Daunomycin-induced Unfolding and Aggregation of Chromatin

Abstract

Using equilibrium dialysis and sedimentation velocity analysis, we have characterized the binding of the antitumor drug daunomycin to chicken erythrocyte chromatin before and after depletion of linker histones and to its constitutive DNA under several ionic strengths (5, 25, and 75 mM NaCl). The equilibrium dialysis experiments reveal that the drug binds cooperatively to both the chromatin fractions and to the DNA counterpart within the range of ionic strength used in this study. A significant decrease in the binding affinity was observed at 75 mM NaCl. At any given salt concentration, daunomycin exhibits higher binding affinity for DNA than for linker histone-depleted chromatin or chromatin (in decreasing order). Binding of daunomycin to DNA does not significantly affect the sedimentation coefficient of the molecule. This is in contrast to binding to chromatin and to its linker histone-depleted counterpart. In these instances, preferential binding of the drug to the linker DNA regions induces an unfolding of the chromatin fiber that is followed by aggregation, presumably because of histone-DNA interfiber interactions.Daunomycin is an anthracycline antibiotic widely used as a potent chemotherapeutic agent in the treatment of various cancers (1, 2). Studies on its mechanism of action indicate that nuclear DNA is an important target for daunomycin. The structure of this pharmacologically active drug consists of two distinct domains ( Fig. 1): a planar aglycon chromophore that intercalates between adjacent base pairs of DNA and an amino sugar ring that lies in the minor groove of the DNA double helix (3, 4). Binding of daunomycin to DNA results in the inhibition of both DNA replication and RNA transcription (5-7).The binding of daunomycin to DNA has been studied in detail in the past (8 -11). However, in the cell, DNA does not exist as a naked structure but is associated with histones and other nuclear proteins in a complex that is known as chromatin (12-13). Thus, chromatin, not DNA, is the major target for daunomycin in vivo. How the presence of chromosomal proteins might modulate the binding of this drug to DNA and how in turn chromatin structure is affected by the binding of the drug are important questions that need to answered to understand the biological mechanism of action of daunomycin. Although several attempts in this direction have already been carried out using isolated nucleosome (14, 15) and crude chromatin fractions (16 -18), the mechanism of daunomycin binding to chromatin and the conformational transitions involved are still poorly understood. In this paper we report several experiments designed to address these questions in more detail.We have studied the ionic strength dependence of the binding of daunomycin to a well defined fraction of chicken erythrocyte chromatin consisting of an average number of 43 nucleosomes. The results are compared with those obtained with the same chromatin fraction upon depletion of linker histones and with the purified constitutive DNA counterpart. EXPERIMENTAL PROC...

NAP1 Modulates Binding of Linker Histone H1 to Chromatin and Induces an Extended Chromatin Fiber Conformation

“…The chromatin fibers studied were isolated from HeLa cells and showed hydrodynamic and structural properties comparable with earlier studies (38,47,58,74). Incubation with NAP1 changed the chromatin sedimentation profile significantly as apparent from the 30 -45% decrease of the sedimentation coefficient distribution.…”

The anthracycline antibiotics: antitumor drugs that alter chromatin structure

“…(19,20) However, our results and those of others using the same technique have demonstrated the occurrence of positive cooperative binding isotherms. (22)(23)(24) These controversial results could be due to differences in the DNA sequence and/or experimental conditions used. The magnitude of the binding constant of daunomycin to DNA is strongly dependent on the base pair composition of DNA.…”

“…(66) Concluding remarks As has been described in the previous sections, DNA intercalation of anthracyclines leads to changes in chromatin supercoiling that eventually result in chromatin aggregation. (23) DNA intercalation still remains one of the main targeting sites for the binding of these drugs to DNA. Therefore, in closing, we would like to propose that such aggregation, in addition to the chromatin structural changes described above, may contribute in a concerted way to the apoptotic cell death induced by these drugs.…”

“…Therefore, in closing, we would like to propose that such aggregation, in addition to the chromatin structural changes described above, may contribute in a concerted way to the apoptotic cell death induced by these drugs. (58,59) Chromatin aggregation (23) may facilitate or promote the DNA fragmentation (68,69) by specific apoptotic endonucleases. Indeed, it has been recently shown that chromatin aggregation precedes oligonucleosomal fractionation (70) and that DNA is supercoiled in apoptotic chromatin.…”

Exploration of Electrochemical Intermediates of the Anticancer Drug Doxorubicin Hydrochloride Using Cyclic Voltammetry and Simulation Studies with an Evaluation for Its Interaction with DNA

“…In the cell, nuclear DNA is associated with a variety of proteins forming a nucleoprotein complex called chromatin [18]. Accumulating evidence suggests these drugs interact with chromatin leading to chromatin unfolding and aggregation that causes a change in structure and function of chromatin [19,20]. The structural disruption is likely to interfere with metabolic processes of DNA (replication and transcription) contributing to apoptosis induced by these drugs in cancer cells [19,20].…”