Grant S, Freemerman A J, Gregory P C, Martin H A, Turner A J, Mikkelsen R, Chelliah J, Yanovich S, Jarvis W D
Department of Medicine, Medical College of Virginia, Richmond 23298, USA.
Oncol Res. 1995;7(7-8):381-92.
The permeant Ca2+ chelator acetoxymethyl-1,2-bis(2-aminopheoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA/AM), an agent previously used to characterize drug-induced apoptosis in neoplastic cells, has been examined with respect to induction of DNA fragmentation and cytotoxicity in the human leukemia cell lines HL-60 and U937. Exposure of cells to various concentrations of BAPTA/AM for 6 h resulted in a biphasic induction of internucleosomal DNA cleavage, with maximal damage occurring at 10-microM concentrations. Higher BAPTA/AM concentrations were associated with the loss of internucleosomal cleavage products, but with the appearance of larger (i.e., 50-kilobase) fragments on pulsed-field gel electrophoresis. Cells exposed to 10 microM BAPTA/AM exhibited classic apoptotic morphology, whereas cells exposed to 50-microM concentrations displayed atypical features (e.g., cell swelling, chromatin clumping); in each case, substantial cytotoxicity was noted. The actions of BAPTA/AM did not depend upon the presence of extracellular Ca2+, nor were they affected by impermeant Ca2+ chelators. Measurement of cytosolic Ca2+ by Fura-2/AM or Indo-1 revealed late but not early increases in intracellular Ca2+ in BAPTA/AM-treated cells. Finally, BAPTA/AM-induced apoptosis was accompanied by the concentration-dependent downregulation of the immediate early response gene c-jun. These findings suggest a complex role for Ca2+ chelators such as BAPTA/AM in the regulation of human myeloid leukemic cell apoptosis, and indicate that this agent may selectively antagonize internucleosomal DNA fragmentation without interfering with other aspects of the apoptotic response and/or cell lethality.
渗透性钙离子螯合剂乙酰氧基甲基-1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸（BAPTA/AM），一种先前用于表征肿瘤细胞中药物诱导凋亡的试剂，已针对其在人白血病细胞系HL-60和U937中诱导DNA片段化和细胞毒性进行了研究。将细胞暴露于不同浓度的BAPTA/AM中6小时，导致核小体间DNA切割呈双相诱导，最大损伤发生在10微摩尔浓度时。更高的BAPTA/AM浓度与核小体间切割产物的丢失相关，但在脉冲场凝胶电泳上出现更大（即50千碱基）的片段。暴露于10微摩尔BAPTA/AM的细胞表现出典型的凋亡形态，而暴露于50微摩尔浓度的细胞则表现出非典型特征（如细胞肿胀、染色质聚集）；在每种情况下，均观察到明显的细胞毒性。BAPTA/AM的作用不依赖于细胞外钙离子的存在，也不受非渗透性钙离子螯合剂的影响。用Fura-2/AM或Indo-1测量胞质钙离子显示，BAPTA/AM处理的细胞中细胞内钙离子在后期而非早期增加。最后，BAPTA/AM诱导的凋亡伴随着即时早期反应基因c-jun的浓度依赖性下调。这些发现表明，诸如BAPTA/AM之类的钙离子螯合剂在人髓系白血病细胞凋亡调节中具有复杂作用，并表明该试剂可能选择性地拮抗核小体间DNA片段化，而不干扰凋亡反应的其他方面和/或细胞致死性。
