Jarvis W D, Fornari F A, Traylor R S, Martin H A, Kramer L B, Erukulla R K, Bittman R, Grant S
Department of Medicine, Medical College of Virginia, Richmond, 23298-0230, USA.
J Biol Chem. 1996 Apr 5;271(14):8275-84. doi: 10.1074/jbc.271.14.8275.
Prior studies demonstrated that ceramide promotes apoptotic cell death in the human myeloid leukemia cell lines HL-60 and U937 (Jarvis, W. D., Kolesnick, R. N., Fornari, F. A., Jr., Traylor, R. S., Gewirtz, D. A., and Grant, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 73-77), and that this lethal process is potently suppressed by diglyceride (Jarvis, W. D., Fornari, F. A., Jr., Browning, J. L., Gewirtz, D. A., Kolesnick, R. N., and Grant, S. (1994) J. Biol. Chem. 269, 31685-31692). The present findings document the intrinsic ability of sphingoid bases to induce apoptosis in HL-60 and U937 cells. Exposure to either sphingosine or sphinganine (0. 001 10 microM) for 6 h promoted apoptotic degradation of genomic DNA as indicated by (a) electrophoretic resolution of 50-kilobase pair DNA loop fragments and 0.2-1.2-kilobase pair DNA fragment ladders on agarose gels, and (b) spectrofluorophotometric determination of the formation and release of double-stranded fragments and corresponding loss of integrity of bulk DNA. DNA damage correlated directly with reduced cloning efficiency and was associated with the appearance of apoptotic cytoarchitectural traits. At sublethal concentrations (</=750 nM), however, sphingoid bases synergistically augmented the apoptotic capacity of ceramide (10 microM), producing both a leftward shift in the ceramide concentration-response profile and a pronounced increase in the response to maximally effective levels of ceramide. Thus, sphingosine and sphinganine increased both the potency and efficacy of ceramide. The apoptotic capacity of bacterial sphingomyelinase (50 milliunits/ml) was similarly enhanced by either (a) acute co-exposure to highly selective pharmacological inhibitors of protein kinase C such as calphostin C and chelerythrine or (b) chronic pre-exposure to the non-tumor-promoting protein kinase C activator bryostatin 1, which completely down-modulated total assayable protein kinase C activity. These findings demonstrate that inhibition of protein kinase C by physiological or pharmacological agents potentiates the lethal actions of ceramide in human leukemia cells, providing further support for the emerging concept of a cytoprotective function of the protein kinase C isoenzyme family in the regulation of leukemic cell survival.
先前的研究表明，神经酰胺可促进人髓系白血病细胞系HL-60和U937的凋亡性细胞死亡（贾维斯，W.D.，科尔斯尼克，R.N.，福尔纳里，F.A.，小，特雷勒，R.S.，格维尔茨，D.A.，和格兰特，S.（1994年）美国国家科学院院刊91，73 - 77），并且这种致死过程被甘油二酯强烈抑制（贾维斯，W.D.，福尔纳里，F.A.，小，布朗宁，J.L.，格维尔茨，D.A.，科尔斯尼克，R.N.，和格兰特，S.（1994年）生物化学杂志269，31685 - 31692）。目前的研究结果证明了鞘氨醇碱在HL-60和U937细胞中诱导凋亡的内在能力。暴露于鞘氨醇或鞘氨醇胺（0.001 - 10 microM）6小时可促进基因组DNA的凋亡性降解，这表现为：（a）在琼脂糖凝胶上50千碱基对DNA环片段和0.2 - 1.2千碱基对DNA片段梯带的电泳分辨率，以及（b）双链片段形成和释放的荧光分光光度测定以及大量DNA完整性相应丧失。DNA损伤与克隆效率降低直接相关，并与凋亡细胞结构特征的出现有关。然而，在亚致死浓度（≤750 nM）下，鞘氨醇碱协同增强了神经酰胺（10 microM）的凋亡能力，使神经酰胺浓度 - 反应曲线向左移动，并使对最大有效水平神经酰胺的反应显著增加。因此，鞘氨醇和鞘氨醇胺增加了神经酰胺的效力和效果。细菌鞘磷脂酶（50毫单位/毫升）的凋亡能力同样通过以下两种方式增强：（a）急性共同暴露于蛋白激酶C的高度选择性药理抑制剂，如钙泊三醇C和白屈菜红碱，或（b）长期预先暴露于非肿瘤促进性蛋白激酶C激活剂苔藓抑素1，其完全下调了可检测的总蛋白激酶C活性。这些发现表明，生理或药理试剂对蛋白激酶C的抑制增强了神经酰胺在人白血病细胞中的致死作用，为蛋白激酶C同工酶家族在调节白血病细胞存活中的细胞保护功能这一新兴概念提供了进一步支持。
