NIH-3T3 cells transformed by the EJ-ras oncogene exhibit reduced platelet-derived growth factor-mediated Ca2+ mobilization.

Abstract

NIH-3T3 cells transformed by the EJ-ras oncogene synthesize only 10-15% as much inositol 1,4,5-trisphosphate (InsP3) as control cells after stimulation with platelet-derived growth factor (PDGF). This is despite the fact that the basal (unstimulated) levels of InsP3 synthesized in control and EJ-ras-transformed cells are not significantly different. Using the fluorescent indicator fura-2 and digitalimaging techniques, we have visualized and quantified changes in intracellular Ca2+ concentrations in control and EJ-rastransformed NIH-3T3 cells in response to PDGF. Within 3 min after exposure of control cells to PDGF, intracellular Ca2+ levels are increased 3-to 9-fold, paralleling the increase in InsP3. In contrast, the majority (>90%) of the EJ-rastransformed cells show no increase in Ca2+ levels after PDGF exposure and the few that did respond exhibited only a small transient increase. Pronounced differences in the intracellular localization of Ca2+ increases in control and the responding EJ-ras-transformed cells were also observed. Despite the inhibition of InsP3 synthesis and subsequent Ca2+ mobilization, the EJ-ras-transformed cells respond mitogenically to PDGF. These data do not support the hypothesis that the EJ-ras gene product (p21) stimulates a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in NIH-3T3 cells; instead they suggest that the EJ-ras p21 may uncouple the PDGF receptor from phospholipase C resulting in inhibition of PDGFstimulated activity of phospholipase C, InsP3 synthesis, and Ca2+ mobilization.creased PLC activity (14,15 (16). In this report we show by digital-imaging techniques and fura-2 fluorescence measurements that PDGF-stimulated Ca2 + mobilization is diminished in EJ-rastransformed cells, indicating that the entire sequence of PDGF-stimulated PtdInsP2 hydrolysis, InsP3 synthesis, and Ca2+ mobilization is inhibited. These data appear to dissociate inositol phospholipid turnover and Ca2+ mobilization from PDGF-stimulated mitogenesis in these transformed cells.

Repression of Platelet-Derived Growth Factor β-Receptor Expression by Mitogenic Growth Factors and Transforming Oncogenes in Murine 3T3 Fibroblasts

“…Similarly, previous reports from this laboratory (40,41) and others (3,4,29,35) have shown significant decreases in PDGF-induced second messenger synthesis and immediate-early gene induction in v-ras-transformed fibroblasts relative to untransformed cells. Recent studies in this laboratory have shown that v-ras-transformed cells contain a potent inhibitor of PDGF␤R phosphorylation (41,61); the present study indicates that the decreased responsiveness of v-rastransformed cells to PDGF may additionally result from reduced expression of the PDGF␤R.…”

Antisense-fos RNA causes partial reversion of the transformed phenotypes induced by the c-Ha-ras oncogene.

“…Thus, c-fos induction by ras-regulated signal transduction pathways appears to be desensitized in cells that continuously overexpress ras oncogenes. Desensitization of phospholipase C (5,6) and protein kinase C (50) activation has also been observed in ras-transformed cells, further implicating the involvement of these activities in signal transduction pathways that are regulated by ras proteins and that result in c-fos gene expression.…”

Cells transformed by ODC, c-Ha-ras and v-src exhibit MAP kinase/Erk-independent constitutive phosphorylation of Sos, Raf and c-Jun activation domain, and reduced PDGF receptor expression

“…In addition, PLCg-1 has been reported to associate with the PDGF receptor and become phosphorylated almost normally in ras-transformed cells (Kaplan et al, 1990), in spite of the documented loss or reduction of the PLC activity. Further, there are con¯icting results as to whether the mitogenic response to PDGF is altered (Molloy et al, 1992;Parries et al, 1987) or not (Benjamin et al, 1988;Lin et al, 1988) following ras transformation. Controversial results also exist regarding the speci®city of these changes to ras transformation.…”

“…Cells transformed by ras, the most commonly activated oncogene in human cancers, have been found to become refractory to PDGF stimulation and display loss or suppression of the PDGFstimulated phospholipase C activity (Alonso et al, 1988;Parries et al, 1987), Ca 2+ mobilization (Benjamin et al, 1988) and expression of growth regulatory genes, like c-myc, c-fos and ornithine decarboxylase (ODC) (HoÈ lttaÈ et al, 1988;Lin et al, 1988;Zullo and Faller, 1988). The ras-transformed cells have, however, been found to display relatively normal levels of PDGF receptors (Parries et al, 1987;Rake et al, 1991;Zullo and Faller, 1988).…”