Growth state-dependent regulation of protein kinase C in normal and transformed murine cells.

We determined whether growth state can influence the action of protein kinase C by measuring protein kinase C activity in growing and stationary cultures of normal and transformed cells. Two approaches were used to measure protein kinase C: assay of intact cells for inhibition of epidermal growth factor (EGF) binding in response to phorbol dibutyrate (PDBu); and assay of detergent extracts for total calcium, phospholipid-dependent kinase activity. In extracts of growing and stationary Swiss 3T3 cells, the total amount of protein kinase C activity was similar, indicating that growth state does not alter the level of enzyme in the cell. The short-term response of Swiss 3T3 cells to an activator of protein kinase C also appeared to be independent of growth state, since the 50% effective dose for PDBu inhibition of EGF binding to its receptor was approximately 7 nM for both growth conditions. In contrast, the response of cells to long-term treatment with PDBu was significantly different depending upon the initial growth state of the cells. In both growth states, PDBu caused loss of protein kinase C activity, which reflected a loss in protein mass as determined by immunoblotting with antiserum to protein kinase C. However, the maximum decrease approached 100% in stationary cultures versus approximately 75% in growing cells. Protein kinase C levels in several transformed cell lines were subject to down modulation in a similar growth state-dependent manner. Further, the inhibition of EGF binding by tumor promoters following long-term treatment of Swiss 3T3 cells with PDBu also varied with growth state. In down modulated growing cells, PDBu caused almost complete inhibition of EGF binding, whereas in down modulated stationary cells, minimal inhibition of EGF binding by PDBu was observed. These results suggest that prolonged treatment with tumor promoters alters the sensitivity of cells to activators of protein kinase C in a growth state-dependent manner.