Activation of signal transduction pathways protects quiescent Balb/c‐3T3 fibroblasts against death due to serum deprivation

Abstract

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 doseresponse 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‐lsobutyl‐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 diacylglycerols 1,2‐dioctanoylglycerol (DiC₈) and 1‐oleoyl‐2‐acetylglycerol (OAG) and the calcium ionophore ionomycin show low activity. Supplemation 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.