Flamigni F, Faenza I, Marmiroli S, Stanic' I, Giaccari A, Muscari C, Stefanelli C, Rossoni C
Dipartimento di Biochimica 'G.Moruzzi', Università di Bologna, via Irnerio 48, 40126 Bologna, Italy.
Biochem J. 1997 Jun 15;324 ( Pt 3)(Pt 3):783-9. doi: 10.1042/bj3240783.
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.
神经酰胺已成为细胞生长和凋亡中的一种新型脂质介质。在从静止状态被刺激生长的二氟甲基鸟氨酸抗性L1210细胞中，神经酰胺N - 乙酰鞘氨醇（C2 - 神经酰胺）和N - 己酰鞘氨醇（C6 - 神经酰胺）的细胞渗透性类似物分别以8.3和1.5 microM的IC50抑制鸟氨酸脱羧酶（ODC）活性的诱导。这种作用与抑制细胞生长和[3H]胸苷掺入的能力密切相关。细胞生长的抑制也与细胞凋亡有关。添加细菌鞘磷脂酶导致ODC诱导和[3H]胸苷掺入显著但有限的降低。可能作为神经酰胺类似物的细菌脂多糖也抑制该酶的诱导。此外，C6 - 神经酰胺在很大程度上阻止了伴随ODC活性诱导的ODC mRNA及其前体ODC不均一核RNA的积累。还观察到ODC周转略有增加。在相同实验条件下，通过凝胶迁移率变动分析研究了一些已知与ODC基因结合并使其反式激活的转录因子的DNA结合活性。然而，只有Myc/Max的结合受到C6 - 神经酰胺处理的负面影响。此外，在细胞被刺激生长后增加的免疫反应性c - Myc的量被C6 - 神经酰胺强烈降低。这些结果表明，抑制c - Myc和ODC表达可能是白血病细胞对神经酰胺反应中的早期事件。
