Swiss Mouse 3T3 Cells Research Articles

Mitogen-activated S6 kinase is stimulated via protein kinase C-dependent and independent pathways in Swiss 3T3 cells.

Soluble extracts prepared from quiescent Swiss mouse 3T3 cells that had been briefly exposed to various mitogens exhibited a 2- to 3-fold elevation in phosphorylating activities toward ribosomal protein S6 and a synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), patterned after a phosphorylation site sequence from S6. Optimal activation of the phosphorylating activity occurred within 15-20 min of exposure of the cells to platelet-derived growth factor (10 ng/ml), epidermal growth factor (100 nM), and insulin (100 nM), and 2-5 min after 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nM) treatment. Fractionation of the cytosolic extracts from mitogen- or TPA-treated cells on Sephacryl S-300, TSK-400, and DEAE-Sephacel columns gave results suggesting that a single stimulated kinase accounted for the enhanced S6 and RRLSSLRA phosphorylating activities. The mitogen-activated kinase had an apparent Mr of about 85,000 as determined with Sephacryl S-300, but eluted with an apparent Mr of 26,000 from a TSK-400 high pressure liquid chromatography column. The S6 kinase was also stimulated in cytosols from insulin-like growth factor 1- (100 nM), vasopressin- (250 nM), prostaglandin F2 alpha- (250 nM), and 10% fetal calf serum-treated cells but not from quiescent cells exposed to beta-transforming growth factor (2 ng/ml). TPA, vasopressin and prostaglandin F2 alpha appeared to stimulate this kinase via a protein kinase C-dependent mechanism, since the responses to these hormones, but not to platelet-derived growth factor, epidermal growth factor, and insulin, were lost in protein kinase C-depleted cells.

A c-ras-Ki oncogene is activated, amplified and overexpressed in a human osteosarcoma cell line.

We present a characterization of an activated oncogene which we found to be present in DNA of the OHA osteosarcoma cell line. We identify this tumor oncogene which transforms Swiss mouse 3T3-cells, with c-ras-Ki 2, one of two known members of the Kirsten ras family of human proto-oncogenes. Its structural outlines are given and we show that: 1) a single point mutation causing a substitution of valine for glycine in codon 12 was found by DNA sequencing; 2) the c-ras-Ki gene is amplified and overexpressed in the original OHA tumor cells and its transformants and 3) the gene product is an abnormal form of the p21 protein.

Insulin-like synergistic stimulation of DNA synthesis in Swiss 3T3 cells by the BSC-1 cell-derived growth inhibitor related to transforming growth factor type beta.

A cell growth inhibitor (GI), purified from BSC-1 cell-conditioned medium, has little if any effect on DNA synthesis when added alone to monolayer cultures of quiescent Swiss mouse 3T3 cells in serum-free medium. However, the inhibitor, which is closely related to transforming growth factor type beta (TGF-beta), exhibits a pronounced synergistic stimulation of DNA synthesis in combination with certain peptide (bombesin, vasopressin) or polypeptide (platelet-derived growth factor) mitogens. A similar synergistic response has been demonstrated for TGF-beta purified from human platelets. In the presence of 3 nM bombesin, a half-maximal stimulation of DNA synthesis was obtained at a GI concentration of approximately 60 pg/ml, with a maximal response at approximately 600 pg/ml. The synergistic interactions demonstrated by GI or TGF-beta in stimulating Swiss 3T3 cells closely resemble those previously shown for insulin, and we have observed that GI does not synergize with insulin to stimulate DNA synthesis in these cells. Like insulin, and in contrast to bombesin, vasopressin, and platelet-derived growth factor, GI does not activate cellular inositolphospholipid hydrolysis, calcium mobilization, or cross-regulation of epidermal growth factor receptor affinity. These results raise the possibility that the biochemical pathways activated by GI/TGF-beta and insulin converge at a post-receptor stage.

A tumour promoter, 12-O-tetradecanoylphorbol 13-acetate, increases cellular 1,2-diacylglycerol content through a mechanism other than phosphoinositide hydrolysis in Swiss-mouse 3T3 fibroblasts.

Treatment of Swiss-mouse 3T3 fibroblasts with a tumour-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), increases the absolute amount of 1,2-diacylglycerol (1,2-DG) in a time- and dose-dependent manner. Within 1 min of TPA (0.1 microM) addition to confluent monolayer cells, cellular 1,2-DG content increases significantly, and reaches a value nearly 4-fold above control at 10 min. The content of 1,2-DG then falls, but still remains at twice the basal value for up to 60 min. The effect of TPA on 1,2-DG content is dose-dependent, with a half-maximal effect obtained with 6 nM. Studies with a series of tumour promoters have revealed that stimulating effect on 1,2-DG formation is closely related to their relative potency as a tumour promoter, as well as their mitogenic effect on 3T3 cells. TPA does not have any stimulatory effect on phosphoinositide turnover, which is a major mechanism of 1,2-DG formation by natural agonists, including a peptide mitogen, bombesin, but significantly inhibits bombesin-induced phosphoinositide turnover. Nevertheless, the content of 1,2-DG is even higher in cells treated with both bombesin and TPA than in cells treated with bombesin alone. In addition, a diacylglycerol kinase inhibitor, R59022, increases 1,2-DG content synergistically with bombesin, but not with TPA. Measurements of the absolute amounts of various phospholipids show that TPA does not increase phosphatidic acid, despite a remarkable increase in 1,2-DG. Further studies on phospholipid metabolism reveal that TPA stimulates metabolic turnover of phosphatidylcholine (PC), without changing the mass of PC. TPA stimulates the incorporation of both [3H]choline and [32P]Pi into PC, and the release of [3H]choline metabolites from prelabelled cells. These findings suggest that TPA increases cellular 1,2-DG content mainly via increased PC turnover and diacylglycerol kinase inhibition. The present study provides evidence for a novel effect of TPA on the metabolism of 1,2-DG in Swiss-mouse 3T3 cells.

Regulation of glycogen synthase activity by growth factors. Relationship between synthase activation and receptor occupancy.

Platelet-derived growth factor (PDGF) was found to stimulate the activity of glycogen synthase, an enzyme subjected to regulation by phosphorylation-dephosphorylation reactions. In Swiss mouse 3T3 cells, the time course of enzyme activation by PDGF is very similar to that of epidermal growth factor (EGF) and insulin. A 3-fold maximal stimulation was observed by 30 min, and the enzyme activity ratio returned to basal levels by 100 min. The PDGF effect was maximal at 1 ng/ml (30 pM) and half-maximal stimulation was observed at 0.2 ng/ml (6 pM). Parallel measurements of 125I-PDGF binding indicate that binding was maximal by 10 min and thus should not be rate-limiting for enzyme activation. In addition, presaturation of the receptors with PDGF at 4 degrees C did not expedite subsequent enzyme activation at 37 degrees C. Removal of PDGF in the media after the 4 degrees C pretreatment did not affect the enzyme activation response, indicating that the continued presence of PDGF in the medium is not necessary after receptor binding is saturated. Results of sequential addition of PDGF, EGF, and insulin indicated a refractory period in the response system. This property is evident even when the second addition involved a different growth factor and is independent of the sequence of addition of the factors. There was little additivity in the actions of the three growth factors in effecting enzyme activation and suggests a common intermediate element in the three signalling pathways.

Translational control of mRNA expression during the early mitogenic response in Swiss mouse 3T3 cells: identification of specific proteins.

Addition of serum or epidermal growth factor to quiescent Swiss mouse 3T3 cells in culture leads to a number of specific changes in the pattern of protein synthesis. Earlier experiments with actinomycin D suggested that the altered expression of these proteins was controlled at either the pretranslational or translational level. Here we have identified and further characterized the regulation of mRNA expression for ten of these proteins, including protein synthesis elongation factor eEF-1 alpha, poly A binding protein, vimentin, the multiple forms of the actin protein family, and alpha- and beta-tubulin. Using an in vitro translation system, we determined the change in the level of mRNA encoding for each of these proteins after serum stimulation. The results showed that the amount of mRNA coding for eEF-1 alpha, poly A binding protein, vimentin, and alpha- and beta-tubulin remains unchanged during this time, whereas that of the actin family increases. Thus, with the exception of the actin family, the results argue that the expression of all the proteins identified is regulated at the translational level. The importance of this latter group of proteins in cell growth and the abundance of their cognate mRNAs should prove them useful tools in elucidating the mechanisms involved in the activation of translationally repressed mRNA during the mitogenic response.

Effects of bombesin and insulin on inositol (1,4,5) trisphosphate and inositol (1,3,4) trisphosphate formation in Swiss 3T3 cells

The effects of bombesin and insulin, separately and in combination, have been studied in Swiss mouse 3T3 cells. Bombesin caused a rapid transfer of 3H from the lipid inositol pool of prelabeled cells into inositol phosphates. Label in inositol tetrakisphosphate (InsP 4) and in Ins1,4,5P 3 and Ins1,3,4P 3 rose within 10 sec of stimulation and that in Ins1,4P 2, another InsP 2 and InsP 1, more slowly. Insulin, which had little effect on its own, increased the turnover of inositol lipids due to acute bombesin stimulation and also enhanced the DNA synthesis evoked by prolonged bombesin treatment. The results suggest that bombesin acting as a growth factor, uses inositol lipids as part of its transduction mechanism and that insulin acts synergistically to enhance both inositol phosphate formation and DNA synthesis.

Specificity of inositol phosphate-stimulated Ca2+ mobilization from Swiss-mouse 3T3 cells.

Pure samples of inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and inositol 1,2-cyclic 4,5-trisphosphate were prepared and tested for their ability to mobilize calcium from intracellular stores in a permeabilized Swiss mouse 3T3 cell preparation. In this system inositol 1,4,5-trisphosphate mobilizes Ca2+ with a half-maximal dose of 0.3 microM. Inositol 1,2-cyclic 4,5-trisphosphate mobilized Ca2+ to the same extent with a half-maximal dose of 0.3 microM, whereas inositol 1,3,4-trisphosphate required a half-maximal dose of approx. 9 microM to give the same effect. Inositol 1,3,4,5-tetrakisphosphate was ineffective up to 20 microM and at that concentration did not antagonize the mobilization induced by inositol 1,4,5-trisphosphate. The relevance of these findings to the function of the inositol tris/tetrakis-phosphate pathway is discussed.

Bovine brain-derived growth factor. Purification and characterization of its interaction with responsive cells.

A large-scale purification procedure for brain-derived growth factors, without chromatography in 0.1% trifluoracetic acid, has been developed. Two related brain-derived growth factors have been purified to homogeneity from bovine brain using this procedure involving ammonium sulfate fractionation, followed by chromatography on CM-Sephadex C-50, sulfated Sephadex G-50, and heparin-Sepharose 4B. Brain-derived growth factor A (BDGF-A, molecular weight approximately 16,000) and brain-derived growth factor B (BDGF-B, molecular weight approximately 17,000) were eluted from heparin-Sepharose 4B at 1.2 and 1.6 M NaCl, respectively. Both BDGF-A and BDGF-B have a pI value of 5.7, have the same specific mitogenic activity, and react with mouse anti-BDGF-A antiserum. Both growth factors have a broad spectrum of mitogenic activity (vascular and aorta endothelial cells, chondrocytes, osteoblasts, epithelial cells, glial cells, fibroblasts, and smooth muscle cells), with a half-maximum effect at 10-20 pM. The binding of BDGF to bovine aorta endothelial cells and Swiss mouse 3T3 cells has been characterized. Binding of 125I-BDGF-A to these cells reached equilibrium in less than 15 min. Scatchard plot analysis of the binding of 125I-BDGF-A to endothelial cells and 3T3 cells showed a single class of high-affinity receptor with Kd values of 20 +/- 5 and 13 +/- 3 pM and receptor numbers/cell of 7,000 +/- 1,000 and 20,000 +/- 3,000, respectively. BDGF-B competed for 125I-BDGF-A binding in the same manner as unlabeled BDGF-A, suggesting that BDGF-A and BDGF-B bind to the same receptor. Basic pituitary fibroblast growth factor appeared to be a weak inhibitor, whereas platelet-derived growth factor and epidermal growth factor had no effect on 125I-BDGF-A binding. Protamine, histone, polylysine, and polyarginine are potent inhibitors of the mitogenic activity of BDGF-A and BDGF-B and of binding of 125I-BDGF-A to responsive cells. The half-time (t1/2) of internalization and degradation of cell surface-bound 125I-BDGF is 3 h.

Metabolic turnover of proliferation-related nuclear proteins in serum-stimulated Swiss mouse 3T3 cells

Stimulation of quiescent Swiss mouse 3T3 cells either by serum or by pure growth factors induces DNA synthesis after a lag period of about 15 h. Following restimulation by serum or by growth factors there is an overall increase of 2–4-fold in the rate of biosynthesis of nuclear proteins. Two nuclear polypeptides show specific temporal correlations with the transition from quiescence to proliferation. The synthesis of p30 (30 kDa, p I 5.2) is at a maximum within 5 h of restimulation, while the synthesis of p36 (36 kDa, p I 4.25) is first seen at 10–12 h after restimulation. The synthesis of p36 correlates well with the initiation of DNA biosynthesis. The metabolic turnover of both of these proteins has been estimated by pulse-chase and by cycloheximide inhibition experiments. They both have a half-life of 10–15 h and appear to be cell-cycle related.

Association of bovine brain-derived growth factor receptor with protein tyrosine kinase activity.

Bovine brain-derived growth factor (BDGF) is very similar to endothelial cell growth factor and brain-derived acidic fibroblast growth factor in terms of pI (5.7) and molecular weight (approximately 17,000). BDGF has a wide spectrum of cell specificity, including bovine aorta endothelial cells and Swiss mouse 3T3 cells. BDGF stimulates the phosphorylation of a 135-kDa protein in plasma membranes of 3T3 cells. The optimal concentration for stimulation of phosphorylation is close to the Kd of 125I-BDGF binding to receptor, suggesting that the BDGF-stimulated 32P-labeled 135-kDa protein may be the BDGF receptor. The alkaline stability of this 32P-labeled 135-kDa phosphoprotein and phosphoamino acid analysis of the acid hydrolysates indicate that the phosphorylation occurs at tyrosine residues. The molecular size of BDGF receptor is estimated as approximately 135 kDa by cross-linking 125I-BDGF to its receptor in 3T3 cells, using a bifunctional reagent, ethylene glycolbis(succinimidylsuccinate). Both BDGF-stimulated phosphorylation and 125I-BDGF binding to receptor can be inhibited by protamine. These results suggest that the BDGF receptor is a 135-kDa protein which is associated with a protein tyrosine kinase activity.

Induction of cellular DNA synthesis by purified adenovirus E1A proteins

The purified Escherichia coli-expressed products of the human subgroup-C adenovirus E1A 13 S and 12 S mRNAs are shown to induce cellular DNA synthesis when introduced by microinjection into quiescent, G 0-arrested mammalian cells from immortalized cell lines. The E1A proteins stimulated cellular DNA synthesis in mouse Swiss 3T3 cells, when microinjected either individually or in combination. A truncated EIA protein, in which 169 carboxyl terminal residues of the 289-amino acid E1A 13 S mRNA product are deleted, was unable to induce cellular DNA synthesis in these cells. Our results provide evidence that E1A proteins can function, independent of other viral functions, in the stimulation of cellular DNA synthesis in certain cell types. The present results are consistent with the E1A gene products acting to modulate the expression of the cellular genes which control cell cycle progression into S phase.

Biosynthesis of nuclear proteins after stimulation of quiescent Swiss mouse 3T3 cells.

Stimulation of quiescent Swiss mouse 3T3 fibroblasts either by serum or by the low molecular weight hormones, prostaglandin F2 alpha and insulin, induces DNA synthesis after a lag period of about 15 h. Following restimulation by serum or these pure hormones there is an overall increase of two- to fourfold in the rate of biosynthesis of nuclear proteins. In addition, there is a relative decrease in some proteins (Mr = 200 X 10(3), pI 6.0-6.5), while others increase (e.g. actin). Two polypeptides show specific correlations with the exit from G0. The synthesis of p30 (Mr = 30 X 10(3), pI 5.2) is at a maximum within 5 h of restimulation, while the synthesis of p36 (Mr = 36 X 10(3), pI = 4.25) is first seen at 10-20 h after restimulation. Synthesis of p36 correlates well with the initiation of DNA synthesis. The synthesis of both proteins is stimulated by serum and by the hormones. Thus there are common biosynthetic responses to different stimuli indicating convergent pathways leading to DNA biosynthesis. Addition of hydrocortisone with the growth-stimulatory hormones inhibits both entry into the S phase and biosynthesis of p36. In contrast, hydrocortisone does not alter the biosynthesis of p30. This 'early' protein, p30, is different from the products of both c-fos and c-myc. Therefore, we have identified two specific components that might participate in the regulation of cell proliferation.

31P NMR analysis of intracellular pH of Swiss Mouse 3T3 cells: effects of extracellular Na+ and K+ and mitogenic stimulation.

Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional 31P and 19F probes of intracellular pH (pHc) were found to be impracticable. Cells were therefore superfused with 1 to 4 mM 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pHc on external Na+ concentration (CoNa). PHc also depended on intracellular Na+ concentration (CcNa). Increasing ccNa by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducing CoNa produced a larger acid shift in pHc than with external K+ present. Comparison of separate preparations indicated that pHc was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pHc of Swiss mouse 3T3 cells using 31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.

Antiviral and antiproliferative effects of interferons in quiescent fibroblasts are dissociable

Quiescent mouse Swiss 3T3 cells or human fibroblasts treated with interferon for only 2 hr before the addition of serum are effectively protected against virus infection for up to 24 hr after the addition of serum and the removal of interferon. In sharp contrast, exposure of quiescent fibroblasts to interferon only before addition of serum or the peptides epidermal growth factor and insulin, has no inhibitory effect on cell proliferation. Further, even when interferon is added after the addition of growth factors, an exposure of several hours is required to inhibit stimulation of DNA synthesis. These findings strongly suggest that the antiviral action of interferons is dissociable from their ability to inhibit cell proliferation. The implications of these findings for elucidating the molecular events leading to the biological responses elicited by interferon are discussed.

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.

Permeability change in transformed mouse fibroblasts caused by ionophores, and its relationship to membrane permeabilization by exogenous ATP.

Electrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [3H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanide m-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Non-electrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP+) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (delta psi). The data could be explained by a delta psi threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K+, influx of Na+ and reduction of delta psi preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of delta psi induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP acts via specific sites on the surface of transformed cells.

Epidermal growth factor-mediated activation of an S6 kinase in Swiss mouse 3T3 cells.

Extracts from epidermal growth factor (EGF)-stimulated Swiss mouse 3T3 cells are up to 10 times more potent in phosphorylating ribosomal protein S6 than extracts from quiescent cells. Preparation of extracts in the absence of phosphatase inhibitors leads to a time-dependent loss of kinase activity. In order of potency, the most efficient phosphatase inhibitors in protecting the S6 kinase activity are phosphotyrosine followed by p-nitrophenyl phosphate, beta-glycerol phosphate, and phosphoserine. The kinetics of kinase activation following EGF treatment are rapid and transient. The maximum increase is observed between 15 and 30 min with only 20-30% of the activity remaining after 2 h. Phosphorylation of S6 in the intact cell follows a similar pattern of activation, reaching a maximum between 30 and 60 min and then slowly returning to basal levels by approximately 3 h. The activation of protein synthesis is also rapid; however, in contrast to the transient activation of the S6 kinase and S6 phosphorylation, it remains persistently high for at least 6 h following EGF treatment. Comparison of these events with EGF binding shows that about 50% of the cell surface binding sites are lost within 10 min of exposure to EGF, and about 25% remain after 2 h. Finally, sodium orthovanadate, which is known to mimic the mitogenic effect of EGF, also leads to activation of the S6 kinase, however, with distinct kinetics and by an apparent EGF receptor-independent pathway.

Epidermal growth factor stimulates glycogen synthase activity in cultured cells.

Addition of epidermal growth factor (EGF) to quiescent cultured cells was found to stimulate the activity of glycogen synthase (UDPglucose:glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11), an enzyme subjected to regulation by covalent modification. In Swiss mouse 3T3 cells, the activation by EGF paralleled the effect seen with insulin; the time course and dose-response curves of the two polypeptide factors were similar. Stimulation of enzyme activity ratio [(activity in the absence of glucose 6-phosphate)/(activity in the presence of glucose 6-phosphate)] was maximal after 20-30 min of incubation. Both factors caused a maximal stimulation of 2.5-fold in synthase activity ratio at approximately equal to 10 nM, and the half-maximal effect was observed at 0.1-1 nM. Insulin and EGF exhibited partial additivity in effecting this enzyme activation. In contrast, human A431 cells showed no response to insulin. Although quantitatively different, the EGF effect in the latter cells was time dependent, reaching a maximum at 90 min, and dose dependent, with a maximal stimulation of 4-fold in synthase activity ratio at 10 nM. Half-maximal effect was observed at 0.3 nM EGF. Direct quantitation of allosteric effectors (glucose 6-phosphate, adenine nucleotides, and Pi) present in the enzyme assay mixtures indicated that the observed activation was not simply a consequence of changes in metabolite concentrations. These results suggest that EGF may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms.

Increase of epidermal growth factor-stimulated cell-cycle progression and induction of thermotolerance by heat shock: Temperature and time relationship

When quiescent confluent cultures were incubated at increased temperature and then incubated at 37 degrees C prior to a second increase of temperature (46 degrees C) it appeared that heat-induced morphological alteration and ability to proliferate could be influenced by the previous thermal history of the cells. Incubations for 20 min in a temperature range of 41-46 degrees C caused cells to develop thermo-tolerance within 3 h of incubation at 37 degrees C. Confluent quiescent Swiss mouse 3T3 cells were incubated at 41.8, 43.7 or 45.6 degrees C and then reincubated at 37 degrees C to determine the effects of heat shock on the mitogenic effects of epidermal growth factor (EGF). Preincubation at 43.7 degrees C or 45.6 degrees C enhanced stimulation of G1-S progression by EGF. Preincubation at 43.7 degrees C markedly increased the rate at which cells enter the S phase without changing the length of the lag phase. A comparison of the duration of incubation at 43.7 degrees C for potentiation of EGF-induced DNA synthesis and that for induction of thermotolerance showed that a similar time interval for induction of effect could be implied.