The effects of the anti-cancer anthracyclines doxorubicin and daunorubicin on the activity of protein kinase C (PKC) were examined in intact Swiss 3T3 cells. The 2 drugs stimulated the phosphorylation of an 80K phosphoprotein found to be identical to that generated in response to the PKC activator 12-0-tetradecanoylphorbol-13-acetate as indicated by gel electrophoresis and peptide mapping. The effect of doxorubicin was dose-dependent in the range to lo-' M and was not associated with a detectable translocation of PKC activity from cytorol to the cell membrane. Doxorubicin and daunorubicin were found to increase the incorporation of phosphate into phosphatidic acid, phosphatidylinositol4-monophosphate and phosphatidyl inositol 4,5-bisphosphate. In addition, the anthracyclines induced a rise in inositol phosphates, thus indicating a stimulation of the breakdown of phosphoinositider. These data are consistent with an indirect mechanism of PKC activation by anthracyclines. We propose that diacylglycerol, which is derived from the hydrolysis of phospholipids, (including the phosphoinositides), by activation of phospholipases, could mediate PKC activation. The described effects, involving cell-signal-transducing pathways, emphasize a new aspect of the cellular actions of these anti-tumor agents.
X-100). Heat-stable proteins, which include the phosphorylated 80K protein (Blackshear et al., 1986), were recovered by boiling the detergent-soluble material for 10 min, cooling on ice and centrifuging at 12,000 g for 15 min at 4Β°C. Supernatants were mixed with half their volume of 3X Laemmli sample-solution (Laemmli, 1970) containing 3 m phenylmethylsulfonyl fluoride, 375 pg/ml leupeptin, 300 KIU/ml aprotinin, 3 nm Na3V04 and 3 nm Na2Mo04 and were boiled for 2 min.
Phosphoproteins were analyzed by sodium dodecylsulphate 'To whom correspondence and reprint requests should be addressed.