Regulation of phosphoinositide phosphorylation in Swiss 3T3 cells stimulated by platelet-derived growth factor

The regulation of phosphoinositide phosphorylation was studied in Swiss 3T3 cells that were stimulated by platelet-derived growth factor (PDGF). Studies with intact cells showed that the mitogen increased the incorporation of 32P into phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns-P), and phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) during the cell cycle, with distinct peaks of incorporation for all three phosphoinositides after 1 h, and for PtdIns and PtdIns-P2 after 20 h. Direct measurements of the activities of PtdIns kinase and PtdIns-P kinase in freeze-thawed cells revealed that the activity of PtdIns kinase was rate-limiting for the synthesis of PtdIns-P2. Maximal activities of PtdIns kinase and PtdIns-P kinase, with exogenous substrates, were unchanged during the 1st h of PDGF treatment, but doubled during the next 24 h. The increase in PtdIns kinase activity began within 2-4 h, exceeded the increase in cell protein, and was abolished by cycloheximide, which suggests that the enzyme was induced specifically in response to PDGF. The increase in activity of PtdIns-P kinase paralleled the increase in cell protein. Dose-response curves for PDGF showed that the activities of PtdIns kinase and PtdIns-P kinase at 24 h increased in proportion to the extent of mitogenic stimulation of the cells. Our results support the conclusion that the activities of PtdIns kinase and PtdIns-P kinase increase in response to PDGF, but only after several hours of cell cycle traverse.     

Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor.

Swiss 3T3 cells incubated for 60 h with [3H]inositol incorporated radioactivity into phosphatidylinositol (PI) and the two polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). On stimulation with platelet-derived growth factor (PDGF) there were significant increases in the levels of inositol 1-phosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). The effect of PDGF and IP3 on Ca2+ mobilization was studied in both intact cells and in 'leaky' cells that had been permeabilized with saponin. In intact cells, PDGF stimulated the efflux of 45Ca2+, whereas IP3 had no effect. Conversely, IP3 stimulated 45Ca2+ efflux from 'leaky' cells, which were insensitive to PDGF. 'Leaky' cells, which accumulated 45Ca2+ to a steady state within 20 min, were found to release approx. 40% of the label within 1 min after addition of 10 microM-IP3. This stimulation of 45Ca2+ release by IP3 was reversible and was also dose-dependent, with a half-maximal effect at approx. 0.3 microM. It seems likely that an important action of PDGF on Swiss 3T3 cells is to stimulate the hydrolysis of PIP2 to form IP3 and diacylglycerol, both of which may function as second messengers. Our results indicate that IP3 mobilizes intracellular Ca2+, and we propose that diacylglycerol may act through C-kinase to activate the Na+/H+ antiport. By generating two second messengers, PDGF can simultaneously elevate the intracellular level of Ca2+ and alkalinize the cytoplasm by lowering the level of H+.     

Inhibition of early platelet-derived growth factor responses in BALB/c-3T3 cells by interferon

Stimulation of total inositol phosphate production, alteration of cytosolic free calcium [( Ca++]i), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGF were examined in normal and INF pretreated density arrested BALB/c-3T3 fibroblasts. In normal cells, PDGF caused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect on PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALB/c-3T3 cells into G1 by inhibiting PDGF-induced release of Ca++ from intracellular stores.     

Early changes in inositol lipids and their metabolites induced by platelet-derived growth factor in quiescent Swiss mouse 3T3 cells.

Inositol lipid turnover was studied in quiescent Swiss mouse 3T3 cells stimulated by platelet-derived growth factor (PDGF). Stimulation of the cells by PDGF for 10 min at 37 degrees C induced the following changes in lipids: in cells prelabelled with [32P]Pi, a 28% decrease in [32P]phosphatidylinositol 4,5-bisphosphate, a 41% decrease in [32P]phosphatidylinositol 4-phosphate and a 1.7-fold increase in the 32P-labelling of phosphatidic acid; in cells prelabelled with [3H8]arachidonic acid, a 17.9-fold increase in [3H]phosphatidic acid, a 20% decrease in [3H]phosphatidylinositol (PtdIns), an 8.6-fold increase in [3H]arachidonic acid released into the medium, a 57-fold increase in [3H]prostaglandin E2 in the medium, and a 5.3-fold increase in [3H]monoacylglycerol released into the medium (the last was identified as the 2-acyl derivative); in cells prelabelled with [2-3H]glycerol, a 1.7-fold increase in [3H]diacylglycerol, a 6.7-fold increase in [3H]phosphatidic acid, a 1.6-fold increase in [3H]lysophosphatidylcholine (lysoPtdCho), a 9% decrease in [3H]PtdIns, and a 1.6-fold increase in [3H]monoacylglycerol released into the medium. PDGF stimulated the formation of inositol tris-, bis- and mono-phosphates in the cells prelabelled with myo-[2-3H]inositol. These results indicate that, in Swiss 3T3 cells stimulated by PDGF, diacylglycerol produced by the hydrolysis of inositol lipids is partly degraded to 2-acylglycerol and partly converted into phosphatidic acid. The increase in lysoPtdCho indicates that a portion of arachidonic acid released from the stimulated cells is formed by the hydrolysis of PtdCho with a phospholipase A2. Different values of half-maximal doses of the partially purified PDGF used in this study were found for the various responses of quiescent Swiss 3T3 cells to PDGF. The values for half-maximal doses suggest that activation of a fraction of the cell-surface receptor for PDGF is sufficient for mitogenesis and for an increase in the cytoplasmic free Ca2+ concentration, and that the PGDF-stimulated lipid metabolism is probably proportional to the number of receptor sites activated by PDGF.     

Cell cycle-dependent changes in arachidonic acid and glycerol metabolism in Swiss 3T3 cells stimulated by platelet-derived growth factor

Quiescent Swiss 3T3 cells stimulated to divide by human platelet-derived growth factor (PDGF) were used to investigate cell cycle-dependent changes in arachidonic acid, stearic acid, and glycerol metabolism. PDGF at 12 ng/ml stimulated incorporation of labeled arachidonic and stearic acid into phosphatidic acid and phosphatidylinositol within 60 min. With similar kinetics PDGF stimulated glycerol incorporation into phosphatidic acid and phosphatidylinositol indicating early growth factor-dependent stimulation of de novo phosphatidylinositol synthesis. This early effect of PDGF was specific for the phosphatidylinositol synthesis pathway since no comparable changes were noted in other glycerolipids. After a lag of 4-6 h, PDGF strongly stimulated arachidonic acid incorporation into triacylglycerol: at 6 h, arachidonate radioactivity in triacylglycerol exceeded that in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. This effect of PDGF was not associated with de novo triacylglycerol synthesis since no increase in the rate of glycerol incorporation into this lipid was noted. Finally, PDGF stimulated incorporation of glycerol into all major phospholipids and triacylglycerol during S-phase. These results disclose three novel effects of PDGF on glycerolipid metabolism in Swiss 3T3 cells: 1) early selective activation of the phosphatidylinositol synthesis pathway; 2) delayed strong stimulation of arachidonic acid incorporation into triacylglycerol; and 3) late induction of de novo phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol synthesis. These PDGF effects are likely to play important roles in phosphatidylinositol metabolism, membrane biosynthesis, and fatty acid turnover in rapidly growing cells.     

Effect of the different dimeric forms of the platelet-derived growth factor on cellular responses in mouse Swiss 3T3 fibroblasts

PDGF consists of two polypeptide chains, A and B, and all three possible dimers have been isolated from different sources. Human PDGF, essentially AB, porcine PDGF (BB) and recombinant PDGF-AA were tested on Swiss 3T3 fibroblasts for their ability to stimulate mitogenesis, phosphoinositide turnover and tyrosine phosphorylation of the PDGF receptor. When used in saturating amounts, the three isoforms were equally active in inducing mitogenesis. However, PDGF-AA was less active than AB and BB to induce the phosphorylation of the receptor and the turnover of phosphoinositides (30% and 50%, respectively). These findings suggest that, in Swiss 3T3 fibroblasts, PDGF receptors of the alpha-type are present in a slightly lower amount than beta-type. In addition, the two types of receptor appear to have similar physiological functions.     

Diacylglycerol treatment rapidly decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells

The synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37 degrees C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C after a 1 h incubation with a saturating dose of OAG at 37 degrees C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I-EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells.     

Bombesin and platelet-derived growth factor induce association of endogenous focal adhesion kinase with Src in intact Swiss 3T3 cells.

Stimulation of quiescent Swiss 3T3 cells with bombesin induces a rapid increase in the formation of complexes between focal adhesion kinase (FAK) and Src family members, which can be extracted with a buffer containing Triton, deoxycholate, and SDS but not with a buffer containing Triton alone. An increase in complex formation between FAK and Src in response to bombesin could be detected within 1 min, reached a maximum after 10 min, and declined toward base-line levels after 60 min of bombesin treatment. Bradykinin, endothelin, and lysophosphatidic acid also stimulated FAK-Src complex formation. Bombesin stimulated FAK/Src association through a Ca(2+) and phosphatidylinositol 3'-kinase-independent pathway that requires the integrity of the actin filament network and is partly dependent on functional protein kinase C. Treatment with the selective Src kinase inhibitor PP-2 inhibited both FAK activation and phosphorylation of FAK at Tyr(577) induced by bombesin in intact cells. Platelet-derived growth factor at low concentrations (1-10 ng/ml) also induced FAK-Src complex formation via a pathway that depended on the integrity of the actin cytoskeleton and phosphatidylinositol 3'-kinase. Thus, G protein-coupled receptor agonists and platelet-derived growth factor promote complex formation between endogenous FAK and Src in attached cells through different signal transduction pathways.     

Modulation of the epidermal growth factor receptor by brain-derived growth factor in Swiss mouse 3T3 cells

Incubation of Swiss mouse 3T3 cells at 37 degrees C with bovine brain-derived growth factor (BDGF) decrease the cell surface 125I-EGF binding activity of these cells by 70-80%. This down-modulation of the EGF receptor by BDGF was time, temperature, and dose dependent. Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high-affinity EGF receptors. The BDGF-induced down-modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF. BDGF down-modulated the EGF receptor in phorbol myristic acetate (PMA)-pretreated cells, as well as in control cells. Furthermore, PMA-pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA-pretreated cells. This BDGF-induced down-modulation of the EGF receptor in PMA-desensitized cells suggests that BDGF down-regulates the EGF receptor by a mechanism distinct from that of PMA. Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down-modulation induced either by BDGF or by platelet-derived growth factor (PDGF) but had no effect on the PMA-induced down-modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF-induced down-modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down-modulation of the EGF receptor by increasing the internalization of cell surface high-affinity receptors and that the internalization process may not be required for down-modulation induced by PMA.     

Effects of platelet-derived growth factor on Ca in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca²⁺ was examined in BALB/c-3T3 cells. PDGF induced:

1. 1. A decrease in cell ⁴⁵Ca²⁺ content.

2. 2. An apparent increased rate of efflux of preloaded ⁴⁵Ca²⁺.

3. 3. A decrease in residual intracellular ⁴⁵Ca²⁺ remaining after rapid efflux.

4. 4. When added after the rapid phase of efflux of ⁴⁵Ca²⁺ had occurred, an immediate decrease in post-efflux residual intracellular ⁴⁵Ca²⁺.

All of the observed changes in ⁴⁵Ca²⁺ induced by PDGF are consistent with a rapid release of Ca²⁺ from an intracellular Ca²⁺ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca²⁺ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca²⁺ stores and CTC fluorescence intensity were identical with the effects of the Ca²⁺ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca²⁺ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca²⁺ stores. Release of Ca²⁺ from intracellular sequestration sites may be a mechanism by which PDGF Stimulates Cell growth.     

Bombesin and platelet-derived growth factor stimulate formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms.

Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.     

Epidermal growth factor and bombesin differ strikingly in the induction of early responses in Swiss 3T3 cells

Swiss 3T3 cells express receptors for both the polypeptide epidermal growth factor (EGF) and the tetradecapeptide bombesin and respond mitogenically to these substances. These cells thus provide a system to analyze potential signal transduction pathways involved in mitogenic stimulation. Here we have determined and compared the early ionic responses elicited by EGF and bombesin and their relation to diacylglycerol (DG) and inositolphosphate (InsPn) production. Whereas EGF fails to cause any significant change in intracellular Ca2+, bombesin effectively induces prompt and transient Ca2+ mobilization from intracellular stores. Further support of the idea that these receptors utilize distinct signalling pathways comes from the measurements of cytoplasmic pH (pHi). As in most target cells, EGF induces a delayed (1 min) but sustained intracellular alkalinization that reaches a new steady state after approximately 10 min. Bombesin, in contrast, elicits a biphasic response; within seconds, a rapid but transient rise in pHi is observed, followed by a further slower sustained alkalinization. Inhibition of the Na+/H+ exchanger prevents both EGF as well as bombesin-induced alkalinization. However, under these conditions, bombesin evokes a rapid and sustained acidification related to the Ca2+ response. Apparently, bombesin initiates a Ca2(+)-dependent acidifying process immediately after binding of the hormone to its receptor. Furthermore, we could demonstrate that the bombesin-induced alkalinization depends on protein kinase C activation whereas the EGF response does not. Determination of the total DG and InsPn accumulation revealed that EGF is ineffective in stimulating phospholipase C-mediated production of these second messengers. In contrast, bombesin causes a rapid DG and InsPn production coinciding with the Ca2+ response and the first phase of the rise in pHi followed by a slower DG accumulation coinciding with the second alkalinization phase. Our results show that in Swiss 3T3 cells the bombesin receptor activates the hydrolysis of inositol lipids as a mechanism of signal transduction, which consequently causes changes in Ca2+i and pHi. Clearly, the EGF receptor utilizes different pathways to evoke mitogenesis and stimulates Na+/H+ exchange independently of DG production and protein kinase C activation.     

Effects of platelet-derived growth factor on Ca2+ in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca2+ was examined in BALB/c-3T3 cells. PDGF induced: A decrease in cell 45Ca2+ content. An apparent increased rate of efflux of preloaded 45Ca2+. A decrease in residual intracellular 45Ca2+ remaining after rapid efflux. When added after the rapid phase of efflux of 45Ca2+ had occurred, an immediate decrease in post-efflux residual intracellular 45Ca2+. All of the observed changes in 45Ca2+ induced by PDGF are consistent with a rapid release of Ca2+ from an intracellular Ca2+ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca2+ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca2+ stores and CTC fluorescence intensity were identical with the effects of the Ca2+ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca2+ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca2+ stores. Release of Ca2+ from intracellular sequestration sites may be a mechanism by which PDGF stimulates cell growth.     

Purification and properties of porcine platelet-derived growth factor.

The purification to homogeneity of a potent growth factor from porcine platelets is described. This cationic mitogen is named porcine platelet-derived growth factor (PDGF) on the basis of close structural, functional and immunological similarities to human PDGF. Porcine PDGF, like its human homologue, is a hydrophobic, disulphide cross-linked protein, which is stable to heat, acid, sodium dodecyl sulphate (SDS), and guanidine. The purified protein has an apparent mol. wt. on SDS-polyacrylamide gels of 38 000, similar to those reported for human PDGF (27 500-35 000). Amino terminal sequence analysis of native porcine PDGF gave a single 15 amino acid residue sequence, of which 11 residues were identical to the amino terminal sequence of the B chain of human PDGF. Gel permeation h.p.l.c. in guanidine solutions of the reduced protein revealed a single species of mol. wt. 17 000 suggesting that native porcine PDGF may be a homodimer of a 17 000 mol. wt. chain. Since porcine PDGF can be purified at low cost from large quantities of fresh platelets, it provides an alternative source of PDGF for structural and functional studies, and could be of use in preparing defined media for cell culture.     

Inhibition of epidermal growth factor binding to Swiss 3T3 cells by human platelet release-products

Human platelet ionophore release-products (IRP) inhibit the binding of 125I-labelled epidermal growth factor (125I-EGF) to its receptors on Swiss 3T3 cells. The inhibition appears to be caused by platelet-derived growth factor (PDGF) in the IRP and results from a decrease in the apparent affinity of cellular receptors for 125I-EGF. However, our results indicate that PDGF does not bind directly to EGF receptors, since (1) PDGF does not down-regulate EGF receptors; (2) the PDGF-mediated inhibition of 125I-EGF binding is temperature-dependent; (3) cells which possess EGF receptors but lack PDGF receptors do not exhibit a PDGF-mediated inhibition of 125I-EGF binding.     

Vasopressin rapidly stimulates protein kinase C in quiescent Swiss 3T3 cells

Addition of vasopressin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an acidic molecular weight 80,000 cellular protein (termed 80K). The effect was concentration- and time-dependent; enhancement in 80K phosphorylation could be detected as early as 30 sec after the addition of the hormone. Recently, a rapid increase in the phosphorylation of an 80K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact Swiss 3T3 cells. Here we show that the 80K phosphoproteins generated in response to vasopressin and phorbol 12,13-dibutyrate (PBt2) were identical as judged by one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) and peptide mapping following partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with PBt2 which leads to the disappearance of protein kinase C activity blocked the ability of vasopressin to stimulate the phosphorylation of 80K. The effect of vasopressin on 80K phosphorylation and mitogenesis was selectively blocked by the vasopressin antagonist (Pmp1-O-Me-Tyr2-Arg8) vasopressin suggesting that these responses are mediated by its specific receptor in these cells. The removal of vasopressin leads to dephosphorylation (within minutes) of the 80K phosphoprotein. We conclude that vasopressin rapidly stimulates protein kinase C activity in intact 3T3 cells.     

Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells.

We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.     

Expression of acidic fibroblast growth factor cDNA confers growth advantage and tumorigenesis to Swiss 3T3 cells.

Acidic fibroblast growth factor (aFGF), a polypeptide with a mol. wt of approximately 16,000, is a potent mitogen for a variety of cells and shares 55% amino acid sequence identity with basic FGF. The recent isolation of three new oncogenes which share 35-45% amino acid sequence similarity with the FGFs suggests that the coding sequences for the FGFs themselves may be oncogenic under certain circumstances. To test this hypothesis, we cotransfected 3T3 NR6 cells with factors expressing the aFGF coding sequence and the bacterial neomycin gene. The aFGF produced by cotransfected cells was found only in the cellular homogenate and not in medium conditioned by the cells. Cells expressing aFGF grew to 10 times the density of control cells at saturation and were multilayered and disorganized, similar to transformed cells. The cotransfected cells do not grow in soft agar, but show enhanced soft agar growth relative to controls in the presence of added aFGF and heparin. The aFGF-producing cells formed small, non-progressive tumors when injected subcutaneously into nude mice. Our data suggest that expression of aFGF in NR6 cells results in enhanced growth, and that several traits characteristic of the transformed phenotype are partially expressed.     

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding

Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an Mr 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent; enhancement in 80k phosphorylation could be detected as early as 10 s after the addition of peptide. Recently, a rapid increase in the phosphorylation of an 80k cellular protein after treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts (Rozengurt, E., A. Rodriguez-Pena, and K. A. Smith, 1983, Proc. Natl. Acad. Sci. USA., 80:7244-7248; Rozengurt, E., A. Rodriguez-Pena, M. Coombs, and J. Sinnett-Smith, 1984, Proc. Natl. Acad. Sci. USA., 81:5748-5752). The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125I-labeled epidermal growth factor (125I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. Peptides structurally related to bombesin, including gastrin-releasing peptide, also stimulated 80k phosphorylation and inhibited 125I-EGF binding; both effects were selectively blocked by a novel bombesin antagonist. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca2+ concentration does not mediate the bombesin inhibition of 125I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125I-EGF to its cellular receptor.