Inhibition of the expression of ornithine decarboxylase and c-Myc by cell-permeant ceramide in difluoromethylornithine-resistant leukaemia cells.
Ceramide has emerged as a novel lipid mediator in cell growth and apoptosis. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, the cell-permeant analogues of ceramide N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide) inhibited the induction of ornithine decarboxylase (ODC) activity with IC50 of 8.3 and 1.5 microM respectively. This effect was strictly related to the ability to inhibit cell growth and [3H]thymidine incorporation. 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 [3H]thymidine incorporation. Bacterial lipopolysaccharide, which may act as a ceramide analogue, also inhibited the induction of the enzyme. Moreover, C6-ceramide largely prevented the accumulation of ODC mRNA and its precursor, ODC heterogeneous nuclear RNA, that accompanied the induction of ODC activity. A slight increase in ODC turnover was also observed. The DNA-binding activity of some transcription factors known to bind and transactivate 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 C6-ceramide. Furthermore, the amount of immunoreactive c-Myc, which increased after stimulation of the cells to growth, was strongly reduced by C6-ceramide. These results suggest that the inhibition of c-Myc and ODC expression may be early events in the response of leukaemia cells to ceramide.
Flamigni
F
F
Dipartimento di Biochimica 'G.Moruzzi', Università di Bologna, via Irnerio 48, 40126 Bologna, Italy.
Faenza
I
I
Marmiroli
S
S
Stanic'
I
I
Giaccari
A
A
Muscari
C
C
Stefanelli
C
C
Rossoni
C
C
D000818:Animals; D002463:Cell Membrane Permeability; D002518:Ceramides / Q000494:pharmacology*; D000518:Eflornithine / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D016259:Genes, myc; D007939:Leukemia L1210 / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D051379:Mice; D009955:Ornithine Decarboxylase / Q000235:genetics*; D014407:Tumor Cells, Cultured
D002518:Ceramides; D009955:Ornithine Decarboxylase; D000518:Eflornithine