INHIBITION OF THE EXPRESSION OF ORNITHINE DECARBOXYLASE AND C-MYC BY CELL-PERMEANT CERAMIDE IN DIFLUOROMETHYLORNITHINE-RESISTANT LEUKEMIA-CELLS

Ceramide has emerged as a novel lipid mediator in cell growth and apop tosis. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, the cell-permeant analogues of ceramide N-acet ylsphingosine (C-2-ceramide) and N-hexanoylsphingosine (C-6-ceramide) inhibited the induction of ornithine decarboxylase (ODC) activity with IC50 of 8.3 and 1.5 mu M respectively. This effect was strictly relat ed to the ability to inhibit cell growth and [H-3]thymidine incorporat ion. The suppression of cell growth was also associated with apoptosis . The addition of bacterial sphingomyelinase resulted in a significant , but limited, reduction of ODC induction and [H-3]thymidine incorpora tion. Bacterial lipopolysaccharide, which may act as a ceramide analog ue, also inhibited the induction of the enzyme. Moreover, C-6-ceramide largely prevented the accumulation of ODC mRNA and its precursor, ODC heterogeneous nuclear RNA, that accompanied the induction of ODC acti vity. A slight increase in ODC turnover was also observed. The DNA-bin ding activity of some transcription factors known to bind and transact ivate the ODC gene was investigated by gel mobility-shift assay under the same experimental conditions. However, only the binding of Myc/Max was negatively affected by the treatment with C-6-ceramide. Furthermo re, the amount of immunoreactive c-Myc, which increased after stimulat ion of the cells to growth, was strongly reduced by C-6-ceramide. Thes e results suggest that the inhibition of c-Myc and ODC expression may be early events in the response of leukaemia cells to ceramide.