Jarvis W D, Fornari F A, Browning J L, Gewirtz D A, Kolesnick R N, Grant S
Department of Medicine, Medical College of Virginia, Richmond 23298.
J Biol Chem. 1994 Dec 16;269(50):31685-92.
Prior studies demonstrated that increased intracellular availability of ceramide induces apoptotic DNA degradation and cell death in the human leukemia cell lines HL-60 and U937 (Jarvis, W. D., Kolesnick, R. N., Fornari, F. A., Traylor, R. S., Gewirtz, D. A., and Grant, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 73-77). The present findings show that diglyceride opposes ceramide-related apoptosis in HL-60 and U937 cells. Acute (6-12-h) exposure to sphingomyelinase (100 milliunits/ml) or synthetic ceramide (10 microM) promoted apoptotic degradation of genomic DNA as indicated by (a) the appearance of both approximately 50-kilobase pair (kbp) DNA fragments and approximately 0.2-1.2-kbp DNA fragment ladders on agarose gels, (b) formation and release of small double-stranded DNA fragments, and (c) loss of integrity of bulk DNA. DNA damage was associated with reduced clonogenicity and expression of apoptotic morphology. In contrast, exposure to phospholipase C (0.001-100 milliunits/ml) or synthetic diglyceride (10 microM) failed to promote apoptosis and abolished the lethal actions of ceramide as defined by each of the indices outlined above. Ceramide-related apoptosis was also reduced by acute (6-h) exposure to tumor promoters such as phorbol dibutyrate and mezerein and the non-tumor-promoting agent bryostatin 1; conversely, chronic (24-h) pretreatment with these agents failed to modify ceramide-mediated cytotoxicity, but abolished the protective actions of diglyceride. These findings demonstrate that diglyceride and pharmacological protein kinase C activators reduce or abolish ceramide-mediated apoptosis in human leukemia cells and support the concept of a cytoprotective function for protein kinase C in the regulation of leukemic cell survival. In addition, the capacity of diglyceride to prevent very early genomic lesions (e.g. generation of 50-kbp DNA fragments) suggests that acute activation of protein kinase C arrests apoptosis at an initial stage.
先前的研究表明，神经酰胺细胞内可用性的增加会诱导人白血病细胞系HL-60和U937发生凋亡性DNA降解和细胞死亡（贾维斯，W.D.，科尔斯尼克，R.N.，福尔纳里，F.A.，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）。目前的研究结果表明，甘油二酯可对抗HL-60和U937细胞中与神经酰胺相关的凋亡。急性（6 - 12小时）暴露于鞘磷脂酶（100毫单位/毫升）或合成神经酰胺（10微摩尔）会促进基因组DNA的凋亡性降解，这表现为：（a）在琼脂糖凝胶上出现约50千碱基对（kbp）的DNA片段和约0.2 - 1.2 kbp的DNA片段梯带；（b）小双链DNA片段的形成和释放；（c）大量DNA完整性的丧失。DNA损伤与克隆形成能力降低和凋亡形态的表达相关。相反，暴露于磷脂酶C（0.001 - 100毫单位/毫升）或合成甘油二酯（10微摩尔）未能促进凋亡，并消除了上述各项指标所定义的神经酰胺的致死作用。急性（6小时）暴露于肿瘤促进剂如佛波酯二丁酸酯和大戟二萜醇酯以及非肿瘤促进剂苔藓抑素1也可减少与神经酰胺相关的凋亡；相反，用这些药物进行慢性（24小时）预处理未能改变神经酰胺介导的细胞毒性，但消除了甘油二酯的保护作用。这些研究结果表明，甘油二酯和药理学蛋白激酶C激活剂可减少或消除人白血病细胞中神经酰胺介导的凋亡，并支持蛋白激酶C在调节白血病细胞存活中具有细胞保护功能的概念。此外，甘油二酯预防非常早期基因组损伤（例如产生50 kbp DNA片段）的能力表明，蛋白激酶C的急性激活在初始阶段阻止了凋亡。
