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 3H- or 14C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G1, S, G2, 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 3H-uridine incorporation and RNA content provided the means to distinguish quiescent G0 from cycling G1 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.

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips

“…The mild conditions and rapid detection of EdU by click chemistry (Salic and Mitchison, 2008;Darzynkiewicz et al, 2011) are an improvement to the classical BrdU labeling protocol we used previously (Lee et al, 2010). The use of EdU also enables another level of purification of S phase nuclei by two-color sorting before immunoprecipitation of labeled DNA.…”

“…EdU offers a substantial improvement over classical replication timing assays, which have typically used 5-bromo-2'-deoxyuridine (BrdU) to label and immunoprecipitate newly replicated DNA (Hiratani et al, 2008;Schwaiger et al, 2009;Chen et al, 2010;Hansen et al, 2010;Lee et al, 2010;Ryba et al, 2011). A key benefit of using EdU is that a heat or acid denaturation step is not required for immunoprecipitation (reviewed in Darzynkiewicz et al, 2011). Hence, it is possible to visualize EdU-labeled DNA while maintaining native subnuclear structure (Kotogany et al, 2010;Bass et al, 2014) and to separate labeled from unlabeled nuclei by flow cytometry prior to DNA isolation .…”

Assessment of Cell Cycle Inhibitors by Flow Cytometry

“…It is the case of EdU (5-ethynyl-2'-deoxyuridine) used instead of BrdU for DNA synthesis analysis in FCM. EdU is incorporated into the replication forks of new DNA during Sphase; exposed alkynyl residues can be identified with "click chemistry" reaction using fluorescent azides (AlexaFluor™ 488 Click-IT assay supply by Invitrogen Corp) in presence of copper (I) and cells could be visualized (Darzynkiewicz et al, 2011). One modification of this assay is by replacing dye azide, with a BrdU azide and to detect by BrdU antibody (Fig.…”

The Use of Flow Cytometric DNA Ploidy Analysis of Liver Biopsies in Liver Cirrhosis and Hepatocellular Carcinoma

“…Modern flow cytometers are able to analyze several thousand particles every second (analyzer flow cytometry) and can actively separate and isolate particles having specified properties; cell sorter flow cytometry (Darzynkiewicz et al, 2011). A flow cytometer is similar to a microscope, except that, instead of producing an image of the cell, flow cytometry offers "high-throughput" (for a large number of cells) automated quantification of set parameters .…”

“…To assess DNA ploidy of the tumor sample one has to compare DNA content of the G0/G1 cells population of the presumed tumor cells with that of normal (control) cells. Towards this end most frequently the peak value of the integrated fluorescence (peak channel) of G0/1 population of normal cells is being considered to be DI 1.0 and DNA ploidy of the tumor cells is expressed as a ratio of the peak value (channel) of fluorescence intensity of these cells with respect to that of the normal G0/1 cells (Darzynkiewicz et al, 2011). It is also common to express DI of the tumor as a ratio of modal rather than the peak value of fluorescence intensity representing DNA content of G0/G1 population tumor cells to modal value of G0/1 population of normal cells.…”