IDENTIFICATION OF A DEFECT IN THE PHOSPHOLIPASE-D DIACYLGLYCEROL PATHWAY IN CELLULAR SENESCENCE

Normal cells become senescent in culture after a limited number of pop ulation doublings becoming unable to respond to mitogens. This raises the possibility of defects in mitogenic signaling pathways in cellular senescence. In contrast to young human diploid fibroblasts (HDF), the ir senescent counterparts failed to undergo protein kinase C transloca tion in response to serum stimulation. On the other hand, phorbol 12-m yristate 13-acetate was equally active in inducing protein kinase C tr anslocation in young and senescent HDF. This suggested a defect in gen eration of the endogenous activator of protein kinase C, diacylglycero l. Stimulation of young HDF with serum resulted in 3-4-fold generation of diacylglycerol (DAG). In contrast, senescent cells displayed insig nificant DAG: formation in response to serum. The mechanism of DAG gen eration was investigated next. In young HDF, serum induced a 5-fold ac tivation of the phospholipase D (PLD) pathway as measured by the incor poration of exogenous ethanol into phosphatidylethanol, which is a mea sure of the transphosphatidylation reaction of PLD. In contrast, PLD i n senescent cells was not activated by serum. Since senescent cells de monstrate significant elevations in the level of endogenous ceramide, the impact of ceramide on the PLD/DAG pathway was also investigated. A soluble analog of ceramide, C-6-ceramide, was found to inhibit serum- stimulated DAG accumulation and PLD activation in young cells. These d ata demonstrate for the first time a defect in PLD activation in cellu lar senescence and suggest that ceramide may be responsible for the in hibition of this pathway.