Abstract
C3H10T1/2 fibroblasts transformed by the minimal expression of oncogenic Ha-Ras (V12H10 cells) or N-Ras (K61N10 cells) have constitutive mitogen-activated protein kinase (MAPK) activity and proliferate in serum-free medium. The constitutive MAPK activity and serum-independent proliferation of V12H10 cells are sensitive to the growth factor antagonist, suramin (Hamilton, M., and Wolfman, A. (1998) Oncogene 16, 1417-1428), suggesting that Ha-Ras-mediated regulation of the MAPK cascade is dependent upon the action of an autocrine factor. Serum-free medium conditioned by V12H10 cells contains an activity that stimulates MAPK activity in quiescent fibroblasts. This MAPK stimulatory activity could be specifically blocked by the epidermal growth factor receptor (EGFR) inhibitors, PD153035 and PD158780. These inhibitors also blocked the serum-independent proliferation of V12H10 cells. Immunodepletion of conditioned medium with antibodies to transforming growth factor alpha and EGF significantly inhibited its ability to stimulate MAPK activity. Stable transfection of EGFR-negative NR6 and EGFR-positive Swiss3T3 cells with oncogenic (G12V)Ha-Ras demonstrated that only the Ha-Ras-transfected Swiss 3T3 cells possessed constitutive MAPK activity, and this activity was sensitive to PD153035. These data suggest that autocrine activation of the EGFR is required for the regulation of the MAPK cascade in cells minimally expressing oncogenic Ha-Ras.
Highlights
We recently reported that mouse fibroblasts transformed by the minimal expression of activated (G12V)Ha-Ras (V12H10 cells) were unable to proliferate in serum-free medium in the presence of suramin, a growth factor antagonist [13,14,15]
Detection of a mitogen-activated protein kinase (MAPK)-stimulating Activity in Conditioned Medium—To determine whether Ha-Ras-transformed V12H10 cells secrete a factor that is responsible for regulating their constitutive MAPK activity, sub-confluent V12H10 cells were incubated in serum-free DMEM for 72 h
The expression of activated Ras proteins is transforming as measured by alterations in cell morphology, the ability to grow in soft agar, constitutive MAPK activity, and a diminished requirement for exogenous growth factors or serum
Summary
We recently reported that mouse fibroblasts transformed by the minimal expression of activated (G12V)Ha-Ras (V12H10 cells) were unable to proliferate in serum-free medium in the presence of suramin, a growth factor antagonist [13,14,15]. The failure of oncogenic Ha-Ras to associate with Raf-1 is contrary to current dogma, which suggests that it is the direct association of an oncogenic Ras with Raf-1 that is responsible for regulating the MAPK cascade. These observations imply that the mechanism through which Ha-Ras regulates MAPK activity was through the action of an autocrine growth factor(s) rather than through a direct association with Raf-1. We sought to determine which receptor pathway activity was required to regulate MAPK activity and the serum-independent proliferation of Ha-Ras-transformed mouse fibroblasts
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