|Budget Amount *help
¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1993 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1992 : ¥900,000 (Direct Cost : ¥900,000)
EGF is known to pull the trigger of signal transduction of growth. Using A-341 cells and their transfectants. we studied the relation between EGF high-affinity binding sites and rounding induced by EGF and the roles of protein kinase C (PKC) and protein kinase A (PKA) in EGF-induced cell rounding.
Elevation of intracellular Ca^<2+> concentration ([Ca^<2+>]_1) and rounding took place when A-431 cells were sitimulated with EGF(10^<-8>M), Pretreatment with 12-0-tetradecanoyl-phorbol-13-acetate (TPA, 100ng/ml), an activator of PKC, for 30 min inhibited rounding, elevation of [Ca^<2+>] _1, and expression of high-affinity binding sites for EGF.But, when pretreatment with TPA(100ng/ml) was carry out for 20 h, which treatment is known to result in down-regulation of PKC, all three of the above responses to EGF occured. Preteatment with forskolin (100mu M), an activator of PKA, for 30 min gave the same result as that with TPA (100ng/ml) for 20 h. On the other hand, in the case of the An-10 cells, lacking high-affinity binding sites for EGF, stimulated with EGF (10^<-8>M) did not kead to rounding, but elevationof [Ca^<2+>] _i did occur. Pretreatment of TPA (100ng/ml) for 30 min inhibited rounding, elevationof [Ca^<2+>] _i and expression of high-affinity binding sites for EGF same as in A-431 cells. But, pretreatment of the cells with TPA (100ng/ml) for 20 h caused cell rounding, elevationof [Ca^<2+>] _i and expression of high-affinity binding sites for EGF.However rounding and high-affinity binding sites were not found in the case of no pretreat ment of Am-10 cells. Forskolin (100mu M) pretreatment for 30 min gave about the same results as TPA (100ng/ml) pretreatment for 20 hours.
these results suggest that founding of A-431 cells induced by EGF was injibited by activation of PKC bia PLC, but accelerated by activation of PKA.Furthermoer, the rounding response apearsto correlate with the presense of high-affinity binding sites for EGF.