Inhibition of CTP:phosphocholine cytidylyltransferase by C2-ceramide and its relationship to apoptosis

Apoptosis induced by antitumor phospholipid analogs takes place after the inhibition of the CTP: phosphocholine cytidylyltransferase (CCT; EC 2.7.7.15) catalyzed step of phosphatidylcholine (PtdCho) biosynthesis. Exposure of cells to synthetic short-chain ceramide analogs also triggers apoptosis concomitant with decreased PtdCho biosynthesis, and the present study was undertaken to ascertain whether C-2-ceramide inhibition of PtdCho synthesis is direct or secondary to other ceramide-mediated cellular responses. The exposure of COS-7 cells to either C-2-ceramide, ET-18-OCH3, or farnesol resulted in time- and dose-dependent apoptotic cell death. Cells treated with C-2-ceramide or ET-18-OCH3 selectively and immediately accumulated phosphocholine, whereas CDP-choline increased with farnesol treatment. In vitro assays of CCT activity demonstrated that C-2-ceramide directly inhibited CCT. Comparison of different N-linked sphingosine derivatives suggests an inverse relationship between the length of the N-linked carbon chain and the derivatives ability to trigger apoptosis and inhibit CCT. Taken together, our results suggest CCT as a primary target for C-2-ceramide inhibition that accounts for its cytotoxic effects.