Leung M F, Sokoloski J A, Sartorelli A C
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Cancer Res. 1992 Feb 15;52(4):949-54.
The cytoskeleton is composed mainly of microtubules (MT), microfilaments, and intermediate filaments (IF) that form a structural network which connects cellular membranes, cytoplasmic organelles, and the nucleus. Since the cytoskeleton may be involved in modulating signal transduction and in the morphological and structural changes that occur during cellular proliferation and differentiation, cytoskeletal changes were measured by immunofluorescence microscopy and fluorescence-activated cell sorter analysis during the differentiation of HL-60 leukemia cells induced by retinoic acid (RA). Differentiated HL-60 cells exhibited increased staining intensity and altered organization of MT and IF, as visualized by immunofluorescence microscopy with anti-tubulin monoclonal antibody and anti-vimentin antibody, respectively. A new procedure was developed and used to measure the content of the cytoskeletal components of HL-60 cells during the process of maturation. HL-60 cells were fixed with formaldehyde in an MT-stabilizing buffer, permeabilized using L-lysophosphatidylcholine, stained for immunofluorescent measurement with antibodies specific for particular cytoskeletal components, and analyzed by flow cytometry. Terminally differentiated cells produced by exposure to RA contained larger amounts of MT and the IF vimentin. During the course of the maturation process, a transient increase in the amounts of the microtubule-associated proteins, (MAPs) MAP2 and tau, occurred. An RA-supersensitive clone, designated HL-60/S4, and an RA-resistant clone, designated HL-60/R3, were developed by mutagenization and selection. Use of these clones supported the concept that the observed changes in MT, MAPs, and vimentin were associated with the differentiation process rather than being due to other effects produced by the retinoid. Thus, the findings suggest that changes in MT, MAPs, and IF are important to the terminal maturation of leukemia cells.
细胞骨架主要由微管（MT）、微丝和中间丝（IF）组成，它们形成一个连接细胞膜、细胞质细胞器和细胞核的结构网络。由于细胞骨架可能参与调节信号转导以及细胞增殖和分化过程中发生的形态和结构变化，因此在用视黄酸（RA）诱导HL - 60白血病细胞分化的过程中，通过免疫荧光显微镜和荧光激活细胞分选分析来检测细胞骨架的变化。分别用抗微管蛋白单克隆抗体和抗波形蛋白抗体进行免疫荧光显微镜观察，结果显示，分化后的HL - 60细胞微管和中间丝的染色强度增加且排列改变。开发了一种新方法并用于测量HL - 60细胞在成熟过程中细胞骨架成分的含量。HL - 60细胞在MT稳定缓冲液中用甲醛固定，用L - 溶血磷脂酰胆碱使其通透，用针对特定细胞骨架成分的抗体进行免疫荧光染色测量，然后通过流式细胞术进行分析。暴露于RA产生的终末分化细胞含有大量的微管和中间丝波形蛋白。在成熟过程中，微管相关蛋白（MAPs）MAP2和tau的量出现短暂增加。通过诱变和筛选得到了一个RA超敏感克隆，命名为HL - 60/S4，以及一个RA抗性克隆，命名为HL - 60/R3。使用这些克隆支持了以下观点：观察到的微管、MAPs和波形蛋白的变化与分化过程相关，而不是由于类视黄醇产生的其他效应。因此，这些发现表明微管、MAPs和中间丝的变化对白血病细胞的终末成熟很重要。
