Activation of signal transduction pathways protects quiescent Balb/c-3T3 fibroblasts against death due to serum deprivation.
Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin protect density-inhibited murine Balb/c-3T3 fibroblasts against death by distinctive mechanisms. Determination of the cell survival-enhancing activity of growth factors by cell enumeration and neutral red uptake measurement gives equivalent results. PDGF displays a steep dose-response relationship in the 1-5 ng/ml range. The other factors display shallow log-linear relationships in the following ranges: EGF: 0.2-5 ng/ml; IGF-1: 2-80 ng/ml; and insulin: 57-4,500 ng/ml. Agonists that lead to the activation of protein kinase A, including forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate (Br-cAMP) and N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (db-cAMP), markedly increase both short-term (5-h) and long-term (20-h) survival of cells. 2-Isobutyl-1-methylxanthine (IBMX) markedly enhances short-term survival, but its effect decays with time. The protein kinase C agonist 12-O-tetradecanoyl phorbol-13-acetate (TPA) has a moderate protective effect at concentrations of 16-32 nM, and 64 nM TPA is highly effective. The synthetic diaclglycerols 1,2-dioctanoylglycerol (DiC8) and 1-oleoyl-2-acetylglycerol (OAG) and the calcium ionophore ionomycin show low activity. Supplementation of EGF with a protein kinase A or C agonist results in a varying additive increase in short-term (5-h) cell survival and supplementation of EGF + insulin or PDGF + EGF + insulin increases further the already high level of protection given by the growth factor combinations. Combining a protein kinase A and a protein kinase C agonist in the absence of growth factors gives an approximately additive increase in cell survival. Results obtained with kinase, RNA, and protein synthesis inhibitors suggest that: 1) activated protein kinase C catalyzes one or more phosphorylation events in quiescent Balb/c-3T3 cells that lead to gene expression with the protein product(s) mediating protection of quiescent cells against death, and 2) phosphorylation events catalyzed by protein kinase A largely serve to protect cells by a mechanism not requiring de novo RNA and protein biosynthesis.
Tamm
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Rockefeller University, New York, New York 10021.
Kikuchi
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D015075:2-Aminopurine / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology*; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D009499:Neutral Red / Q000493:pharmacokinetics; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D016196:S Phase / Q000502:physiology; D015290:Second Messenger Systems / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors
D000470:Alkaloids; D007328:Insulin; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D009499:Neutral Red; D015075:2-Aminopurine; D004815:Epidermal Growth Factor; D012313:RNA; D007334:Insulin-Like Growth Factor I; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate