Effects of anthracycline antibiotic, daunomycin on thymus chromatin: The role of chromosomal proteins

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Gen. Pharmac. Vol. 25, No. 4, pp. 787-793, 1994 Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0306-3623/94$7.00+ 0.00

0306-3623(93)E0027-6

Effects of Anthracycline Antibiotic, Daunomycin on Thymus Chromatin: The Role of Chromosomal Proteins A Z R A RABBANI* and J A M S H I D D A V O O D I Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384 Tehran, Iran [Fax 9821 6404680] (Received 10 September 1993)

Abstract--l. We have examined the effect of antitumour antibiotic daunomycin on calf thymus chromatin employing u.v./vis spectroscopic and hydrodynamic techniques. The experiments were undertaken to determine the influence of added drug on DNA-protein complex. 2. The results show that the binding of drug to chromatin is dose dependent and a DNA to drug ratio below 1: 100 leaves small oligonucleotidesin the supernatant, however, at higher ratios chromatin occurs aggregation. 3. Analysis of both the proteins and DNA reveals that daunomycin induces chromatin condensation by cross linking between its components suggesting that chromosomal proteins play a significant role in this process.

KeyWords:Daunomycin, thymus chromatin, DNA

INTRODUCTION Daunomycin is an anthracycline antibiotic widely used as a chemotherapeutic agent for the treatment of various cancers (Wiernik, 1980; Gianni et al., 1983). Numerous studies on the interaction of daunomycin with DNA have been undertaken and all indicate that the drug exerts its biological function via intercalation into DNA double strands and subsequently inhibits DNA replication and RNA transcription (Neidle and Sanderson, 1983; Goodman et al., 1977; Scheilinx et aL, 1979). The structure of daunomycin--oligonucleotide or DNA complexes are known at the atomic resolution level (Quigley et al., 1980; Wang et al., 1987; Frederick et al., 1990) and its interaction with nucleosomes (Chaires et al., 1983) and left handed Z-DNA (Chaires, 1983) have been explored. It has also been shown that by using microccocal nuclease, DNA binding drugs unfold and disrupt chromatin structure (Grimmond and Beerman, 1982). All these investigations suggest that daunomycin function primarily at the DNA level. In the cell nucleus DNA is complexed with a series *To whom correspondence should be addressed.

of proteins called histones and nonhistone chromosomal proteins (Johns, 1982). Histones interact with DNA and make nucleosomes (Bradbury et al., 1981; Kornberg, 1974). To explore whether the presence of these proteins affect the binding of drug to DNA, we have examined the interaction of daunomycin with the DNA--histone complex in solution and shown that these proteins also contribute in DNA-drug interaction processes (Rabbani et al., 1994). In this study we attempted to examine the effect of daunomycin on isolated chromatin to provide further insights into the action of this drug on the possible release, displacement or other alterations in chromatin composition, MATERIALS AND METHODS Chemicals and experimental conditions

Calf thymus was obtained from Ziaran slaughter house, transferred to the lab in liquid nitrogen and stored at until use. The experiments were performed at two temperatures, 23 and 37°C. All reagents were of analytical grade. Daunomycin was purchased from Sigma Chemical Co. and used without further purification. It was dissolved in

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AZRA RABBANI and JAMSHID DAVOODI

distilled water at a stock concentration of 1 mg/ml, divided into equal portions and stored at - 2 0 ° C prior to use. Dilutions were made on the day using 25 mM triethanolamine hydrochloride (TEA) pH 7.4 containing I mM EDTA or 50 mM Tris-HCl buffer pH 7.4. TKMC buffer consisted of 15 mM NaCI, 60mM KC1, 5 mM MgC12, 15 mM Tris-HCl and I mM CaCI2 pH 7.4. EcoR1 digested DNA (Sigma) with 125-5148 base pairs was used as a DNA molecular weight marker. Nuclei preparation Nuclei from frozen calf thymus (10g) were prepared essentially according to the procedure of Burgoyne et al. (1970). All steps were carried out at 4°C and phenylmethylsulfonyl fluoride (PMSF) at a final concentration of 0.5 mM was added to TKMC buffer immediately before use as a proteolytic inhibitor. Pure and intact nuclei were washed twice with TKMC buffer and then suspended in 10ml of the same buffer, and 0.1 ml was mixed with 3 ml of N NaOH--the DNA content was determined by measuring the absorbance at 260 nm. Binding measurements The nuclei suspension was diluted with the reaction buffer (25 mM TEA pH 7.4) and after brief homogenization, was divided into several portions each containing 1 mg/ml of DNA. To the samples, daunomycin with a serial concentrations of 2, 4, 8, 15, 25, 35, 50 and 100/tg/ml was added and the volumes adjusted to 1.5 ml with TEA buffer. An equal volume of buffer was added to the control and the samples were incubated in the dark at 23°C or 37°C for 30 min with occasional mixing. After incubation, experimental and the controls were centrifuged for 5 rain at 10000 r.p.m. (Ependorf) at 4"C and the clear supernatants and the pellets were both kept for analysis. Spectroscopy The supernatants obtained from the treated and controls were subjected to spectroscopic analysis using double beam Schimadzu UV 260 spectrophotometer. Absorbances at 480, 260, 230 and 280 nm were monitored and the absorption and difference spectra were recorded between 190 and 550 nm using buffer, controls, or the same concentration of drug at each point as a reference. DNA and protein extraction The proteins released in the supernatants were precipitated with 10-12% trichloroacetic acid (TCA) with respect to 100% TCA and the pellets dissolved in SDS gel sample solvent for electrophoresis (see below). Also the pellets after drug binding were

incubated with 0.25N HC1 (Johns, 1971), 0.35M NaCI (Goodwin and Johns 1973) and 5% perchloric acid procedure of Sanders (1977) to isolate histones and high mobility group nohistone proteins respectively. DNA was isolated from the supernatants by the method of Britten et al. (1974) with some modifications. Briefly, to 400 p l of the samples 0.1 vol of 5 M sodium perchlorate and 0.5 vol of isoamylalcohol:chloroform (1: v/v) were added and vortexed for 10 min at room temperature. The samples were then centrifuged for 15 min at 2000g at 4°C. The aqueous phases were separated and the extraction was repeated twice as above. To the final combined aqueous phases, 2 vol of ethanol were added and DNA pellets were collected by centrifugation, washed with ethanol followed by acetone and dried. Gel electrophoresis The proteins were analysed on 15% SDS polyacrylamide gel as described by Laemmli (1970). Electrophoresis was carried out for 2hr at 180V. Histones from calf thymus were used in parallel as protein markers. DNA samples were analysed on 4% Tris-borate-EDTA (TBE)-acrylamide slab gel system (Maniatis, 1975). The gels were run at a constant voltage of 150V for 1.5hr at ambient temperature. The gels were stained with either ethidium bromide (0.5 ~g/ml) in distilled water or by "stain all" and photographed. Stained gels were scanned on a quick scan densitometer Beckman model R-112. EcoRl digested DNA was used as a DNA molecular weight marker. RESULTS The effect of daunomycin on isolated chromatin was investigated at drug to DNA ratios between 0.1-0.001 w/w and after set periods of incubation the supernatants were analysed for protein and DNA changes and undound drug contents. Figure 1 illustrates the 480 nm absorbance measurements of the treated samples compared to the controls. Serial concentrations of daunomycin were made in buffer and used as a control. It is shown that raising drug concentration from 2#g/ml to 100/~g/ml in buffer and in the absence of chromatin, produce a straight upward line but when drug is added to the chromatin samples, a considerable decrease in the characteristic drug absorbance at 480 nm is observed. The results indicate that, in the drug treated samples, most of the daunomycin has bound to the chromatin and coprecipitated with it during centrifugation. Also the

Effects of daunomycin on chromatin

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Fig. 1. The effect of daunomycin on thymus chromatin. Absorbances at 480 nm of the supernatants obtained from the incubation of chromatin with various concentrations of drug at 25"C (O) and at 37°C (©). Absence of chromatin O; different concentrations of drug in buffer pH 7.5. The results are means of at least five separate experiments.

experiments performed at two different temperatures, 25 and 37°C, show similar binding properties. The amount of proteins and D N A released by drug action, was also determined by measuring the optical density of the samples at 260, 230 and 280 nm and subtracting the corresponding drug absorbance using standard drug solutions of the same concentrations at each point. Interestingly, it is found that addition of daunomycin to chromatin alters the absorbances in a rather unusual manner as is seen in Fig. 2. Under conditions where the levels of chromatin were held constant and increasing amounts of drug were added to the binding reactions it seems that drug binding to the chromatin is apparently dose dependent, since at a very low concentration (less than 4/~g/ml) the

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Fig. 2. Changes in the absorbances of chromatin solutions in the presence of various concentrations of daunomycin. O, absorbances at 260 nm,/~ at 230 nm and O; at 280 nm. Results are means of 5 experiments (P < 0.01).

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Fig. 3. Absorption spectra (A) and difference spectra (B) of the samples treated with zero (b), 4#g/ml, 20,ug/ml and 50 #g/ml daunomycin. Spectra were made against the interaction buffer pH 7.5. For difference spectra the control (chromatin without drug) was used as a reference.

absorbance is increased to a value exceeding those of control. However, gradual increase in drug concentration reduces the absorbances to a minimum of 0.3 when 100 Itg/ml daunomycin is used. Absorption spectra of the chromatin solutions treated with various concentrations of daunomycin [Fig. 3(A)] are in agreement with the above observations. As is seen, absorbance changes occur at both 260 and 210-230nm, however, differences at 260 nm are considerably higher. Drawing difference spectra against the control (chromatin with no drug) produce a negative peak for 40 #g/ml daunomycin and higher drug values (more than 25/~g/ml but at 4/lg/ml daunomycin, a positive spectra is observed [Fig. 3(B)] revealing different behaviour of drug at two concentration ranges. In an attempt to more critically analyse the possible displacement and nature of the chromosomal proteins released into the supernatants after drug binding, the proteins were precipitated with T C A and then an equal amount of each was loaded onto the SDS gel. The gel pattern [Fig. 4(A)] shows that exposure of chromatin to buffer alone results in the

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release of some proteins which are mostly core histories as analysed parallel to thymus core histones. This spontaneous release of loosely bond proteins has also been observed by previous workers (Comings and Okada, 1976; Bartkowiak et al., 1989) and represents the background of the experiments. At very low levels of drug the proteins remaining in the supernatants are also core histones but an increase in drug concentration causes chromatin aggregation and thereby disappearance of the histones from the gel pattern. Microdensitometric tracing of the bands also clearly demonstrates the differences. The nature of the DNA released in to the supernatant after drug treatments was analysed. In this case DNA was isolated by a phenol extraction procedure explained in the method section and then were run on TBE gels parallel to a DNA maker with 125-5148 base pairs. The result is given in Fig. 5. It is shown that in the control and at low concentrations of daunomycin, DNA represents a ladder pattern and a smear of various DNA sizes but when higher concentrations of drug (more than 25 #g/ml) are used DNA patterns are changed and only DNA sizes at 5148 bp position are visible implying DNA aggregation by drug binding. In an attempt to access whether the antibiotic used in this work produced cross linking, the histones, nonhistone chromosomal proteins and DNA were extracted from the chromatin pellets after drug treatment. It was found, that only a trace of histones H3 and H4 appears on the gels hence nearly 90% of histones and all of 0.35 M NaC1 or 5% perchloric acid soluble proteins (HMG proteins) are completely unextractable. It is therefore suggested that the reaction of daunomycin with thymus chromatin produces cross links which are not accessible to the extraction procedures employed in this study.

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Fig. 4. (A) 15% SDS polyacrylamide gel electrophoresis pattern of daunomycin treated and control samples. Lane 1 is whole histone prepared from thymus as a comparison. Lanes 2 9 are 0, 2, 4, 10, 20, 30, 50 and 100/~g/ml daunomycin respectively. (B) Densitometric scan of the same gel at 560 nm.

Recognition of the mechanism of anthracycline DNA-binding drugs interaction as a antitumour agent is a problem which still demands further investigation. Although the mechanism of the interaction is often inferred from studies of the affinity of these drugs to free DNA as a main target (Chaires et al., 1982, 1987; Gabbay et al., 1976), the environment of nucleic acids in the cell, especially DNA-protein complexes, may significantly modulate these interactions. The present study represents an attempt to characterize the binding of anthracycline antibiotic, daunomycin, to isolated chromatin to elucidate the possible effect and function of chromosomal proteins in drug binding process. Our results suggest that daunomycin affects the chromatin

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structure in a dose dependent manner. At very low concentrations, it seems to unfold the structure and somehow produces an increased level of soluble oligonucleotides and core histories in the supernatant compared to the controls. However, at higher concentrations it induces aggregation and condensation of chromatin possibly via forming cross links between the chromatin components. This is evidently shown by the disappearance of small DNA sizes and core GP 25/4--M

histones from the gel patterns when drug concentrations higher than 25/~g/ml are applied. Furthermore absorption and difference spectra of chromatin samples treated with daunomycin also clearly demonstrate the considerable changes in the nucleoprotein structure of chromatin by drug action. Concerning the effect of anthracyclines on chromatin, conflicting results have been reported according to the experimental design employed. Grimmond and

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AZRA RABBANI and JAMSHIDDAVOODI

Beerman (1982) have shown that anthracyclines and ethidium bromide produce some unwinding of chromatin which leads to the increased accessibility of core particle DNA to nucleases. Chaires et al. (1983) have also studied the binding properties of daunomycin to HI depleted nucleosomes and shown both unfolding and aggregation of nucleosomes. In our laboratory extensive work has been carried out on the interaction of anthracycline antibiotics (adriamycin and daunomycin) with the chromosomal proteins, histones and high mobility group (HMG) nonhistone proteins, and their complex with DNA in solution. According to our observations the interaction of adriamycin with thymus core histones reduce their binding affinity to DNA (Rabbani et al., 1993). Also both drugs bind firmly to H M G proteins and affect their thermal denaturation behaviour (manuscript in preparation). Therefore the above findings together with the results presented in this report confirm the participation of chromosomal proteins in drug DNA binding process. To date several reports have shown the alteration and condensation of chromatin by different antitumour agents (Waldes and Center 1982; Cera and Palumbo 1990; Grimmond and Beerman, 1982). Bartkowiak et al. (1989) have also studied the selective release of nuclear proteins by various antitumour drugs (but not daunomycin). In our experiments resistance of histones and H M G proteins of the chromatin pellets after drug treatment to the relevant extraction procedures employed clearly reveal that apart from D N A - D N A cross links, the cross linking of proteins to DNA or to itself is also produced by drug binding. Therefore it is suggested that the structural changes of chromatin under daunomycin influence is related to a dose of drug used, thus at a very low concentration, it induces a slight unfolding of chromatin structure and possibly release of some proteins and DNA but at higher values produces various kinds of cross links. Thus in this process, bridging of proteins and DNA through the drug may play a basic and important role in the antitumour action of the drug. It should be noted that in our experimental conditions and methods used any protein-protein cross links could not be detected, however this effect cannot be ignored completely.

CONCLUSION In conclusion, studies characterizing the binding sites of antitumor agents in the cell are important because they can provide clues needed for future drug efficacy and design. Although it is generally accepted that the biological activity of drugs is due to the

strong DNA binding properties which leads to the inhibition of cellular replication and transcription process, it is believed that in eukaryotes, DNA is complexed essentially with histones which protect it in regular structures called nucleosomes. Therefore, chromosomal proteins could also be suitable sites for drug binding and alteration of chromatin structure. In this case participation of both DNA and its attached proteins in drug action, possibly by cross linking, proceeds its antitumour effect. Acknowledgements--Authors would like to thank Mrs Sh.

Rezaei for her excellent technical assistant. This work was supported by the grant number 721 of the Research Council of the University of Tehran. REFERENCES Bartkowiak J., Kapuscinski J., Melamed M. R. and DarzynkiewiczZ. (1989) Selectivedisplacementof nuclear proteins by antitumor drugs having affinity for nucleic acids. 86, 5151-5154. Britten R. S., Graham D. E. and Neufeld B. R. (1974) Meth. Enzym. 29E, 263-418. Bradbury E. M., Maclean N. and Matthews H. R. (1981) DNA Chromatin and Chromosomes. Blackwell, Oxford. Burgoyne L. A., Wagar M. A. and Atkinson M. R. (1970) Calcium-dependentpriming of DNA synthesisin isolated rat liver nuclei. Biochem. biophys Res. Commun. 39, 254-259. Cera C. and Palumbo M. (1990) Anti-cancer activity of anthracycline antibiotics and DNA condensation. Anticancer Drug Des. 5, 265-271. Chaires J. B., Dattagupta N. and Crothers D. M. (1982) Studies on interaction of anthracycline antibiotics and DNA: equilibrium binding studies on interaction of daunomycin with DNA. Biochemistry 21, 3933-3940. Chaires J. B. (1983) Daunomycin inhibits the B-Z transition in poly (G.C). Nucleic Acids Res. 11, 8485-8494. Chaires J. B., Dattagupta N. and Crothers D. M. (1983) Binding of daunomycin to calf thymus nucleosomes. Biochemistry 22, 284-292. Chaires J. B., Fox K. R., Herrera J. E., Britt M. and Waring M. J. (1987) Site and sequence specificity of the daunomycin-DNA interactions. Biochemistry 26, 8227 8236. Comings D. E. and Okada T. A. (1976) Nuclear proteins III. The fibrillar nature of the nuclear matrix. Exp. Cell Res. 103, 341-360. Frederick C. A., Williams L. D., Ughetto G., Van der Marel G. A., Van Boom J. H., Rich R. and Wang A. H. J. (1990) Structural comparison of anticancer drug-DNA complexes: adriamycin and daunomycin. Biochemistry 29, 2538-2549. Gabbay E. J., Grier D., Fingerle R. E., Reimer R., Levy R., Pearce S. W. and Wilson W. D. (1976) Interaction specificity of the anthracyclines with DNA. Biochemistry 15, 2062-2069. Gianni L., Cordon B. J. and Myers C. E. (1983) The biochemicalbasis of anthracycline toxicity and antitumor action. In Reviews in Biochemical Toxicology (Edited by Hodgson E., Bend J. R.,Philpot R.M.) Vol. 5, pp. 1-82. Elsevier, Amsterdam. Goodman F. M., Lee G. M. and Bachur N. R. (1977) Adriamycin interactions with T4 DNA polymerase. J. biol. Chem. 252, 2670-2674. Goodwin G. H. and Johns E. W. (1973) Isolation and characterization of two calf thymus chromatin nonhistone proteins with high contents of acidic and basic amino acids. Eur. J. Biochem. 40, 215-219. Grimmond H. E. and Beerman T. (1982) Alteration of

Effects of daunomycin on chromatin chromatin structure induced by the binding of adriamycin-daunomycin and ethidium bromide. Biochem. Pharmac. 31, 3379-3386. Johns E. W. (1982) The HMG Chromosomal Proteins. Academic Press, London. Johns E. W. (1971) The preparation and characterization of histones. In Histones and Nucleohistones (Edited by Phillips D. M. P.), p. 1. Plenum Press, London. Kornberg R. D. (1974) Chromatin structure. Science 184, 868-87 I. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Maniatis T., Jeffrey A. and Van de Sande (1975) Chain length determination of small double and single stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14, 3787-3794. Neidle S. and Sanderson M. R. (1983) The interaction of daunomycin and adriamycin with nucleic acids. In Molecular Aspects of Anticancer Drug Action (Edited by Neidle S. and Waring M. J.), pp. 35-37. Verlag Chemie, Weinlein. Quigley G. J., Wang A. H. J., Ughetto G., Van der Marel G., Van Boom J. H. and Rich A. (1980) Molecular

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structure of an anticancer drug-DNA complex daunomycin plus d (CpGpTpapCpG). Proc. natn. Acad. Sci. U.S.A. 77, 7204-7208. Rabbani A., Taghavi M. H. and Goliaei B. (1994) Binding of the antitumor drug, adriamycin, to DNA-histone complexes. Med. J. IRI. In press. Schellinx J. A. A., Dijkwel P. A. and Wanka F. (1979) Inhibition of DNA synthesis in mammalian cells by daunomycin. Eur. J. Biochem. 102, 409-418. Sanders C. (1977) A method for the fractionation of the HMG nonhistone chromosomal proteins. Biochem. biophys. Res. Commun. 78, 1034-1042. Waldes H. and Center M. S. (1982) Adriamycin induced compaction of isolated chromatin. Biochem. Pharmac. 31, 1057 1061. Wang A. H. J., Ughetto G., Quigley G. J. and Rich A. (1987) Interactions between an anthracycline antibiotic and DNA molecular structure of daunomycin complexed to d (CpGpTpapCpG) at 1.2 resolution. Biochemistry 26, 1152 1163. Wiernik P. H. (1980) Current status of adriamycin and daunomycin in cancer treatment. In Anthracyclines: Current Status and New Development (Edited by Crooke S. T. and Reich E.), p. 273. Academic Press, New York.