽  
Kinase C ◽  
Conditional Expression ◽  
T1 Gene ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

A cDNA clone, T1, has been isolated whose corresponding mRNA was transiently expressed at highly elevated levels after conditional expression of the Ha-ras(EJ) gene and after mitogenic activation of quiescent NIH 3T3 cells. Glucocorticoid hormone stimulated substantial T1 expression as well but only in proliferating cells. At least two different signaling pathways participate in the regulation of the T1 gene: a protein kinase C-dependent signal is involved in the response of proliferating NIH 3T3 cells to glucocorticoid in the absence but not the presence of p21ras, whereas a protein kinase C-independent mechanism mediates the response to serum factors. Treatment of cells with the protein kinase inhibitor 2-aminopurine blocked induction of expression of the T1 gene. T1 mRNA accumulation is regulated at the transcriptional level.

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 Induction of a mitogen-responsive gene after expression of the Ha-ras oncogene in NIH 3T3 fibroblasts.

1989 ◽  
Vol 9 (11) ◽  
pp. 5207-5214 ◽  
Author(s):  
A K Werenskiold ◽  
S Hoffmann ◽  
R Klemenz
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Transcriptional Level ◽  
3T3 Cells ◽  
Serum Factors ◽  
Kinase C ◽  
Conditional Expression ◽  
T1 Gene ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

A cDNA clone, T1, has been isolated whose corresponding mRNA was transiently expressed at highly elevated levels after conditional expression of the Ha-ras(EJ) gene and after mitogenic activation of quiescent NIH 3T3 cells. Glucocorticoid hormone stimulated substantial T1 expression as well but only in proliferating cells. At least two different signaling pathways participate in the regulation of the T1 gene: a protein kinase C-dependent signal is involved in the response of proliferating NIH 3T3 cells to glucocorticoid in the absence but not the presence of p21ras, whereas a protein kinase C-independent mechanism mediates the response to serum factors. Treatment of cells with the protein kinase inhibitor 2-aminopurine blocked induction of expression of the T1 gene. T1 mRNA accumulation is regulated at the transcriptional level.

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.

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.

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