Protein kinase C inhibition by UCN-01 induces apoptosis in human glioma cells in a time-dependent fashion
Protein kinase C inhibition by UCN-01 induces apoptosis in human glioma cells in a time-dependent fashion
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DOI：
10.1023/a:1006047025425
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发表时间：
1999-01-01
影响因子：
3.9
通讯作者：
Lazo, JS
中科院分区：
文献类型：
--
作者：
Bredel, M;Pollack, IF;Lazo, JS
本实验室最近的研究表明，UCN-01(7-羟基丝氨酸菌素)是一种非选择性蛋白激酶抑制剂星形孢菌素的衍生物，对蛋白激酶C(PKC)具有相对选择性，在体外和体内模型中对胶质瘤生长具有有效的抑制作用。这种药物具有细胞毒性和细胞抑制作用，这取决于药物暴露的时间段。在本研究中，我们检测了UCN-01诱导的细胞毒性是否与诱导细胞凋亡有关，并进一步表征了这一过程的时间进程，作为UCN-01应用于临床试验的前奏。我们首次证明了UCN-01的细胞毒作用与诱导细胞凋亡的形态特征有关。其次。我们利用场翻转凝胶电泳法在一系列时间点半定量地鉴定了细胞凋亡的电泳学特征。这些研究表明，在药物治疗3-6天后，高分子量DNA片段化的诱导达到顶峰。第三，我们用末端转移酶催化的原位末端标记技术测量了不同时间点发生凋亡的细胞的百分比，证实了凋亡细胞数量随时间和浓度的增加而增加。这与台盼蓝排除法评估的存活细胞百分比的进行性下降有关。细胞杀伤在UCN-01治疗后2-4天内达到高峰，但在接下来的几天继续保持在较低的水平。综上所述，这些研究表明，为了最佳地表现出对胶质瘤细胞的细胞毒作用，需要延长UCN-01的暴露时间，这是在设计这种药物治疗恶性胶质瘤的未来临床试验时必须考虑的一个因素。
Recent studies in our laboratory have shown that UCN-01 (7-hydroxystaurosporine), which is a derivative of the non-selective protein kinase inhibitor staurosporine that exhibits relative selectivity for protein kinase C (PKC), is a potent inhibitor of glioma growth in in vitro and in vivo models. This agent exhibits both cytotoxic and cytostatic effects, depending on the time period of drug exposure. In the present study, we examined whether UCN-01-induced cytotoxicity correlated with the induction of apoptosis, and characterized further the time course of this process as a prelude to application of UCN-01 in clinical trials. We first demonstrated that the cytotoxic effects of UCN-01 were associated with the induction of morphological features of apoptosis. Secondly. we identified electrophoretic features of apoptosis semiquantitatively at a series of time points using field inversion gel electrophoresis. These studies showed a peak in the induction of high-molecular-weight DNA fragmentation after 3-6 days of drug treatment. Thirdly, we measured the percentage of cells undergoing apoptosis at various time points using a terminal transferase-catalyzed in situ end-labeling technique, which confirmed a time- and concentration-dependent increase in apoptotic cell numbers. This correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. Cell killing peaked within 2-4 days after beginning UCN-01 treatment, but continued at a lower level in the ensuing days. Taken together, these studies demonstrated that extended periods of exposure to UCN-01 are needed for optimal manifestation of cytotoxic effects against glioma cells, a factor that must be taken into consideration in the design of future clinical trials with this agent for malignant gliomas.