Induction of Apoptosis by Sphingosine in Human Leukemic HL-60 Cells: A Possible Endogenous Modulator of Apoptotic DNA Fragmentation Occurring during Phorbol Ester-induced Differentiation

The present studies were undertaken to characterize the potential role of sphingosine in the regulation of apoptosis in HL-60 promyelocyte leukemia cells. A 6-h exposure of HL-60 cells to sphingosine or its methylated derivative, N,N-dimethylsphingosine, caused internucleosomal DNA fragmentation and stereotypical morphological changes characteristic of apoptosis (i,e., cell shrinkage, nuclear condensation, and the formation of apoptotic bodies), as well as that to pharmacological inhibitors of protein kinase C such as l-(5-isoquinolinesulfonyl)-2-methylpiperazine and staurosporine. Apoptosis by sphingosine and N,N-dimethylsphin-gosine was measured using a flow cytometric method. The percentages of apoptotic cells in cultures treated with sphingosine (10 μm) and N,N-dimethylsphingosine (10 μm) for 6 h were 55.6 ± 7.8% and 84.2 ± 11.6%, respectively. HL-60 cells were induced to differentiate toward macrophages by treatment with 5 nM 4β-phorbol 12-myristate 13-acetate (PMA). Internucleosomal DNA fragmentation, which was a hallmark of apoptosis, was first detected after 10-h exposure to PMA and increased with longer treatment Sphingosine concentrations in the cells increased concomitantly with the increasing proportion of apoptotic cells during cell differentiation. Sphingosine level in HL-60 cells differentiated by treatment with PMA for 48 h was about 3.3-fold greater than that in untreated cells. Differentiated HL-60 cells exhibited a markedly increased conversion of exogenously added [³H]ceramide to [³H]sphingosine, indicating elevation of ceramidase activity. Moreover, exposure to sphingosine resulted in down-regulation of c-myc mRNA. These observations suggest the possible role of sphingosine in induction of apoptotic DNA fragmentation during PMA-induced differentiation in myeloid leukemia cells. Sphingosine may function as an endogenous modulator mediating the apoptotic signal.