Stimulation of DNA synthesis by natural ceramide 1-phosphate

We found that natural (long-chain) ceramide l-phosphate can be dispersed into aqueous solution when dissolved in an appropriate mixture of methanol/dodecane (49:1, v/v). This solvent mixture facilitates the interaction of this phosphosphingolipid with cells. Under these conditions, incubation of EGFR T17 fibroblasts with natural ceramide I-phosphate caused a potent stimulation of DNA synthesis. This effect was accompanied by an increase in the levels of proliferating-cell nuclear antigen. Concentrations of natural ceramide I-phosphate that stimulated the synthesis of DNA did not inhibit adenylate cyclase activity, nor did they stimulate phospholipase D. Natural ceramide l-phosphate did not alter the cellular phosphorylation state of tyrosine residues or of mitogen-activated protein kinase. Furthermore, natural ceramide l-phosphate failed to induce the expression of the proto-oncogenes c-myc and c-fos, Both the stimulation of DNA synthesis and the induction of proliferating-cell nuclear antigen by natural ceramide l-phosphate were inhibited by natural ceramides. This work suggests that the use of methanol and dodecane to deliver natural ceramide I-phosphate to cells may be useful for elucidation of the biological function(s) and mechanism(s) of action of ceramide l-phosphate.