12-O-TETRADECANOYLPHORBOL-13-ACETATE-INDUCED APOPTOSIS IN LNCAP CELLSIS MEDIATED THROUGH CERAMIDE SYNTHASE

Protein kinase C (PKC) activation is often antiapoptotic, although in a few cell types PKC initiates apoptosis by an unknown mechanism. Rece nt investigations showed that activation of PKC alpha by 12-O-tetradec anoylphorbol 13-acetate (TPA) induced apoptosis in LNCaP prostate canc er cells. The present studies examine the mechanism of this effect and show that de novo ceramide generation through the enzyme ceramide syn thase is required. TPA induced rapid ceramide generation, which was de tectable by Ih and increased linearly for 12 h. TPA-induced apoptosis was measurable by 12 h and was progressive for 48 h. Investigations in to the mechanism of TPA-induced ceramide generation revealed that acid and neutral sphingomyelinase activities were not enhanced. However, T PA induced an increase in ceramide synthase activity that persisted fo r at least 16 h. Treatment with fumonisin B-1, a specific natural inhi bitor of ceramide synthase, abrogated both ceramide production and TPA -induced apoptosis. Ceramide analogues bypassed fumonisin B-1 inhibiti on to initiate apoptosis directly. Thus, ceramide appears to be a nece ssary signal for TPA-induced apoptosis in LNCaP cells. This represents the first description of a pathway by which PKC may signal apoptosis.