Current approaches have limitations in providing insight into the functional properties of particular nucleosomes in their native molecular environment. Here we describe a simple and powerful method involving elution of histones using intercalators or salt, to assess stability features dependent on DNA superhelicity and relying mainly on electrostatic interactions, respectively, and measurement of the fraction of histones remaining chromatin-bound in the individual nuclei using histone type- or posttranslational modification- (PTM-) specific antibodies and automated, quantitative imaging. The method has been validated in H3K4me3 ChIP-seq experiments, by the quantitative assessment of chromatin loop relaxation required for nucleosomal destabilization, and by comparative analyses of the intercalator and salt induced release from the nucleosomes of different histones. The accuracy of the assay allowed us to observe examples of strict association between nucleosome stability and PTMs across cell types, differentiation state and throughout the cell-cycle in close to native chromatin context, and resolve ambiguities regarding the destabilizing effect of H2A.X phosphorylation. The advantages of the in situ measuring scenario are demonstrated via the marked effect of DNA nicking on histone eviction that underscores the powerful potential of topological relaxation in the epigenetic regulation of DNA accessibility.

We observed prominent effects of doxorubicin (Dox), an anthracycline widely used in anticancer therapy, on the aggregation and intracellular distribution of both partners of the H2A-H2B dimer, with marked differences between the two histones. Histone aggregation, assessed by Laser Scanning Cytometry via the retention of the aggregates in isolated nuclei, was observed in the case of H2A. The dominant effect of the anthracycline on H2B was its massive accumulation in the cytoplasm of the Jurkat leukemia cells concomitant with its disappearance from the nuclei, detected by confocal microscopy and mass spectrometry. A similar effect of the anthracycline was observed in primary human lymphoid cells, and also in monocyte-derived dendritic cells that harbor an unusually high amount of H2B in their cytoplasm even in the absence of Dox treatment. The nucleo-cytoplasmic translocation of H2B was not affected by inhibitors of major biochemical pathways or the nuclear export inhibitor leptomycin B, but it was completely diminished by PYR-41, an inhibitor with pleiotropic effects on protein degradation pathways. Dox and PYR-41 acted synergistically according to isobologram analyses of cytotoxicity. These large-scale effects were detected already at Dox concentrations that may be reached in the typical clinical settings, therefore they can contribute both to the anti-cancer mechanism and to the side-effects of this anthracycline.

Unexpectedly, the widely used anticancer agents cisplatin (cis-pt) and Daunorubicin (Dauno) exhibited cell type-and concentration-dependent synergy or antagonism in vitro. We attempted to interpret these effects in terms of the changes elicited by the drugs in the chromatin, the target held primarily responsible for the cytotoxicity of both agents. We measured the effect of Cis-Pt on the levels of Dauno in different cell compartments, the effect of Cis-Pt on Dauno-induced nucleosome eviction, and assessed the influence of Dauno on DNA platination in flow-and laser scanning cytometry as well as in laser ablation-inductively coupled plasma-mass spectrometry assays. We show that the two drugs antagonize each other through a decrease of interstrand crosslinks upon co-treatment with Dauno, and also via the diminished Dauno uptake in the presence of Cis-Pt, and both effects are observed already at low Dauno concentrations. At high Dauno concentrations synergy becomes dominant because histone eviction by Dauno intercalation into the DnA is enhanced in the presence of co-treatment with cis-pt. These interactions may have an impact on the efficacy of combination treatment protocols, considering the long retention time of DnA adducts formed by both agents. Anticancer drugs that target DNA are some of the most effective agents in combating cancer. Anthracyclines and platinum-based drugs have been exploited in combination chemotherapy to treat a broad variety of different types of cancer, such as ovarian carcinoma 1,2 , small cell lung cancer 3 , and in endometrial carcinoma 4 , among others. Although many of the cellular targets of these anticancer agents are known, their involvement in toxicity is poorly understood. Therefore, how they interact with each other when used in combination is difficult to predict. Daunorubicin (Dauno) 5 , the first discovered anthracycline compound 6 , affects a broad range of biochemical processes and a number of different mechanisms have been proposed to be responsible for Dauno-elicited cytotoxicity. These include inhibition of DNA and RNA synthesis (mainly due to binding of the drug to the DNA), Topoisomerase II poisoning (by trapping the enzyme at cleavage sites), oxidative stress (due to formation of reactive oxygen species), involving also lipid peroxidation (by chelating iron), as reviewed in 7. Dauno binds the H1 family of histones and causes chromatin aggregation thereby influencing higher-order chromatin conformation 8. As a DNA intercalator, Dauno unwinds the double-stranded DNA by 8° per each intercalated molecule 8,9 , thereby extending it, and also increasing its melting temperature 10. Dauno will relax, and at higher concentrations overwind the DNA 9,11. Simultaneously with relaxation, nucleosomes become evicted 9,12. Thus there is ample reason to assume that the binding of other DNA targeting drugs may be altered when Dauno is simultaneously applied influencing DNA topology. Since Dauno and many other DNA targeting drugs such as Cisplatin (Cis-Pt) form rather stable adducts in ...

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