Laboratory of Cytobiochemistry, Faculty of Biotechnology, University of Wrocław, S. Przybyszewskiego 63-77, 51-148 Wrocław, Poland.
Mol Cell Biochem. 2010 Jun;339(1-2):63-77. doi: 10.1007/s11010-009-0370-4. Epub 2010 Jan 8.
It has been shown that changes in spectrin distribution in early apoptosis preceded changes in membrane asymmetry and phosphatidylserine (PS) exposure. PKCtheta was associated with spectrin during these changes, suggesting a possible role of spectrin/PKCtheta aggregation in regulation of early apoptotic events. Here we dissect this hypothesis using Jurkat T and HL60 cell lines as model systems. Immunofluorescent analysis of alphaIIbetaII spectrin arrangement in Jurkat T and HL60 cell lines revealed the redistribution of spectrin and PKCtheta into a polar aggregate in early apoptosis induced by fludarabine/mitoxantrone/dexamethasone (FND). The appearance of an alphaIIbetaII spectrin fraction that was insoluble in a non-ionic detergent (1% Triton X-100) was observed concomitantly with spectrin aggregation. The changes were observed within 2 h after cell exposure to FND, and preceded PS exposure. The changes seem to be restricted to spectrin and not to other cytoskeletal proteins such as actin or vimentin. In studies of the mechanism of these changes, we found that (i) neither changes in apoptosis regulatory genes (e.g., Bcl-2 family proteins) nor changes in cytoskeleton-associated proteins were detected in gene expression profiling of HL60 cells after the first hour of FND treatment, (ii) caspase-3, -7, -8, and -10 had minor involvement in the early apoptotic rearrangement of spectrin/PKCtheta, and (iii) spectrin aggregation was shown to be partially dependent on PKCtheta activity. Our results indicate that spectrin/PKCtheta aggregate formation is related to an early stage in drug-induced apoptosis and possibly may be regulated by PKCtheta activity. These findings indicate that spectrin/PKCtheta aggregation could be considered as a hallmark of early apoptosis and presents the potential to become a useful diagnostic tool for monitoring efficiency of chemotherapy as early as 24 h after treatment.
已经表明，在早期细胞凋亡中，血影蛋白分布的变化先于膜不对称性和磷脂酰丝氨酸（PS）暴露的变化。在这些变化过程中，PKCθ与血影蛋白相关联，这表明血影蛋白/ PKCθ聚集可能在调节早期凋亡事件中起作用。在这里，我们使用 Jurkat T 和 HL60 细胞系作为模型系统来剖析这一假设。使用 Jurkat T 和 HL60 细胞系的 alphaIIbetaII 血影蛋白排列的免疫荧光分析表明，在 fludarabine/mitoxantrone/dexamethasone（FND）诱导的早期细胞凋亡中，血影蛋白和 PKCθ重新分布到极性聚集体中。在与血影蛋白聚集同时观察到 alphaIIbetaII 血影蛋白部分变得不溶于非离子洗涤剂（1％Triton X-100）。这些变化在细胞暴露于 FND 后 2 小时内观察到，并且先于 PS 暴露。这些变化似乎仅限于血影蛋白，而不是其他细胞骨架蛋白，如肌动蛋白或波形蛋白。在对这些变化的机制进行研究时，我们发现（i）在 FND 处理后第一个小时，HL60 细胞的基因表达谱中未检测到凋亡调节基因（例如 Bcl-2 家族蛋白）或细胞骨架相关蛋白的变化，（ii）caspase-3、-7、-8 和 -10 在血影蛋白/ PKCθ的早期凋亡重排中仅起次要作用，（iii）血影蛋白聚集部分依赖于 PKCθ的活性。我们的结果表明，血影蛋白/ PKCθ聚集体的形成与药物诱导的细胞凋亡的早期阶段有关，并且可能受到 PKCθ活性的调节。这些发现表明，血影蛋白/ PKCθ聚集可以被认为是早期凋亡的标志，并有可能成为在治疗后 24 小时内监测化疗效率的有用诊断工具。
