Activation of c-myc and c-K-ras oncogenes in primary rat tumors induced by ionizing radiation

1987 ◽  
Vol 7 (2) ◽  
pp. 932-935
Author(s):  
M J Sawey ◽  
A T Hood ◽  
F J Burns ◽  
S J Garte
Keyword(s):  
Ionizing Radiation ◽  
Tissue Specificity ◽  
3T3 Cells ◽  
Rat Skin ◽  
Oncogene Activation ◽  
Myc Gene ◽  
Ras Oncogenes ◽  
Restriction Polymorphisms ◽  
Rat Tumors ◽  
Nih 3T3

An activated K-ras oncogene was detected by transfection in NIH 3T3 cells and by Southern blot analysis in 6 of 12 rat skin tumors induced by ionizing radiation. The DNA from 10 of the 12 tumors also showed c-myc gene amplification and restriction polymorphisms. Evidence for tissue specificity was observed in patterns of oncogene activation, with each of three clear cell carcinomas exhibiting activation of both c-myc and K-ras oncogenes.

scholarly journals Activation of c-myc and c-K-ras oncogenes in primary rat tumors induced by ionizing radiation.

1987 ◽  
Vol 7 (2) ◽  
pp. 932-935 ◽  
Author(s):  
M J Sawey ◽  
A T Hood ◽  
F J Burns ◽  
S J Garte
Keyword(s):  
Ionizing Radiation ◽  
Tissue Specificity ◽  
3T3 Cells ◽  
Rat Skin ◽  
Oncogene Activation ◽  
Myc Gene ◽  
Ras Oncogenes ◽  
Restriction Polymorphisms ◽  
Rat Tumors ◽  
Nih 3T3

An activated K-ras oncogene was detected by transfection in NIH 3T3 cells and by Southern blot analysis in 6 of 12 rat skin tumors induced by ionizing radiation. The DNA from 10 of the 12 tumors also showed c-myc gene amplification and restriction polymorphisms. Evidence for tissue specificity was observed in patterns of oncogene activation, with each of three clear cell carcinomas exhibiting activation of both c-myc and K-ras oncogenes.

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.

Rat c-raf oncogene activation by a rearrangement that produces a fused protein

1987 ◽  
Vol 7 (3) ◽  
pp. 1226-1232
Author(s):  
F Ishikawa ◽  
F Takaku ◽  
M Nagao ◽  
T Sugimura
Keyword(s):  
Long Terminal Repeat ◽  
Rous Sarcoma Virus ◽  
Cell Transfection ◽  
3T3 Cells ◽  
Rous Sarcoma ◽  
Oncogene Activation ◽  
Sarcoma Virus ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Transfection Assay

In a previous study, activated rat c-raf was detected by an NIH 3T3 cell transfection assay, and a rearrangement was demonstrated in the 5' half of the sequence of the gene. In the present study, the cDNAs of normal and activated rat c-raf were analyzed. Results showed that the activated c-raf gene is transcribed to produce a fused mRNA, in which the 5' half of the sequence is replaced by an unknown rat sequence. This mRNA codes a fused c-raf protein. The normal and activated c-raf cDNAs were each connected to the long terminal repeat of Rous sarcoma virus and transfected into NIH 3T3 cells. Only the activated form had transforming activity. We conclude that the rearrangement is responsible for the activation of c-raf.

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.

Alterations of G-protein coupling function in phosphoinositide signaling pathways of cells transformed by ras and other membrane-associated and cytoplasmic oncogenes

1990 ◽  
Vol 10 (6) ◽  
pp. 3117-3124
Author(s):  
T Alonso ◽  
S Srivastava ◽  
E Santos
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Protein S ◽  
3T3 Cells ◽  
Phosphoinositide Signaling ◽  
Common Effect ◽  
Receptor Number ◽  
Ras Oncogenes ◽  
Nih 3T3 ◽  
Functional Alteration

We showed previously that transformation by cytoplasmic and membrane-associated oncogenes including ras results in uncoupling between surface stimulation by platelet-derived growth factor, bombesin, and serum and activation of intracellular phospholipase C (PLC); this uncoupling does not involve alterations at the receptor or effector enzyme levels (T. Alonso, R. O. Morgan, J. C. Marvizon, H. Zarbl, and E. Santos, Proc. Natl. Acad. Sci. USA 85:4271-4275, 1988). In this study, we stimulated normal and oncogene-transformed NIH 3T3 cells with fluoroaluminate (AIF4-), thus directly activating PLC-associated G protein(s) and bypassing the receptor step. A1F4(-)-elicited PLC responses were significantly impaired in transformed cells when compared with those in their normal counterparts, suggesting that the uncoupling of PLC is the result, at least in part, of functional impairment at the G-protein level. Transformation by ras oncogenes has also been reported to result in enhanced PLC response to bradykinin resulting from increased receptor numbers (G. Parries, R. Hoebel, and E. Racker, Proc. Natl. Acad. Sci. USA 84:2648-2652, 1987; J. Downward, J. de Gunzburg, R. Riehl, and R. Weinberg, Proc. Natl. Acad. Sci. USA 85:5774-5778, 1988). We demonstrate here that transformation by other membrane-associated and cytoplasmic oncogenes also results in increased responsiveness to bradykinin ("supercoupling") and enhanced receptor numbers. However, there is no direct correlation between the number of receptors and the enhancement in responsiveness, suggesting that other factors besides receptor number are also involved in the enhanced responses. We propose that a common effect of transformation by cytoplasmic and membrane-associated oncogenes is functional alteration of coupling G proteins and that a similar modification of different kinds of G proteins may account for the pleiotropic alterations of signal transduction (uncoupling and supercoupling) observed.

scholarly journals Alterations of G-protein coupling function in phosphoinositide signaling pathways of cells transformed by ras and other membrane-associated and cytoplasmic oncogenes.

1990 ◽  
Vol 10 (6) ◽  
pp. 3117-3124 ◽  
Author(s):  
T Alonso ◽  
S Srivastava ◽  
E Santos
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Protein S ◽  
3T3 Cells ◽  
Phosphoinositide Signaling ◽  
Common Effect ◽  
Receptor Number ◽  
Ras Oncogenes ◽  
Nih 3T3 ◽  
Functional Alteration

We showed previously that transformation by cytoplasmic and membrane-associated oncogenes including ras results in uncoupling between surface stimulation by platelet-derived growth factor, bombesin, and serum and activation of intracellular phospholipase C (PLC); this uncoupling does not involve alterations at the receptor or effector enzyme levels (T. Alonso, R. O. Morgan, J. C. Marvizon, H. Zarbl, and E. Santos, Proc. Natl. Acad. Sci. USA 85:4271-4275, 1988). In this study, we stimulated normal and oncogene-transformed NIH 3T3 cells with fluoroaluminate (AIF4-), thus directly activating PLC-associated G protein(s) and bypassing the receptor step. A1F4(-)-elicited PLC responses were significantly impaired in transformed cells when compared with those in their normal counterparts, suggesting that the uncoupling of PLC is the result, at least in part, of functional impairment at the G-protein level. Transformation by ras oncogenes has also been reported to result in enhanced PLC response to bradykinin resulting from increased receptor numbers (G. Parries, R. Hoebel, and E. Racker, Proc. Natl. Acad. Sci. USA 84:2648-2652, 1987; J. Downward, J. de Gunzburg, R. Riehl, and R. Weinberg, Proc. Natl. Acad. Sci. USA 85:5774-5778, 1988). We demonstrate here that transformation by other membrane-associated and cytoplasmic oncogenes also results in increased responsiveness to bradykinin ("supercoupling") and enhanced receptor numbers. However, there is no direct correlation between the number of receptors and the enhancement in responsiveness, suggesting that other factors besides receptor number are also involved in the enhanced responses. We propose that a common effect of transformation by cytoplasmic and membrane-associated oncogenes is functional alteration of coupling G proteins and that a similar modification of different kinds of G proteins may account for the pleiotropic alterations of signal transduction (uncoupling and supercoupling) observed.

Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression

1992 ◽  
Vol 12 (12) ◽  
pp. 5355-5362
Author(s):  
S J Langer ◽  
D M Bortner ◽  
M F Roussel ◽  
C J Sherr ◽  
M C Ostrowski
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Semisolid Medium ◽  
Myc Gene ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

The activity of p21ras is required for the proliferative response to colony-stimulating factor 1 (CSF-1), and signals transduced by both the CSF-1 receptor (CSF-1R) and p21ras stimulate transcription from promoter elements containing overlapping binding sites for Fos/Jun- and Ets-related proteins. A sequence encoding the DNA-binding domain and nuclear localization signal of human c-ets-2, which lacked portions of the c-ets-2 gene product necessary for trans activation, was fused to the bacterial lacZ gene and expressed from an actin promoter in NIH 3T3 cells expressing either the v-ras oncogene or human CSF-1R. Nuclear expression of the Ets-LacZ protein, confirmed by histochemical staining of beta-galactosidase, inhibited the activity of ras-responsive enhancer elements and suppressed morphologic transformation by v-ras as well as CSF-1R-dependent colony formation in semisolid medium. When CSF-1R-bearing cells expressing the Ets-LacZ protein were stimulated by CSF-1, induction of c-ets-2, c-jun, and c-fos ensued, but the c-myc response was impaired. Enforced expression of the c-myc gene overrode the suppressive effect of ets-lacZ and restored the ability of these cells to form colonies in response to CSF-1. NIH 3T3 cells engineered to express a CSF-1R (Phe-809) mutant similarly cannot form CSF-1-dependent colonies in semisolid medium and exhibit an impaired c-myc response, but expression of an exogenous myc gene resensitizes these cells to CSF-1 [M. F. Roussel, J. L. Cleveland, S. A. Shurtleff, and C. J. Sherr, Nature (London) 353:361-363, 1991]. The ability of these cells to respond to CSF-1 was also rescued by enforced expression of an endogenous c-ets-2 gene. The ets family of transcription factors therefore plays a central role in integrating both CSF-1R and ras-induced mitogenic signals and in modulating the myc response to CSF-1 stimulation.

scholarly journals Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression.

1992 ◽  
Vol 12 (12) ◽  
pp. 5355-5362 ◽  
Author(s):  
S J Langer ◽  
D M Bortner ◽  
M F Roussel ◽  
C J Sherr ◽  
M C Ostrowski
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Colony Stimulating Factor ◽  
3T3 Cells ◽  
Semisolid Medium ◽  
Myc Gene ◽  
Nih 3T3 ◽  
Stimulating Factor ◽  
Nih 3T3 Cells

The activity of p21ras is required for the proliferative response to colony-stimulating factor 1 (CSF-1), and signals transduced by both the CSF-1 receptor (CSF-1R) and p21ras stimulate transcription from promoter elements containing overlapping binding sites for Fos/Jun- and Ets-related proteins. A sequence encoding the DNA-binding domain and nuclear localization signal of human c-ets-2, which lacked portions of the c-ets-2 gene product necessary for trans activation, was fused to the bacterial lacZ gene and expressed from an actin promoter in NIH 3T3 cells expressing either the v-ras oncogene or human CSF-1R. Nuclear expression of the Ets-LacZ protein, confirmed by histochemical staining of beta-galactosidase, inhibited the activity of ras-responsive enhancer elements and suppressed morphologic transformation by v-ras as well as CSF-1R-dependent colony formation in semisolid medium. When CSF-1R-bearing cells expressing the Ets-LacZ protein were stimulated by CSF-1, induction of c-ets-2, c-jun, and c-fos ensued, but the c-myc response was impaired. Enforced expression of the c-myc gene overrode the suppressive effect of ets-lacZ and restored the ability of these cells to form colonies in response to CSF-1. NIH 3T3 cells engineered to express a CSF-1R (Phe-809) mutant similarly cannot form CSF-1-dependent colonies in semisolid medium and exhibit an impaired c-myc response, but expression of an exogenous myc gene resensitizes these cells to CSF-1 [M. F. Roussel, J. L. Cleveland, S. A. Shurtleff, and C. J. Sherr, Nature (London) 353:361-363, 1991]. The ability of these cells to respond to CSF-1 was also rescued by enforced expression of an endogenous c-ets-2 gene. The ets family of transcription factors therefore plays a central role in integrating both CSF-1R and ras-induced mitogenic signals and in modulating the myc response to CSF-1 stimulation.

scholarly journals Transforming genes in human leukemia cells

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1371-1378
Author(s):  
H Hirai ◽  
S Tanaka ◽  
M Azuma ◽  
Y Anraku ◽  
Y Kobayashi ◽  
...  
Keyword(s):  
Acute Lymphocytic Leukemia ◽  
Bone Marrow Cells ◽  
Lymphocytic Leukemia ◽  
Myelogenous Leukemia ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Oncogenes ◽  
Nih 3T3 ◽  
Transforming Genes ◽  
Transfection Assay

High-molecular weight DNAs of fresh bone marrow cells from 32 patients with fresh leukemia were assayed for the presence of transmissible activated transforming genes by a DNA-mediated gene transfer technique using NIH/3T3 cells. DNAs of bone marrow cells from four of the 32 patients induced transformation of NIH/3T3 cells. Two of the four cases, a chronic myelogenous leukemia and an acute lymphocytic leukemia, contained activated N-ras oncogenes. Molecular cloning and nucleotide sequence analysis revealed that the lesion responsible for the transforming activity was localized to a single nucleotide transition from guanine to thymine in codon 12 of the predicted protein in each of the two cases. These observations indicate that activation of N-ras oncogenes is independent of the specific stage of cell differentiation or the leukemia phenotype. The other two transforming genes associated with an acute myelogenous leukemia and an acute lymphocytic leukemia showed homology neither with members of the ras gene family nor with the human Blym-1 gene. Thus, the NIH/3T3 transfection assay frequently detects activated N-ras oncogenes in human leukemias, while other transforming genes, distinct from the ras gene family, can be detected in some leukemias by the transfection assay.

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