Please do not disturb: Destruction of chromatin structure by supravital nucleic acid probes revealed by a novel assay of DNA-histone interaction

Abstract

The biomarkers designed to be used supravitally are expected to have minimal effect on structure and function of the cell. Unfortunately nearly all fluorochromes developed to probe live cells interact in undesired way with cellular constituents and affect functional pathways. Herein we comment on potential applications of diverse DNA binding probes in view of the recent article by Wojcik & Dobrucki on DRAQ 5 and SYTO 17. The approach used by these authors to assess DNA-histone interactions using the cells having histones tagged with fluorescent proteins offers a valuable tool to study mechanism of action of antitumor drugs targeting DNA. While the effect of many intercalating drugs may be similar to that of DRAQ5, it may be of particular interest to observe the effects induced by intra-strand and inter-strand DNA crosslinking drugs, alkylating agents, histone deacetylase inhibitors or even anti-metabolites. The cells having histones tagged with fluorescent proteins thus may serve as biomarkers to probe mechanism of action of drugs targeting DNA or affecting chromatin structure. In fact, because such gross chromatin changes as revealed by dissociation and segregation of histones from DNA are most likely incompatible with long-term cell survival, the methodology may be applied for rapid screening of investigational antitumor agents.

Introduction

The biomarkers designed to be used supravitally are expected to have minimal effect on structure and function of the cell. Unfortunately nearly all fluorochromes developed to probe live cells interact in undesired way with cellular constituents and affect functional pathways. The DRAQ5 fluorochrome was developed as supravital stoichiometric DNA fluorochrome, providing new probe to assess cell cycle distribution or DNA ploidy in live cells (1). Unlike another DNA fluorochrome Hoechst 33342 DRAQ5 can be excited at long wavelength (1) which markedly extends its applications. DRAQ5 belongs to the class of the DNA intercalating agents of the anthraquinone family, very close in structure to the well characterized anticancer drug mitoxantrone [(1,4-dihydroxy-5,8-bis ((2-((2-hydroxyethyl)amino)ethyl)amino)anthraquinone], often abbreviated DHAQ (2).

In the recent report Wojcik and Dobrucki (3) describe rapidly occurring dramatic changes in nuclear architecture of HeLa cells after exposure to as low as 1 – 3 μM concentration of DRAQ5. The ability to visualize the chromatin changes was enabled by using the cells expressing eGFP-tagged histone H1 and histone H2B. This elegant methodology revealed the DRAQ5 induced aggregation of chromatin in distinct foci most likely prompted by dissociation of histone H1 from DNA, followed by release of histone H2B from nucleosome cores. The observation of such chromatin changes is consistent with the intercalative mechanism of DRAQ binding to DNA, which similar to DHAQ, causes dissociation of histones and condensation of nucleic acids (4,5). The intercalation of aromatic molecules such as DHAQ between the base-pairs raises the torsional stress and leads to unwinding and elongation of the DNA double helix (2). Such change in conformation of DNA in chromatin perturbs DNA-histone interactions and results in histones dissociation separation from DNA (6). Electron microscopy of nuclei of cells treated with DHAQ reveals patchy areas of condensed chromatin with increased electron opacity (5), strikingly resembling the images presented by Wojcik and Dobrucki (3).

In the original publication introducing DRAQ5 (1) the authors cautioned that because of the cytotoxicity the use of this fluorochrome for long-term cell culture and viable cell sorting is inappropriate. Indeed, the observed changes in chromatin after exposure to DRAQ5 (3) are incompatible with their survival. This does not diminish, however, the value of DRAQ5 as an useful plasma membrane permeant DNA dye that can be used in short-term experiments, especially when multiplexed with other fluorochromes (7,8). A caution, however, should be exercised in interpreting the data either pertaining to chromatin structure of the cells stained with DRAQ5 as representative of normal live cells or even to short term functional assays that may involve nuclear activity.

As a search for apoptosis-targeted anticancer therapeutics is ongoing, there is still a dire need for novel bioassays allowing dynamic studies of cell demise. Undoubtedly, development of virtually inert fluorescent probes would open up new opportunities for many functional assays by both flow and imaging cytometry. In this context, a novel class of substituted unsymmetrical cyanine SYTO probes has recently become commercially available (9). Derived from thiazole orange, SYTOs display low intrinsic fluorescence, with strong enhancement upon binding to DNA and/or RNA. Unlike DRAQ5 none of the SYTO probes can be, however, considered as a truly DNA selective marker. Moreover, no stoichiometric incorporation is observed following their cell entry (10). Cell permeability and broad selection of excitation/emission spectra has recently driven implementation of SYTO dyes in many polychromatic protocols with the detection of apoptosis being one of the most prominent applications (9, 10).

In their report Wojcik and Dobrucki (3) study interactions between chromatin and SYTO 17, a minor-groove binding representative of the far-red family (Ex/Em: 621/634 nm). Confocal visualization of eGFP tagged histones H1 and H2B revealed that, in contrast to DRAQ5, no histone detachment follows SYTO incorporation into DNA. Moreover, even at elevated doses of the dye authors observed only an insignificantly low chromatin aggregation. These noteworthy results are consistent with our recent data on non-toxic properties of selected SYTO probes (10). Surprisingly good intracellular retention of many SYTO molecules enables also straightforward tracing of pre-labeled subpopulations for extended periods of time. Importantly, however, no adverse effects on cell cycle progression are usually associated with SYTO incorporation (10). This is in stark contrast to DRAQ5, Hoechst 33342 or Vybrant DyeCycle Orange where substantial cytotoxicity and G2/M cell cycle arrest effectively obliterate their live-cell applications. Results by Wojcik and Dobrucki (3) noticeably support the notion that loading of cells with minor groove binders like SYTO 17 can facilitate long-term cell tracking and/or viable sorting with minimal effects on the structure and function of the cell.

Although SYTO 17 binding does not perturb chromatin structure, an interesting question arises here about the detection of chromatin changes by SYTO probes. We have only recently postulated that nucleolar segregation, may give explanation to attenuation of SYTO fluorescence during caspase-dependent apoptosis (10, 11). Nucleolar segregation has been observed following the treatment with different types of anti-tumor drugs, in particular with DNA intercalators (12). It was initially assumed that nucleolar segregation is a specific cell response in terms of modulation of chromatin and nucleolar structure due to drug-induced nucleic acids condensation (13, 14). Later reports noticed that nucleolar segregation occurs also early during apoptosis where it appears to be a characteristic “hallmark” of this mode of cell demise (15, 16). Several SYTO dyes have also substantial affinity to RNA. While the condensation, segregation and extrusion of RNA occurs early during apoptosis, this can potentially lie behind decreased stainability of apoptotic cells with these cyanine fluorochromes (10, 11). Further investigation whether SYTOs can be successfully implemented as markers of nuclear/nucleolar rearrangements awaits further exploration.

In context of our discussion, it should be mentioned that the bisbenzimidazole dye Hoechst 33342, similar as SYTO 17, is classified as minor grove ligand and it binds with a preference to A+T rich regions (17). The use of Hoechst 33342 vastly expanded in recent years when it became apparent that its binding restriction to live cells provides a marker of the “side population” (SP) deemed stem cells (18). While limited binding of Hoechst to DNA of stem cells indeed may be non-toxic (18), the interactions with non-stem cells was shown to induce severe perturbations, particularly in combination with UV irradiation (19,20). Reportedly, Hoechst is an inhibitor of DNA topoisomerase I (17,21,22) and induces single strand DNA breaks (17). While it causes nuclear condensation it does not re-arrange nucleosomal structure (22). Interestingly, binding of Hoechst 33342 to chromatin was reported to protect cells from radiation-induced damage to DNA, likely by the means of the ROS scavenging properties of this probe (22). The extent of protection, however, varies between different cell lines (23). While Hoechst 33342 appears to be the probe that is more deleterious to chromatin structure and cell viability than SYTO 17, it would be of interest to compare both dyes using the assay developed by Wojcik and Dobrucki (3).

The approach used by these authors to assess DNA-histone interactions using the cells having histones tagged with fluorescent proteins (3) offers a valuable tool to study mechanism of action of antitumor drugs targeting DNA. While the effect of many intercalating drugs may be similar to that of DRAQ5, it may be of particular interest to observe the effects induced by intra-strand and inter-strand DNA crosslinking drugs, alkylating agents, histone deacetylase inhibitors or even anti-metabolites. The cells having histones tagged with fluorescent proteins thus may serve as biomarkers to probe mechanism of action of drugs targeting DNA or affecting chromatin structure. In fact, because such gross chromatin changes as revealed by dissociation and segregation of histones from DNA are most likely incompatible with long-term cell survival, the methodology may be applied for rapid screening of investigational antitumor agents.