Signaling cascades elicited by angiotensin II resemble those characteristic of growth factor stimulation. In this report, we demonstrate that angiotensin II converges with platelet-derived growth factor (PDGF) beta-receptor signaling cascades, independent of PDGF. Stimulation of smooth muscle cells with angiotensin II resulted in tyrosine phosphorylation on Shc proteins and subsequent complex formation between Shc and growth factor receptor binding protein-2 (GRB2). A 180-kDa protein co-precipitating with Shc.GRB2 complexes also demonstrated increased phosphorylation in response to angiotensin II. Immunoblot analyses and proteolytic digests failed to distinguish this 180-kDa protein from authentic PDGF beta-receptors. Corresponding with Shc and PDGF receptor phosphorylation induced by angiotensin II was the recruitment and phosphorylation of c-Src. Autocrine release of platelet-derived growth factor failed to account for Shc complex formation at the PDGF receptor following angiotensin II treatment, and a specific angiotensin II type I receptor antagonist, losartan, abolished the response. These results support a novel model for cross-talk between the G-protein-linked angiotensin II receptor and the PDGF receptor tyrosine kinase in vascular smooth muscle cells. Communication with the PDGF receptor may account for the ability of angiotensin II to elicit responses typical of growth factor signal transduction.

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Studies of the biochemical mechanisms evoked by conventional treatments for neoplastic diseases point to apoptosis as a key process for elimination of unwanted cells. Although the pathways through which chemotherapeutics promote cell death remain largely unknown, caspase proteases play a central role in the induction of apoptosis in response to a variety of stimuli including tumor necrosis factor, fas ligand, and growth factor deprivation. In this article, we demonstrate the induction of caspase protease activity in MCF7 human breast carcinoma cells exposed to the topoisomerase inhibitor, etoposide. Caspase protease activity was assessed by incubating cell lysates with the known caspase substrates,We observed maximal cleavage of acetyl-L-aspartic-L-glutamic-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin within 6 hr following etoposide addition, a time that precedes cell death. In contrast, acetyl-L-tyrosyl-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin was resistant to cleavage activity. This substrate cleavage specificity implies that a caspase-3-like protease is activated in response to DNA damage. Consistent with the lysate protease activity, an intracellular marker of caspase activation, poly-ADP ribose polymerase (PARP), was cleaved in a concentration-and time-dependent manner after etoposide-treatment. PARP cleavage followed caspase activation and reached maximum cleavage between 12 and 16 hr. Incubation of the cells with the peptidic caspase inhibitor z-valine-alanine-asparagine-CH 2 F prevented caspase activation, inhibited PARP cleavage, and inhibited cell death. Thus, etoposide killing of MCF7 cells requires a caspase-3-like protease.

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.

Data indicating that the 21-kDa protein (p21) Harvey-ras gene product shares sequence homology with guanine nucleotide-binding proteins (G proteins) has stimulated research on the influence(s) of p21 on G-protein-regulated systems in vertebrate cells. Our previous work demonstrated that NIH-3T3 mouse cells expressing high levels of the cellular ras oncogene isolated from the EJ human bladder carcinoma (EJ-ras) exhibited reduced hormone-stimulated adenylate cyclase activity. We now report that in these cells another enzyme system thought to be regulated by G proteins is inhibited, namely phospholipases A2 and C. NIH-3T3 cells incubated in plasma-derived serum release significant levels of prostaglandin E2 (PGE2) as determined by radioimmunoassay when exposed to platelet-derived growth factor (PDGF) at 2 units/ ml; the levels of PGE2 released from EJ-ras-transfected cells are only 3% those of controls despite a similar basal (unstimulated) (Ha-ras) and Kirsten-ras (Ki-ras) are related to acutely transforming murine sarcoma retroviruses, and the third, N-ras, was isolated from a human neuroblastoma (1-4). § ras genes encode proteins with mo-