Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York 10595, USA.
Cytometry A. 2011 May;79(5):328-37. doi: 10.1002/cyto.a.21048. Epub 2011 Mar 18.
This review covers progress in the development of cytometric methodologies designed to assess DNA replication and RNA synthesis. The early approaches utilizing autoradiography to detect incorporation of (3) H- or (14) C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G(1) , S, G(2) , and M, and by analysis of the fraction of labeled mitosis (FLM), to precisely define the kinetics of cell progression through these phases. Analysis of (3) H-uridine incorporation and RNA content provided the means to distinguish quiescent G(0) from cycling G(1) cells. Subsequent progress in analysis of DNA replication was based on the use of BrdU as a DNA precursor and its detection by the quenching of the fluorescence intensity of DNA-bound fluorochromes such as Hoechst 33358 or acridine orange as measured by flow cytometry. Several variants of this methodology have been designed and used in studies to detect anticancer drug-induced perturbations of cell cycle kinetics. The next phase of method development, which was particularly useful in studies of the cell cycle in vivo, including clinical applications, relied on immunocytochemical detection of incorporated halogenated DNA or RNA precursors. This approach however was hampered by the need for DNA denaturation, which made it difficult to concurrently detect other cell constituents for multiparametric analysis. The recently introduced "click chemistry" approach has no such limitation and is the method of choice for analysis of DNA replication and RNA synthesis. This method is based on the use of 5-ethynyl-2'deoxyuridine (EdU) as a DNA precursor or 5-ethynyluridine (EU) as an RNA precursor and their detection with fluorochrome-tagged azides utilizing a copper (I) catalyzed [3+2] cycloaddition. Several examples are presented that illustrate incorporation of EdU or EU in cells subjected to DNA damage detected as histone H2AX phosphorylation that have been analyzed by flow or laser scanning cytometry.
本文综述了用于评估 DNA 复制和 RNA 合成的细胞测量方法的进展。早期的方法利用放射性自显影来检测（3）H 或（14）C 标记的胸腺嘧啶的掺入，从而能够识别细胞周期的四个基本阶段 G1、S、G2 和 M，并通过分析有丝分裂标记的分数（FLM），精确地定义细胞通过这些阶段的动力学。（3）H-尿嘧啶掺入和 RNA 含量的分析提供了区分静止 G0 和循环 G1 细胞的方法。随后，DNA 复制分析的进展基于将 BrdU 用作 DNA 前体，并通过流式细胞术测量的荧光染料如 Hoechst 33358 或吖啶橙结合的 DNA 荧光强度的猝灭来检测其掺入。已经设计并使用了几种变体来检测抗癌药物诱导的细胞周期动力学扰动。方法开发的下一阶段，特别是在体内细胞周期研究中，包括临床应用中，依赖于掺入的卤代 DNA 或 RNA 前体的免疫细胞化学检测。然而，这种方法受到需要 DNA 变性的限制，这使得难以同时检测其他细胞成分进行多参数分析。最近引入的“点击化学”方法没有这种限制，是分析 DNA 复制和 RNA 合成的首选方法。该方法基于将 5-乙炔基-2'-脱氧尿苷（EdU）用作 DNA 前体或 5-乙炔基尿苷（EU）用作 RNA 前体，并利用铜（I）催化的[3+2]环加成反应用荧光染料标记的叠氮化物进行检测。本文提供了几个示例，说明了 EdU 或 EU 在经历 DNA 损伤的细胞中的掺入，这些损伤被检测为组蛋白 H2AX 磷酸化，并通过流式细胞术或激光扫描细胞术进行了分析。
