Studies on the binding affinity of anticancer drug mitoxantrone to chromatin, DNA and histone proteins

Mitoxantrone is a potent antitumor drug, widely used in the treatment of various cancers. In the present study, we have investigated and compared the affinity of anticancer drug, mitoxantrone, to EDTA-soluble chromatin (SE-chromatin), DNA and histones employing UV/Vis, fluorescence, CD spectroscopy, gel electrophoresis and equilibrium dialysis techniques. The results showed that the interaction of mitoxantrone with SE-chromatin proceeds into compaction/aggregation as revealed by reduction in the absorbencies at 608 and 260 nm (hypochromicity) and disappearance of both histones and DNA on the gels. Mitoxantrone interacts strongly with histone proteins in solution making structural changes in the molecule as shown by CD and fluorescence analysis. The binding isotherms demonstrate a positive cooperative binding pattern for the chromatin- mitoxantrone interaction. It is suggested higher binding affinity of mitoxantrone to chromatin compared to DNA implying that the histone proteins may play an important role in the chromatin- mitoxantrone interaction process.     

Interaction of mitoxantrone, as an anticancer drug, with chromatin proteins, core histones and H1, in solution

In the present study, for the first time we have investigated the interaction of anticancer drug mitoxantrone with histone H1 and core histone proteins in solution using fluorescence, UV/Vis, CD spectroscopy and thermal denaturation techniques. The results showed that mitoxantrone reduced the absorbencies of H1 and core histone proteins at 210 nm (hypochromicity) and fluorescence emission intensity was decreased in a dose dependent. Binding of mitoxantrone changed secondary structures of the proteins as circular dichroism analysis confirmed it. Also, mitoxantrone increased the melting temperature of core histones at the final step of denaturation. The results suggest higher affinity of mitoxantrone to histone H1 compared to core histones providing histone proteins as a new target for mitoxantrone action at the chromatin level.     

Sensing core histone phosphorylation — A matter of perfect timing

Systematic analysis of histone modifications has revealed a plethora of posttranslational modifications that mediate changes in chromatin structure and gene expression. Histone phosphorylation is a transient histone modification that becomes induced by extracellular signals, DNA damage or entry into mitosis. Importantly, phosphorylation of histone proteins does lead not only to the binding of specific reader proteins but also to changes in the affinity for readers or writers of other histone modifications. This induces a cross-talk between different chromatin modifications that allows the spatio-temporal control of chromatin-associated events. In this review we will summarize the progress in our current knowledge of factors sensing reversible histone phosphorylation in different biological scenarios. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.     

Exchange of H1 histone depends on aggregation of chromatin, not simply on ionic strength

Chromatin solubility was observed at several concentrations of various cations. Spermine and spermidine precipitated (50%) chromatin at about 0.2 mM, Ca2+ and Mg2+ at about 1-2 mM, and Na+ at about 100 mM. Further increases in cation concentration induced more aggregation, but eventually excess cation increased chromatin solubility so that 50% solubility was observed again at 60 mM Mg2+ and 180 mM Na+. H1 histone was 50% released by 80 mM MgCl2 or 425 mM NaCl. Combinations of MgCl2 and NaCl showed that Mg2+ and Na+ are synergistic in the induction of aggregation in lower concentrations (less than 2 mM) of Mg2+ but antagonistic at higher concentrations, and a similar effect of NaCl on spermidine-induced precipitation was shown below and above about 0.2 mM spermidine. At 5 mM, MgCl2 proved capable of precipitating chromatin depleted of H1 histone, but no concentration of NaCl was capable of doing so. These phenomena can be rationalized by supposing that neutralization of chromatin by any cation (including H1 histone) favors aggregation and also that cross-linking of chromatin fibers by multivalent cations (including H1 histone) is also critically important. The exchange of H1 histone between chromatin fragments was tested in various concentrations of different salts. H1 exchange was correlated with chromatin aggregation rather than with ionic strength and thus appears to depend on fiber to fiber contact. Under conditions where H1 exchanges between chromatin fibers that are permitted to make contact with each other, no H1 exchange occurred between chromatin inside the nucleus and chromatin outside, even though H1 histone is capable of passage through the nuclear membrane.     

Distinct effects of histone H1 and non-histone chromosomal proteins on the structure of nucleosomes and the chromatin fibre in solution

In this study we attempt to differentiate between the effects of the non-histone chromosomal proteins and histone H1 on the structure of the nucleosomes and the chromatin fibre in solution. The properties of chromatin preparations with different histone H1 and non-histone protein compositions were compared using circular dichroism and flow linear dichroism and the following conclusions were drawn. When histone H1 is absent the non-histone proteins partially prevent the unfolding of the nucleosomes at low ionic strength. The complete blocking of this unfolding, however, is accomplished only in the presence of histone H1. The non-histone proteins do not affect the orientation of the nucleosomes along the fibre axis. Only histone H1 can maintain the positive anisotropy of the chromatin fibre.     

Role of non-histone proteins in chromatin solubility

Treatment of chromatin gel with low ionic strength solution of tRNA has produced the dioxyribonucleoprotein (dnptRNA) in which only part of non-histone proteins was removed without loss of any major histone fraction. The solubility of DNP in the presence of 0.15 M NaCl and 1 to 5 mM MgCl2 was considerably higher than that of initial untreated chromatin. It has been assumed that the solubility of chromatin depended primarily on some non-histone proteins and not on H1 histone.     

Evidence for two transport systems for nitrate in the acidophilic thermophilic alga Cyanidium caldarium

In the unicellular non-vacuolate red alga Cyanidium caldarium nitrate uptake occurs through two specific permease systems which, on the basis of kinetic constants can be defined as low affinity system and high affinity system. The high affinity system is saturated at very low nitrate concentrations (<1 M), whereas the low affinity system is saturated only at high nitrate concentrations (K _m=0.45±0.10 mM). The low affinity system is present in cells growing under conditions of nitrogen limitation as well as in cells growing in excess nitrate. In contrast, the high affinity system is present only in cells growing under conditions of nitrogen limitation. The high affinity system works only at acid pH and is inactive at neutral pH. The low affinity system is active both at acid and at neutral pH.     

Biosorption of heavy metals by Fucus spiralis

The sorption uptake of cadmium, nickel, zinc, copper and lead by marine brown alga Fucus spiralis was investigated in bimetallic, trimetallic and multimetallic solutions. The experimental data fitted very well to Langmuir model. In bimetallic systems, the affinity of biomass for lead and copper increased and the sorption uptake of these metals was not affected by increasing concentrations of cadmium, nickel or zinc. However, in solutions with both metals there was a significant mutual decrease of their sorption levels at high concentrations of the other metal. The sorption uptake of cadmium, nickel and copper was investigated in trimetallic aqueous systems. Based on the kinetic parameter b, the affinity of F. spiralis for copper was considerably higher than for cadmium or nickel: bCd=6.39, bNi=1.82 and bCu=17.89. In all tests, the maximum sorption uptake remained practically constant around 1 mmol/g, indicating that the number of active sites on the biomass was limited. Tests with four and five metals showed that copper was preferentially adsorbed. The differences between the experimental sorption data and those given by the chemical speciation program PHREEQCI were negligible. In general, the software used provided satisfactory estimated data for each metal and hence can be a useful tool to predict or simulate the real process.     

Fractionation of chromatin fragments on columns of Biogel A50-m at different salt concentrations

Nuclease fragmented chromatin was chromatographed on Biogel at various NaCl concentrations. The yield of eluted chromatin, and its H1/core histone ratio was minimal at 0.18 M NaCl where the ratio of H1 subtypes H1c/H1ab was maximal. Therefore, the eluted material was aggregation-resistant chromatin while aggregatable chromatin remained on the columns. Previous results were interpreted as H1 depletion of chromatin by ion-exchange properties of Biogel, but the primary phenomenon is now seen as a separation of classes of chromatin that differ in sensitivity to salt-induced aggregation. At very low salt concentrations, Biogel chromatography can be used without concern for H1 depletion.     

In vitro interaction of 63-nickel(II) with chromatin and DNA from rat kidney and liver nuclei

The interaction of nickel(II) with chromatin was studied in vitro and in isolated nuclei from rat liver and kidney. Nickel(II) bound to chromatin, polynucleosomes (DNA + histone octamer protein complex), and to deproteinized DNA both in intact nuclei and in vitro. The amount of nickel(II) bound depended on the concentration of nickel(II), the presence of chromosomal proteins and the binding sites on DNA which provide a stable coordination environment for nickel(II). The binding of nickel(II) to chromatin and to DNA in whole nuclei was much slower than in vitro indicating that assessibility of the DNA binding sites was influenced by the presence of the nuclear membrane, nuclear matrix and nuclear proteins and/or by the condensed nuclear structure of chromatin. Since DNA containing bound nickel(II) was isolated from chromatin, nickel(II) directly interacted with stable binding sites on the DNA molecule in chromatin. Nickel(II) was associated with the histone and non-histone nuclear proteins as well as the DNA in rat liver and kidney chromatin. Nickel(II) was found to bind to calf thymus histones in vitro. Nickel(II)-nuclear protein and -DNA interactions were investigated by gel electrophoretic analysis of in vitro incubation products. Although nickel-histone and nickel-non-histone protein interactions were completely disrupted by the electrophoretic conditions, fluorography revealed the presence of inert nickel(II)-DNA and/or nickel(II)-DNA-protein complexes.     

Engineered nickel bioaccumulation in Escherichia coli by NikABCDE transporter and metallothionein overexpression

Mine wastewater often contains dissolved metals at concentrations too low to be economically extracted by existing technologies, yet too high for environmental discharge. The most common treatment is chemical precipitation of the dissolved metals using limestone and subsequent disposal of the sludge in tailing impoundments. While it is a cost-effective solution to meet regulatory standards, it represents a lost opportunity. In this study, we engineered Escherichia coli to overexpress its native NikABCDE transporter and a heterologous metallothionein to capture nickel at concentrations in local effluent streams. We found the engineered strain had a 7-fold improvement in the bioaccumulation performance for nickel compared to controls, but also observed a drastic decrease in cell viability due to metabolic burden or inducer (IPTG) toxicity. Growth kinetic analysis revealed the IPTG concentrations used based on past studies lead to growth inhibition, thus delineating future avenues for optimization of the engineered strain and its growth conditions to perform in more complex environments.     

Chromatin affinity purification and quantitative mass spectrometry defining the interactome of histone modification patterns

DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated.     

Influence of lead,cadmium, and nickel on the growth ofMedicago sativa (L.)

Summary Alfalfa (Medicago sativa L.), cv. Iroquois, was grown in the greenhouse in soils amended with additions of either lead, cadmium, or nickel. Metals, at rates varying from 0–250 ppm, were not uniformly mixed but were placed close to the soil surface so as to simulate surface deposition. In one series of experiments the sulphate salt of each metal and two soils were used. In a second series of experiments the nitrate salts and one soil were used. Neither salt of lead significantly depressed alfalfa yields. Both salts of either cadmium or nickel significantly depressed yields. Additions of all metals to the soil resulted in both increased metal uptake and concentrations in alfalfa tissue, particularly for cadmium and nickel. The highest tissue concentrations of cadmium and nickel were associated with plant stunting and necrosis. However, at rates of 125 ppm and less, substantial increases in cadmium and nickel concentrations were obtained frequently without serious yield reductions. Generally, metal concentrations were greatest in the first harvest following metal application. Concentration and uptake of lead and cadmium were greater when the metal was applied to the soil as nitrate than when applied as the sulphate salt.     

Chromatin aggregation depends on the anion species of the salts

The effects of anions on chromatin aggregation may be classified into three categories. First, monovalent anions, glutamate, acetate, chloride, and thiocyante, follow the lyotropic series in their effects on both H1 histone displacement and chromatin aggregation. Second, alkyl carboxylates and dicarboxylates differ in their ability to induce chromatin aggregation depending on charge density, suggesting possible interference by bulky alkyl chains with neutralization (screening) of closely spaced positive protein charges. Third, the multivalent anions, citrate3- and SO4(2-), bind tightly to histone and disrupt nucleosomes and thus interfere with chromatin aggregation. Substantial differences in chromatin aggregation were observed with different species of anions. At salt concentrations of 0-500 mN and pH 7.0, as much as 70% of the chromatin could be induced to aggregate by monosodium glutamate and sodium acetate, whereas only 10% or less was precipitated by NaSCN, Na2SO4, and Na3citrate. The physiological anion composition of the nucleus is not known; however, the anion effects discussed in the present work suggest a potential for regulation of chromatin condensation in higher eukaryotes.     

Effect of salt on the binding of the linker histone H1 to DNA and nucleosomes

The linker histones are involved in the salt-dependent folding of the nucleosomes into higher-order chromatin structures. To better understand the mechanism of action of these histones in chromatin, we studied the interactions of the linker histone H1 with DNA at various histone/DNA ratios and at different ionic strengths. In direct competition experiments, we have confirmed the binding of H1 to superhelical DNA in preference to linear or nicked circular DNA forms. We show that the electrophoretic mobility of the H1/supercoiled DNA complex decreases with increasing H1 concentrations and increases with ionic strengths. These results indicate that the interaction of the linker histone H1 with supercoiled DNA results in a soluble binding of H1 with DNA at low H1 or salt concentrations and aggregation at higher H1 concentrations. Moreover, we show that H1 dissociates from the DNA or nucleosomes at high salt concentrations. By the immobilized template pull-down assay, we confirm these data using the physiologically relevant nucleosome array template.     

The cytotoxic effect of heavy metals on an L-cell culture

The dependence of some parameters of L-cells culture viability on different concentrations of heavy metals was studied. Considerable cytotoxic effect of low concentrations of nickel (0.025 mcg/ml) and lead (0.05 mcg/ml) was shown. Copper and chrome at concentrations of 0.25-0.5 mcg/ml promote cells proliferation between third and fifth days of cultivation. Nickel at concentration 0.025 mcg/ml and lead at all investigated concentrations synchronize cells division in culture. Increasing of giant polynucleas cells level in culture was characteristic for investigated metals. The maximum levels of this type cells were caused by the action of nickel, chrome and copper.     

Histone H1 and chromatin interactions in human fibroblast nuclei after H1 depletion and reconstitution with H1 subfractions.

BACKGROUND: Linker histones constitute a family of lysine-rich proteins associated with nucleosome core particles and linker DNA in eukaryotic chromatin. In permeabilized cells, they can be extracted from nuclei by using salt concentration in the range of 0.3 to 0.7 M. Although other nuclear proteins are also extracted at 0.7 M salt, the remaining nucleus represents a template that is relatively intact. METHODS: A cytochemical method was used to study the affinity of reconstituted linker histones for chromatin in situ in cultured human fibroblasts. We also investigated their ability to condense chromatin by using DNA-specific osmium ammine staining for electron microscopy. RESULTS: Permeabilized and H1-depleted fibroblast nuclei were suitable for the study of linker histone-chromatin interactions after reconstitution with purified linker histone subfractions. Our results showed that exogenous linker histones bind to chromatin with lower affinity than the native ones. We detected no significant differences between the main H1 and H1 degrees histone fractions with respect to their affinity for chromatin or in their ability to condense chromatin. CONCLUSIONS: Linker histone interactions with chromatin are controlled also by mechanisms independent of linker histone subtype composition.     

Analyses of linker histone--chromatin interactions in situ.

Recent studies, using cytometric techniques based on fluorescence microscopy, have provided new information on how linker histones interact with chromatin in vivo or in situ. In particular, the use of green fluorescent proteins (GFPs) has enabled detailed studies of how individual H1 subtypes, and specific motifs in them, interact with chromatin in vivo. Furthermore, the development of cytochemical methods to study the interaction between linker histones and chromatin using DNA-binding fluorochromes as indirect probes for linker histone affinity in situ, in combination with highly sensitive and specific analytical methods, has provided additional information on the interactions between linker histones and chromatin in several cell systems. Such results verified that linker histones have a substantially higher affinity for chromatin in mature chicken erythrocytes than in frog erythrocytes, and they also indicated that the affinity decreased during differentiation of the frog erythrocytes. Furthermore, in cultured human fibroblasts, the linker histones showed a relatively high affinity for chromatin in interphase, whereas it showed a significantly lower affinity in highly condensed metaphase chromosomes. This method also enables the analysis of linker histone affinity for chromatin in H1-depleted fibroblasts reconstituted with purified linker histones. No consistent correlation between linker histone affinity and chromatin condensation has so far been detected.     

Binding of linker histones to the core nucleosome

Binding of chicken erythrocyte linker histones H1/H5 to the core nucleosome has been studied. Histones H1/H5 bind very efficiently to the isolated core nucleosome in vitro. The binding of linker histones to the core nucleosome is associated with aggregation of the particles. Approximately one molecule of linker histone binds per core nucleosome in the aggregates, irrespective of the concentration of the linker histones and the salt used. Histone H5 shows greater binding affinity to the core nucleosome as compared to H1. The carboxyl-terminal fragment of the linker histones binds strongly to the core nucleosome while the binding of the central globular domain is weak. Each core nucleosome is capable of binding two molecules of carboxyl-terminal fragment of linker histone. The core nucleosome containing one molecule of carboxyl-terminal fragment of linker histone requires higher salt concentration for aggregation while the core nucleosome containing two molecules of carboxyl-terminal fragment of linker histone can self-associate even at lower salt concentrations. On the basis of these results we are proposing a novel mechanism for the condensation of chromatin by linker histones and other related phenomena.