title: Cytometry of DNA replication and RNA synthesis: Historical perspective and recent advances based on "click chemistry".
abstract: 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.
authors.affiliation: Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York 10595, USA. darzynk@nymc.edu
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Darzynkiewicz
authors.forename: Frank
authors.initials: F
authors.lastname: Traganos
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: H Dorota
authors.initials: HD
authors.lastname: Halicka
authors.forename: Jiangwei
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D002453:Cell Cycle; D057930:Click Chemistry / Q000639:trends*; D004261:DNA Replication; D005434:Flow Cytometry; D006801:Humans; D012313:RNA / Q000096:biosynthesis*
chemical_list: D012313:RNA