Badran A, Iwasaki H, Wano Y, Kishi S, Ueda T
First Department of Internal Medicine, Fukui Medical University, Matsuoka, Japan.
Anticancer Res. 2001 May-Jun;21(3B):1987-95.
A wide variety of chemotherapeutic agents including 1-beta-D-arabinofuranosylcytosine can induce cell death by apoptosis. However, an appropriate method for early detection of apoptosis that can be clinically applicable to peripheral blood samples freshly obtained from leukemic patients under chemotherapy has not yet been established. We investigated the chronology of ara-C induced apoptosis in CCRF-CEM cells by monitoring the expression of a number of apoptotic markers in a time- and concentration-dependent manner with the aim of estimating the earliest reliable marker of apoptosis to apply to clinical drug sensitivity assays. We employed the following techniques: 1) Pulsed field gel electrophoresis to detect high molecular weight DNA fragmentation; 2) FACS analysis for evaluation of APO 2.7 expression and phosphatidylserine externalization; 3) May-Grunwald-Giemsa staining for studying gross morphology; and 4) TUNEL assay to detect multitudes of terminal 3'-OH groups of oligonucleosomal DNA fragments. At 10 microM ara-C, high molecular weight DNA fragmentation was observed after 4 hours incubation being the earliest marker to manifest. APO 2.7 expression and phosphatidylserine externalization were detected almost simultaneously at 6 hours, with marked similarity in their kinetics. Emergence of apoptotic bodies then followed after 12 hours incubation and, finally, oligonucleosomal DNA fragments were demonstrated by positive TUNEL assay at 48 hours. The results suggest the time-sequential expression of each individual marker and introduce high molecular weight DNA fragmentation assay as the most suitable candidate for early detection of sensitivity of malignant cells to apoptosis-inducing anticancer agents, including ara-C.
包括1-β-D-阿拉伯呋喃糖基胞嘧啶在内的多种化疗药物可通过凋亡诱导细胞死亡。然而，尚未建立一种适用于临床的早期检测凋亡的方法，该方法可应用于化疗期间从白血病患者新鲜获取的外周血样本。我们通过以时间和浓度依赖性方式监测多种凋亡标志物的表达，研究了阿糖胞苷（ara-C）诱导CCRF-CEM细胞凋亡的时间顺序，目的是评估可应用于临床药物药物敏感性试验的最早可靠的凋亡标志物。我们采用了以下技术：1）脉冲场凝胶电泳检测高分子量DNA片段化；2）流式细胞术分析评估APO 2.7表达和磷脂酰丝氨酸外化；3）May-Grunwald-Giemsa染色研究大体形态；4）TUNEL测定法检测寡核小体DNA片段的多个末端3'-OH基团。在10 microM阿糖胞苷作用下，孵育4小时后观察到高分子量DNA片段化，这是最早出现的标志物。APO 2.7表达和磷脂酰丝氨酸外化在6小时时几乎同时被检测到，其动力学具有显著相似性。孵育12小时后出现凋亡小体，最后，在48小时时通过TUNEL阳性测定法证实存在寡核小体DNA片段。结果表明了每个标志物的时间顺序表达，并引入高分子量DNA片段化测定法作为早期检测恶性细胞对包括阿糖胞苷在内的凋亡诱导抗癌药物敏感性的最合适候选方法。
