Nuclear cytometry and chromatin organization

Abstract

The nuclear-targeting chemical probe, for the detection and quantification of DNA within cells, has been a mainstay of cytometry-from the colorimetric Feulgen stain to smart fluorescent agents with tuned functionality. The level of nuclear structure and function at which the probe aims to readout, or indeed at which a DNA-targeted drug acts, is shadowed by a wide range of detection modalities and analytical methods. These methods are invariably limited in terms of the resolution attainable versus the volume occupied by targeted chromatin structures. The scalar challenge arises from the need to understand the extent and different levels of compaction of genomic DNA and how such structures can be re-modeled, reported, or even perturbed by both probes and drugs. Nuclear cytometry can report on the complex levels of chromatin order, disorder, disassembly, and even active disruption by probes and drugs. Nuclear probes can report defining features of clinical and therapeutic interest as in NETosis and other cell death processes. New cytometric approaches continue to bridge the scalar challenges of analyzing chromatin organization. Advances in super-resolution microscopy address the resolution and depth of analysis issues in cellular systems. Typical of recent insights into chromatin organization enabled by exploiting a DNA interacting probe is ChromEM tomography (ChromEMT). ChromEMT uses the unique properties of the anthraquinone-based cytometric dye DRAQ5™ to reveal that local and global 3D chromatin structures effect differences in compaction. The focus of this review is nuclear and chromatin cytometry, with linked reference to DNA targeting probes and drugs as exemplified by the anthracenediones.

Concurrent detection of lysosome and tissue transglutaminase activation in relation to cell cycle position during apoptosis induced by different anticancer drugs

“…In its most common applications, AO is being used to differentially stain DNA versus RNA or to assess susceptibility of DNA in situ to denaturation . Both these assays require fixed or permeabilized cells.…”

Targeting DNA topoisomerase IIα (TOP2A) in the hypoxic tumour microenvironment using unidirectional hypoxia‐activated prodrugs (uHAPs)

“…Further, the mechanism of action of an activated prodrug needs to cope with the modified cellular phenotypes that make up the hTME 10,14,63 . Even after the successful delivery of an active molecule to a nuclear environment by a DNA‐interactive HAP, for example, chromatin networks 64 can direct or limit drug access to specific sequences or the sites of functional complexes 65 . The mechanism of HAP activation is critical and needs to be selective for the hTME 12,14 .…”

“… 129 TOP2 poison drugs interact with the closed conformation of the DNA‐gate and inhibit the religation of cleaved G‐segment by the occupation of the DNA cleavage site. 129 , 130 The human TOP2 proteins, TOP2A and an isomer TOP2B, are the targets for several anti‐cancer agents 64 , 131 including etoposide, intercalating anthracyclines (doxorubicin and daunorubicin) and the anthraquinone MTX. 132 These drugs act as poisons stalling the enzyme at the 'TOP2 cleavage complex' stage (outlined in Figure 5 ).…”