Both v-Ha-Ras and v-Raf stimulate expression of the vascular endothelial growth factor in NIH 3T3 cells.
Stimulation of NIH 3T3 cells with platelet-derived growth factor (PDGF)-BB and 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances vascular endothelial growth factor (VEGF) gene expression. To address the question of whether Ras and Raf are involved in the induction of VEGF gene expression by PDGF and TPA, we examined the effects of both factors on NIH 3T3 cells stably transfected with v-Ha-ras or v-raf. In serum-starved NIH 3T3 cells, only low levels of mRNA expression can be detected, whereas both ras and raf transformed cell lines express enhanced levels of a 4.3-kilobase VEGF transcript. Stimulation with PDGF or TPA resulted in increased VEGF mRNA in all cell lines, with highest levels found in the transformed cells. Immunofluorescence studies confirmed that the elevated VEGF mRNA expression correlated with enhanced protein levels. Positive immunofluorescence signals could be detected in v-Ha-ras or v-raf transformed cell lines but not in unstimulated NIH 3T3 cells. VEGF from conditioned medium of v-raf transformed NIH 3T3 cells was partially purified by chromatography on heparin-Sepharose. Biological activity of this VEGF protein was demonstrated by competition with binding of recombinant 125I-VEGF165 to human umbilical vein endothelial cells and by its ability to stimulate proliferation of these cells.
Grugel
S
S
Institute for Molecular Medicine, Tumor Biology Center, Freiburg, Germany.
Finkenzeller
G
G
Weindel
K
K
Barleon
B
B
Marmé
D
D
D016475:3T3 Cells; D000818:Animals; D001667:Binding, Competitive; D015152:Blotting, Northern; D015153:Blotting, Western; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D017077:Culture Media, Conditioned; D016228:Endothelial Growth Factors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D008222:Lymphokines / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D027384:Oncogene Proteins v-raf; D011485:Protein Binding; D015682:Retroviridae Proteins, Oncogenic / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D017077:Culture Media, Conditioned; D016228:Endothelial Growth Factors; D008222:Lymphokines; D015682:Retroviridae Proteins, Oncogenic; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D027384:Oncogene Proteins v-raf; D015689:Oncogene Protein p21(ras)