CELL-PERMEABLE CERAMIDE INHIBITS THE GROWTH OF B-LYMPHOMA RAJI CELLS LACKING TNF-ALPHA-RECEPTORS BY INDUCING G0 G1 ARREST BUT NOT APOPTOSIS- A NEW MODEL FOR DISSECTING CELL-CYCLE ARREST AND APOPTOSIS/

We examined the effects of a cell-permeable ceramide analog, C2-cerami de, on the growth of TNF-alpha-resistant B lymphoma Raji cells lacking TNF-alpha-receptors (TNF-R). C2-ceramide inhibited the clonal growth of not only TNF-alpha-sensitive myeloid leukemia cells (HL60 and U937) but also Raji cells. Following stimulation with C2-ceramide, HL60 and U937 cells showed apoptotic cell death, whereas Raji cells did not sh ow a detectable level of apoptosis. However, a cell-cycle arrest in G0 /G1 phase was observed in Raji cells after the treatment with C2-ceram ide, which was accompanied by the dephosphorylation of retinoblastoma (RB) gene products and decreased expression of p53 proteins. Failure o f C2-ceramide to induce apoptosis in Raji cells might be explained by the lack or low expression of apoptosis-inducing proteins by two lines of evidence: (1) Raji cells were resistant to apoptosis induced by ce ramide even in the presence of transcription/translation inhibitors; ( 2) Bax protein expression was not detectable in Raji cells, although B cl-2 protein expression in Raji cells was even less than that in HL60 and U937 cells. Moreover, protein kinase C (PKC), whose activation has been described to inhibit ceramide-induced apoptosis, inhibitor H-7 d id not induce apoptotic cell death in Raji cells, suggesting that an i mbalance between PKC and ceramide pathways is not the reason for the r esistance of Raji cells against ceramide-induced apoptosis. Finally, c eramide-induced activation of nuclear factor kappa B (NF-kappa B) was observed in Raji cells as well as HL60 cells, indicating that activati on of this molecule may not be specific for apoptosis. By using the pr esent model, one can dissect cell-cycle arrest and apoptosis induced b y ceramide.