Tamm I, Kikuchi T
Rockefeller University, New York, New York 10021.
J Cell Physiol. 1991 Jul;148(1):85-95. doi: 10.1002/jcp.1041480111.
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.
血小板衍生生长因子（PDGF）、表皮生长因子（EGF）、胰岛素样生长因子-1（IGF-1）和胰岛素通过独特机制保护密度抑制的小鼠Balb/c - 3T3成纤维细胞免于死亡。通过细胞计数和中性红摄取测量来测定生长因子的细胞存活增强活性，结果相当。PDGF在1 - 5 ng/ml范围内呈现陡峭的剂量反应关系。其他因子在以下范围内呈现浅对数线性关系：EGF：0.2 - 5 ng/ml；IGF-1：2 - 80 ng/ml；胰岛素：57 - 4500 ng/ml。导致蛋白激酶A激活的激动剂，包括福斯可林、8-溴腺苷3':5'-环一磷酸（Br-cAMP）和N6,2'-O-二丁酰腺苷3':5'-环一磷酸（db-cAMP），显著提高细胞的短期（5小时）和长期（20小时）存活率。2-异丁基-1-甲基黄嘌呤（IBMX）显著提高短期存活率，但其效果随时间衰减。蛋白激酶C激动剂12-O-十四酰佛波醇-13-乙酸酯（TPA）在16 - 32 nM浓度下具有中等保护作用，64 nM TPA则非常有效。合成二酰甘油1,2-二辛酰甘油（DiC8）和1-油酰-2-乙酰甘油（OAG）以及钙离子载体离子霉素活性较低。用蛋白激酶A或C激动剂补充EGF会使短期（5小时）细胞存活率有不同程度的累加增加，而补充EGF +胰岛素或PDGF + EGF +胰岛素会进一步提高生长因子组合已经很高的保护水平。在没有生长因子的情况下联合使用蛋白激酶A和蛋白激酶C激动剂，细胞存活率会有近似累加的增加。用激酶、RNA和蛋白质合成抑制剂获得的结果表明：1）活化的蛋白激酶C催化静止的Balb/c - 3T3细胞中的一个或多个磷酸化事件，导致基因表达，其蛋白质产物介导静止细胞免于死亡的保护作用；2）蛋白激酶A催化的磷酸化事件主要通过一种不需要从头合成RNA和蛋白质生物合成的机制来保护细胞。
