30
Biochimica et Biophysica Acta, 1131 (1992) 3/)-34 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05./)0
BBAEXP 92373
8-Methoxycaffeine inhibition of Drosophila DNA topoisomerase II Silvia T o r n a l e t t i a, A n n i H. Andersen b, K e n t C h r i s t i a n s e n b a n d A n t o n i a M. P e d r i n i ~' '~ lstituto di Genetica Biochimica ed El,oluzionistica del C.N.R., Pacia (Italy) and h Institute for Molecular Biology and Plant Physiology, Unicersity of Aarhus, Arhus (Denmark)
(Received 5 November 1991) (Revised manuscript received 10 February 1992)
Key words: 8-Methoxycaffeine; DNA topoisomerase 11
We have investigated the effect of 8-methoxycaffeine on the interaction between Drosophila DNA topoisomerase Il and DNA. We have shown that 8-methoxycaffeine affected the enzyme strand-passing activity by inhibiting decatenation of kinetoplast DNA, and that it interfered with the breakage-reunion reaction by stabilizing a cleavable complex. Treatment of the cleavable complex with protein denaturant resulted in DNA breaks. High resolution mapping of the cleavage sites in the central spacer region of Tetrahymena rDNA revealed that, contrary to what was observed with clinically important DNA topoisomerase II inhibitors, 8-methoxycaffeine did not modify the cleavage pattern observed without the drug. Methylated oxypurines (MOPs) are a class of drugs that induce various alterations on the genetic material in plant, Drosophila and cultured mammalian cells (for reviews see Refs. 1-4). Among these alterations, the capability of caffeine and its derivatives in producing S-independent chromosomal aberrations is well documented. Late G2-phase and prophase are the most sensitive stages, the aberrations produced at this stage being mainly chromatid exchanges. The formation of chromosomal aberrations is strongly conditioned by active oxidative phosphorylation, suggesting that exchange processes are closely related to the ATP level of the cells. To account for the ATP and cell cycle dependence of MOPs induced chromosomal aberrations, Kihlman and Andersson [4] have proposed that at least part of the multiple effects caused by caffeine and derivatives on the genetic material could be due to an interference with DNA topoisomerase II. Eukaryotic DNA topoisomerases type II are ubiquitous enzymes defined by their ability to catalyze ATP dependent DNA topoisomerisation reactions [5]. Central to all DNA topoisomerase II reactions is the enzyme ability to induce transient enzyme-bridged
Abbreviations: DTT, dithiothreitol; mAMSA, amsacrine; 8-MOC, 8-methoxycaffeine; rDNA, ribosomal DNA; Ro 15-0216, 2-dimethylamino-4'[(l-met hyl-2-nitroimidazol-5-yl)methoxy]acetanilide; SDS, sodium dodecyl sulfate; SSC, standard saline citrate. Correspondence: A.M. Pedrini, Istituto di Genetica Biochimica ed Evoluzionistica del C.N.R.. Via Abbiategrasso 207, 27100-Pavia, Italy.
strand breaks, where the enzyme subunits are covalently bound to the 5' ends of the cleaved strands, forming a covalent DNA-enzyme intermediate, termed cleavable complex. Treatment with strong denaturants, such as sodium dodecyl sulfate (SDS), can irreversibly trap the cleavable complex and reveal strand breaks. Inaccurate rejoining by subunits exchange of the cleaved strands may lead to exchanges between duplex DNA. For these properties, several authors have suggested the possible involvement of DNA topoisomerase II in chromosome rearrangements [6-9]. Several observations are consistent with this proposal. Eukaryotic enzymes can catalyze non homologous recombination in vitro [10], and hotspots for enzyme cutting have been found at translocational breakpoints [9]. Furthermore, among the cellular effects of the antineoplastic DNA topoisomerase II inhibitors [11], that stabilize the cleavable complex [12], there is an increased frequency of sister chromatid exchanges and chromatid aberrations. The most sensitive stage of the cell cycle is late G2 phase [13], a phase requiring active DNA topoisomerase II to carry on normal chromosome segregation [14]. The recent finding that 8-methoxycaffeine (8-MOC), the most active caffeine derivative in inducing chromosomal aberrations, can induce protein-linked single and double strand DNA breaks in mouse leukemia L1210 cells and purified nuclei strengthens Kihlman's suggestion that DNA topoisomerase II may be the putative target of methylated oxypurines [15]. In fact, the induced breaks have the same properties (saturability,
31 reversibility and t e m p e r a t u r e dependence) as those produced by the antineoplastic D N A topoisomerase II inhibitor, ellipticine. Prompted by the similarity between the putative target protein of 8-MOC and D N A topoisomerase II, we have carried on a study to test whether topoisomerase II is a target for 8-MOC. We have initiated by investigating the influence of the drug on the formation of cleavable complex between highly purified topoisomerase II from Drosophila melanogaster [16] and D N A isolated from the central spacer region of the r D N A molecule of Tetrahymena thermophila [17,18], containing two symmetric strong recognition sites for Drosophila topoisomerase II [19]. To quantitate the cleavage reaction a filter retention assay, involving the precipitation of covalent e n z y m e - D N A complex, was done. The HinfI r D N A fragment (see map of Fig. 2 and Ref. 17) was 3' end-labelled [20] and than incubated with the enzyme in the presence of increasing concentrations of 8-MOC. The reaction was stopped by SDS and, following alkali denaturation of the cleavage products, the complexes were precipitated on filters. The results, presented in Fig. 1, clearly show that 8-MOC stimulated cleavable complex formation between Drosophila topoisomerase II and rDNA. Furthermore, radioactivity was retained on the filter when a 3' end-labelled fragment was used, strongly indicating that topoisomerase II was covalently linked to the 5' end of the cleaved DNA. If 0.8 M NaC1 was added to the reaction mixture containing 10 m M 8-MOC prior to SDS treatment, the amount of radioactivity retained on the filter dropped to background level, demonstrating that the effect of 8-MOC on the topoisomerase I I - D N A complexes was reversible (data not shown). The results suggest that 8-MOC interacts with the cleavable complexes in a way similar to well known intercalating antitumor drugs such as ellipticine and m A M S A [21]. Although the filter retention assay is a quantitative assay, it measures only the overall stimulation of topoisomerase II mediated cleavage. To study the cleavage specificity of topoisomerase II in the presence of 8MOC, the HinfI r D N A fragment was labelled at the 5' ends [20] and incubated with Drosophila topoisomerase II in the presence of increasing concentrations of 8-MOC. After incubation the reactions were stopped by SDS and, to obtain an enrichment of cleaved material, phenol extractions were performed. D N A was isolated from the phenol interphase [18] and after proteinase K treatment double stranded cleavage sites were m a p p e d by native acrylamide gel electrophoresis. Without drug two sites were recognized by Drosophila topoisomerase II. The two cleavage sites are recognized by eukaryotic topoisomerase II enzymes from heterologous sources, including Tetrahymena, Drosophila, calf thymus and man [19]. The presence of
2.5 a
b
c
d
e
• 0
5
f
• 10
8-MOC, mM Fig. 1. 8-Methoxycaffeine stimulates cleavable complex formation. Plasmid pTtrI, constructed in E. Blackburns laboratory by insertion of the 4 kb palindromic central spacer region from Tetrahymena thermophila rDNA into the HindlII site of pBR322 DNA (see map in Fig. 2), was digested with HinfI (New England Biolab) and the resulting fragments separated by agarose gel electrophoresis. The relevant 2.7 kb fragment, encompassing two strong topoisomerase II cleavage sites [17-19], was electrophoresed onto a 2 mm wide DEAE membrane strip (Schleicher and Schuell, NA45) placed adjacent to the lane. The DNA was eluted with 1 M NaCI, concentrated by ethanol precipitation, and labelled by filling the recessed 3' ends using the Klenow fragment of Escherichia coli DNA polymerase I (New England Biolab) and [a-32p]dATP [20]. 8-Methoxycaffeine (a generous gift of Dr. B.A. Kihlman) was dissolved in prewarmed water (80°C) at a final concentration of 4 mg/ml. The 3' end labelled restriction fragment (0.15 pmol) was incubated with Drosophila topoisomerase II (3 pmol) in 20 mM Tris-HCl (pH 8.0), 3 mM CaCI2, 1 mM MgCI2, 0.1 M sucrose in the presence of increasing concentrations of 8-MOC: a, no drug; b, 10/xM; c, 330 p~M; d, 1
mM; e, 2.5 mM; f, 10 mM (see inset). After incubation for 10 min at 30°C, cleavage and covalent binding of the topoisomerase to DNA was revealed by simultaneus addition of 1% SDS and 10 mM Na2-EDTA. Subsequently, NaCI was added to a final concentration of 0.8 M. In control samples NaCl and Naz-EDTA were added prior to SDS. The samples were made 0.33 M NaOH, 33 mM Naz-EDTA in a final volume of 400/xl and passed through a nitrocellulose filter (Schleicher and Schuell, NA45) preequilibrated in 0.1 M NaOH, 1 mM Na2-EDTA at a flow rate of approx. 0.4 ml/min per cm. The filter was washed three times with 0.1 M NaOH, 1 mM Na2-EDTA and once in 6xSSC, dried and processed for autoradiography or liquid scintillation photometry. The results were visualized by autoradiography (inset) and quantitated by scintillation counting of the autoradiographic spots (graph).
increasing concentrations of 8-MOC in the incubation mixture with Drosophila topoisomerase II (Fig. 3, lanes 3 - 8 ) resulted in a stimulation of double stranded cleavage at the two sites recognized in the absence of drug, but new sites were not generated. Even the highest concentration of 8-MOC (1 mM) did not result in any change in the sequence specificity of topoisomerase II and cleavage was not inhibited although 8-MOC, at the concentrations used, unwinds D N A [22]. The conserved sequence specificity of Drosophila topoisomerase II in the presence of 8-MOC is in contrast to results obtained with mAMSA, where new sites beyond
32
linlI I
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~
1 234567
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!
8
the two shown here, turned up at concentrations as low as 10 tzM (data not shown). The stimulation of cleavable complex formation by 8-MOC is expected to affect the strand passing activity of Drosophila topoisomerase II. To monitor the strand passing activity we employed the decatenation assay [23] u s i n g k i n e t o p l a s t D N A . A s s h o w n in Fig. 3, t h e d e c a t e n a t i n g a c t i v i t y o f Drosophila t o p o i s o m e r a s e II w a s s t r o n g l y i n h i b i t e d by 8 - M O C c o n c e n t r a t i o n s s t i m u l a t i n g t o p o i s o m e r a s e II c l e a v a b l e c o m p l e x f o r m a t i o n . 5 0 % i n h i b i t i o n w a s o b s e r v e d at 5 0 0 / z M . I n s p i t e o f t h e i n t e r c a l a t i v e m o d e o f 8 - M O C b i n d i n g t o D N A [22],
8-MOC, mM
C
0
0,25
0.5
1
kDNA
)
Minicircles
100
e., Q
y /,
50
I Fig. 2. Effect of 8-MOC on the sequence specificity of topoisomerase II. Map of the central spacer of the palindromic rDNA molecule from Tetrahymena: thin line, central spacer region of rDNA; thick line, coding region; arrow, palindrome symmetry axis; arrow heads, sites of DNA topoisomerase II cleavage. The HinfI rDNA fragment was labelled at the 5' ends by T4-polynucleotide kinase (New England Biolab) and [y-32p]ATP [19]. The 5' end labelled restriction fragment was incubated with topoisomerase I1 and 8-MOC, and cleavage was revealed as described in Fig. l. Cleaved DNA molecules were recovered as covalent DNA-protein complexes from interphasc after phenol extraction [18]. Following ethanol precipitation and proteinase K digestion (300 /zg/ml, 60 rain at 37°C), 1 volume of loading buffer containing 0.05% bromophenol blue, 0.03% xylene cyanole, 5 mM Na2-EDTA (pH 8.5) and 30% glycerol was added. Partial digestions with limiting amount of Xbal and Sphl (New England Biolab) were used as markers. Reaction products were analyzed by electrophoresis on a 6% native polyaerilamide gel and cleavage was visualized by autoradiography: lane 1, partial digestion of the fragment with Xbal; lane 2, partial digestion of the fragment with Sphl; lane 3, DNA topoisomerase II mediated cleavage in the absence of 8-MOC; lanes 4--8, DNA topoisomerase II mediated cleavage in the presence of 0.1 ,ttM; 1 /xM; 10 #M; 100 #M and 1 mM 8-MOC.
0
0
!
!
0,5
1
inhibitor, n~M Fig. 3. Effect of 8-MOC on the decatenating activity of DNA topoisomerase II. The strand-passing activity of DNA topoisomerase II was monitored by the decatenation of kinetoplast DNA [23]. A total assay volume of 20 p~l contained 50 mM Tris-HCl (pH 7.5). 120 mM KCI, 10 mM MgCI 2, 0.5 mM DTT, 0.5 mM Na2-EDTA, 1 mM ATP, 200 # g / m l kinetoplast-DNA (a generous gift of Dr. J.H..I. Hoeijmakers) and two units of DNA topoisomerase II. After incubation at 30°C for 30 min, reactions were quenched by addition of 5 ~zl of dye mixture (50% glycerol, 0.5% SDS, 0.025% bromophenol blue). The samples were electrophoresed at room temperature in a 1% agarose gel slab, in TPE-buffer (0.08 M Tris-phosphate, 0.008 M Na2-EDTA) for 3 h at 2 V/cm. Gels, stained with ethidium bromide (1 /zg/ml), were photographed on Polaroid type 55 films. Negatives of gel photographs were scanned by Beckman DU-8 gel scanner. Under the conditions employed, the intensity of the negative was directly proportional to the DNA present. Lane 1, DNA topoisomerase 11, no drug: Lane 2, 3, 4, 5, enzyme and increasing concentration of 8-MOC.
33 relaxation of supercoiled plasmid DNA by topoisomerase I purified from Drosophila was totally unaffected by 8-MOC, suggesting that intercalation per se was unable to cause the observed decrease in decatenation activity (data not shown). Consistent with the studies conducted in nuclei, we have found that 8-MOC stimulates cleavable complex formation between topoisomerase II and DNA. Treatment of the drug-affected complex by SDS results in DNA cleavage, where the enzyme becomes covalently linked to the broken DNA. 8-MOC stimulates the formation of both double and single stranded cleavage by topoisomerase II, where the cleaved DNA in both cases has protein covalently linked. Since the effect of 8-MOC on the cleavable complex formation parallels the inhibitory effect of the drug on the strand-passing activity of topoisomerase II, we suggest that 8-MOC, like the epipodophyllotoxins [24] and the intercalating antitumor drugs mAMSA and ellipticine [21], interferes with the cleavage/religation reaction of topoisomerase II by stabilizing the cleavable complex. In addition, the intercalative type of DNA-drug interaction, analogous to most antitumor intercalators inhibitors of DNA topoisomerase II, favours the possibility of a similar mode of action. Since inhibition of the strandpassing activity of DNA topoisomerase II has been observed not only with 8-MOC but also with caffeine [25,26], it is likely that induction of the cleavable complex might be a general property of methylated oxypurines. In previous experiments, where the effect of 8-MOC has been studied using low resolution gels, 8-MOC stimulation of mouse DNA topoisomerase II cleavable complexes was not visible [15]. The discrepancy between the two results may possibly derive from different properties of the two systems (sensibility of the enzymes or enzyme context), or from a sequence specific effect of 8-MOC inhibition. In this work we have used a highly specific recognition sequence [19] and, by high resolution gel analysis, we have observed that 8-MOC stimulation of the cleavable complex does not interfere with the sequence specificity of the enzyme. Although this property is not unique to this drug, since it has been described also for the trypanosomidal Ro 15-0216 [27]. it is not shared by the antineoplastic DNA topoisomerase II inhibitors that instead modify the sequence selectivity of DNA breakage by DNA topoisomerase II [28]. Comparison between DNA topoisomerase II inhibitors of different chemical classes has shown no correlation between drug cytotoxicity, the level of protein-associated DNA breaks and sequence selectivity. These results have been attributed to the fact that drugs from different chemical classes may produce cleavable complexes with different properties [11]. Therefore, the different stringency of the topoiso-
merase II nucleotide specificity between antineoplastic drugs and 8-MOC might be an important observation for the understanding of the different cellular responses of these two classes of inhibitors (e.g., DNA synthesis, G2-delay inhibition and cytotoxicity). It is possible that 8-MOC stimulated cleavable complexes are formed in the cell at a subset of DNA topoisomerase II sites, as for example those present at the nuclear matrix attachment sites [8], which do not trigger the lethal cellular responses generally observed with the antineoplastic drugs but only modify the normal pattern of DNA replication and stimulate chromatid exchanges. However, before the activities of methylated oxypurines on cell physiology can be fully understood, the mechanism by which these drugs determine the DNA topoisomerase II genomic sites of action must be delineated.
Acknowledgements We wish to thank Dr. B.A. Kihlman and Dr. O. Westergaard for support and collaboration during the course of this work. This work was supported by P.F. Ingegneria Genetica, C.N.R.
References 1 Kihlman, B.A. (1977) Caffeine and chromosome, Elsevier, Amsterdam. 2 Timson, J. (1977) Mut. Res. 47, 1-52. 3 Roberts, J.J. (1984) In DNA Repair and its Inhibition: towards an analysis of mechanisms. Nucleic Acid Symposium. Ser. 13, (Collins, A.R.S., Downs, C.S. and Johnson, R.T., eds.), pp. 193215, IRL Press, Oxford. 4 Kihlman, B.A. and Andersson, H.C. (1990) In Reviews on Environmental Health, Freund Publishing House, Tel Aviv, in press. 5 Wang. J.C. (1987) Biochim. Biophys. Acta 909, 1-9. 6 Painter, R.B. (1980) Mut. Res. 70, 337-341. 7 Cleaver, J.E. (1981) Exp. Cell Res. 136, 27-30. 8 Singh. B. and Gupta. R.S. (1983) Cancer Res. 43, 577-584. 9 Sperry, A.O., Blasquez, V.C. and Garrard, W.T. (1989) Proc. Natl. Acad. Sci. USA 86, 5497-5501. 10 Bae, Y.S., Kavasaki, I., Ikeda, H. and Liu, L.F. (1988) Proc. Natl. Acad. Sci. USA 86, 5497-5501. 11 Zwelling, L.A. (1985) Cancer Metast. Rev. 4, 263-276. 12 Liu, L.F. (1989) Annu. Rev. Biochem. 58, 351-375. 13 Deaven, L.L., Oka, M.S. and Tobey, R.A. (1978) J. Natl. Cancer Inst. 60, 1155-1161. 14 Uemura, Y. and Yanagida, M. (1986) EMBO J. 5, 1060-1064. 15 Russo, P., Poggi, L., Parodi, S., Pedrini, A.M., Kohn, K.W. and Pommier, Y. (1991) Carcinogenesis 12, 1781-1790. 16 Shelten, E.R., Osheroff, N. and Brutlag, D.L. (1983) J. Biol. Chem. 258, 9530-9535. 17 Kjeldsen, E., Bendixen, C., Thomsen, B., Christiansen, K., Bonaven, B.J., Nielsen, O.F. and Westergaard, O. (1991) In DNA Topoisomerases and Cancer (Kohn, K.W. and Potmesil, M., eds.), pp. 249-259, Oxford Press, Oxford. 18 Bonven, B.J., Gocke, E. and Westergaard O. (1985) Cell 41, 541-551. 19 Andersen, A.H., Christiansen. K., Zechiedrich, E.L., Jensen, P.S., Osheroff, N. and Westergaard, O. (1989) Biochemistry 28, 62376244.
34 20 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour. 21 Pornmier, Y., Minford, J.K., Shwartz, R.E., Zwelling, L.A. and Kohn, K. (1985) Biochemistry 24, 6410-6416. 22 Tornaletti, S., Russo, P., Parodi, S. and Pedrini, A.M. (1989) Biochim. Biophys. Acta 1007, 112-115. 23 Miller, K.G., Liu, L.F. and Englund, P.T. (1981) J. Biol. Chem. 245, 5334-5339. 24 Chen, G.L., Yang, L., Rowe, T.C., Halligan, B.D., Tewey, K.W. and Liu, L. (1984) J. Biol. Chem. 259, 13560-13566.
25 Warters, R.L., Lyons, B.W., Kennedy, K. and Mua Li, T. (1989 Mut. Res. 216, 43-55. 26 Shin, C-G, Strayer, J.M., Wani, M.A. and Snapka, R.M. (1990 Terat. Carc. Mutagen. 10, 41-52. 27 S6rensen, B.S., Jensen, P.S., Andersen. A.H., Christiansen, K. Alsner, J., Thomsen, B. and Westergaard, O. (1990) Biochemistr~ 29, 9507-9515. 28 Tewey, K.M., Rowe. T.C., Yang, L., Halligan, B.D. and Liu, L.F (1984) Science 226, 466-468.
Biochimica et Biophysica Acta, 1131 (1992) 3/)-34 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05./)0
BBAEXP 92373
8-Methoxycaffeine inhibition of Drosophila DNA topoisomerase II Silvia T o r n a l e t t i a, A n n i H. Andersen b, K e n t C h r i s t i a n s e n b a n d A n t o n i a M. P e d r i n i ~' '~ lstituto di Genetica Biochimica ed El,oluzionistica del C.N.R., Pacia (Italy) and h Institute for Molecular Biology and Plant Physiology, Unicersity of Aarhus, Arhus (Denmark)
(Received 5 November 1991) (Revised manuscript received 10 February 1992)
Key words: 8-Methoxycaffeine; DNA topoisomerase 11
We have investigated the effect of 8-methoxycaffeine on the interaction between Drosophila DNA topoisomerase Il and DNA. We have shown that 8-methoxycaffeine affected the enzyme strand-passing activity by inhibiting decatenation of kinetoplast DNA, and that it interfered with the breakage-reunion reaction by stabilizing a cleavable complex. Treatment of the cleavable complex with protein denaturant resulted in DNA breaks. High resolution mapping of the cleavage sites in the central spacer region of Tetrahymena rDNA revealed that, contrary to what was observed with clinically important DNA topoisomerase II inhibitors, 8-methoxycaffeine did not modify the cleavage pattern observed without the drug. Methylated oxypurines (MOPs) are a class of drugs that induce various alterations on the genetic material in plant, Drosophila and cultured mammalian cells (for reviews see Refs. 1-4). Among these alterations, the capability of caffeine and its derivatives in producing S-independent chromosomal aberrations is well documented. Late G2-phase and prophase are the most sensitive stages, the aberrations produced at this stage being mainly chromatid exchanges. The formation of chromosomal aberrations is strongly conditioned by active oxidative phosphorylation, suggesting that exchange processes are closely related to the ATP level of the cells. To account for the ATP and cell cycle dependence of MOPs induced chromosomal aberrations, Kihlman and Andersson [4] have proposed that at least part of the multiple effects caused by caffeine and derivatives on the genetic material could be due to an interference with DNA topoisomerase II. Eukaryotic DNA topoisomerases type II are ubiquitous enzymes defined by their ability to catalyze ATP dependent DNA topoisomerisation reactions [5]. Central to all DNA topoisomerase II reactions is the enzyme ability to induce transient enzyme-bridged
Abbreviations: DTT, dithiothreitol; mAMSA, amsacrine; 8-MOC, 8-methoxycaffeine; rDNA, ribosomal DNA; Ro 15-0216, 2-dimethylamino-4'[(l-met hyl-2-nitroimidazol-5-yl)methoxy]acetanilide; SDS, sodium dodecyl sulfate; SSC, standard saline citrate. Correspondence: A.M. Pedrini, Istituto di Genetica Biochimica ed Evoluzionistica del C.N.R.. Via Abbiategrasso 207, 27100-Pavia, Italy.
strand breaks, where the enzyme subunits are covalently bound to the 5' ends of the cleaved strands, forming a covalent DNA-enzyme intermediate, termed cleavable complex. Treatment with strong denaturants, such as sodium dodecyl sulfate (SDS), can irreversibly trap the cleavable complex and reveal strand breaks. Inaccurate rejoining by subunits exchange of the cleaved strands may lead to exchanges between duplex DNA. For these properties, several authors have suggested the possible involvement of DNA topoisomerase II in chromosome rearrangements [6-9]. Several observations are consistent with this proposal. Eukaryotic enzymes can catalyze non homologous recombination in vitro [10], and hotspots for enzyme cutting have been found at translocational breakpoints [9]. Furthermore, among the cellular effects of the antineoplastic DNA topoisomerase II inhibitors [11], that stabilize the cleavable complex [12], there is an increased frequency of sister chromatid exchanges and chromatid aberrations. The most sensitive stage of the cell cycle is late G2 phase [13], a phase requiring active DNA topoisomerase II to carry on normal chromosome segregation [14]. The recent finding that 8-methoxycaffeine (8-MOC), the most active caffeine derivative in inducing chromosomal aberrations, can induce protein-linked single and double strand DNA breaks in mouse leukemia L1210 cells and purified nuclei strengthens Kihlman's suggestion that DNA topoisomerase II may be the putative target of methylated oxypurines [15]. In fact, the induced breaks have the same properties (saturability,
31 reversibility and t e m p e r a t u r e dependence) as those produced by the antineoplastic D N A topoisomerase II inhibitor, ellipticine. Prompted by the similarity between the putative target protein of 8-MOC and D N A topoisomerase II, we have carried on a study to test whether topoisomerase II is a target for 8-MOC. We have initiated by investigating the influence of the drug on the formation of cleavable complex between highly purified topoisomerase II from Drosophila melanogaster [16] and D N A isolated from the central spacer region of the r D N A molecule of Tetrahymena thermophila [17,18], containing two symmetric strong recognition sites for Drosophila topoisomerase II [19]. To quantitate the cleavage reaction a filter retention assay, involving the precipitation of covalent e n z y m e - D N A complex, was done. The HinfI r D N A fragment (see map of Fig. 2 and Ref. 17) was 3' end-labelled [20] and than incubated with the enzyme in the presence of increasing concentrations of 8-MOC. The reaction was stopped by SDS and, following alkali denaturation of the cleavage products, the complexes were precipitated on filters. The results, presented in Fig. 1, clearly show that 8-MOC stimulated cleavable complex formation between Drosophila topoisomerase II and rDNA. Furthermore, radioactivity was retained on the filter when a 3' end-labelled fragment was used, strongly indicating that topoisomerase II was covalently linked to the 5' end of the cleaved DNA. If 0.8 M NaC1 was added to the reaction mixture containing 10 m M 8-MOC prior to SDS treatment, the amount of radioactivity retained on the filter dropped to background level, demonstrating that the effect of 8-MOC on the topoisomerase I I - D N A complexes was reversible (data not shown). The results suggest that 8-MOC interacts with the cleavable complexes in a way similar to well known intercalating antitumor drugs such as ellipticine and m A M S A [21]. Although the filter retention assay is a quantitative assay, it measures only the overall stimulation of topoisomerase II mediated cleavage. To study the cleavage specificity of topoisomerase II in the presence of 8MOC, the HinfI r D N A fragment was labelled at the 5' ends [20] and incubated with Drosophila topoisomerase II in the presence of increasing concentrations of 8-MOC. After incubation the reactions were stopped by SDS and, to obtain an enrichment of cleaved material, phenol extractions were performed. D N A was isolated from the phenol interphase [18] and after proteinase K treatment double stranded cleavage sites were m a p p e d by native acrylamide gel electrophoresis. Without drug two sites were recognized by Drosophila topoisomerase II. The two cleavage sites are recognized by eukaryotic topoisomerase II enzymes from heterologous sources, including Tetrahymena, Drosophila, calf thymus and man [19]. The presence of
2.5 a
b
c
d
e
• 0
5
f
• 10
8-MOC, mM Fig. 1. 8-Methoxycaffeine stimulates cleavable complex formation. Plasmid pTtrI, constructed in E. Blackburns laboratory by insertion of the 4 kb palindromic central spacer region from Tetrahymena thermophila rDNA into the HindlII site of pBR322 DNA (see map in Fig. 2), was digested with HinfI (New England Biolab) and the resulting fragments separated by agarose gel electrophoresis. The relevant 2.7 kb fragment, encompassing two strong topoisomerase II cleavage sites [17-19], was electrophoresed onto a 2 mm wide DEAE membrane strip (Schleicher and Schuell, NA45) placed adjacent to the lane. The DNA was eluted with 1 M NaCI, concentrated by ethanol precipitation, and labelled by filling the recessed 3' ends using the Klenow fragment of Escherichia coli DNA polymerase I (New England Biolab) and [a-32p]dATP [20]. 8-Methoxycaffeine (a generous gift of Dr. B.A. Kihlman) was dissolved in prewarmed water (80°C) at a final concentration of 4 mg/ml. The 3' end labelled restriction fragment (0.15 pmol) was incubated with Drosophila topoisomerase II (3 pmol) in 20 mM Tris-HCl (pH 8.0), 3 mM CaCI2, 1 mM MgCI2, 0.1 M sucrose in the presence of increasing concentrations of 8-MOC: a, no drug; b, 10/xM; c, 330 p~M; d, 1
mM; e, 2.5 mM; f, 10 mM (see inset). After incubation for 10 min at 30°C, cleavage and covalent binding of the topoisomerase to DNA was revealed by simultaneus addition of 1% SDS and 10 mM Na2-EDTA. Subsequently, NaCI was added to a final concentration of 0.8 M. In control samples NaCl and Naz-EDTA were added prior to SDS. The samples were made 0.33 M NaOH, 33 mM Naz-EDTA in a final volume of 400/xl and passed through a nitrocellulose filter (Schleicher and Schuell, NA45) preequilibrated in 0.1 M NaOH, 1 mM Na2-EDTA at a flow rate of approx. 0.4 ml/min per cm. The filter was washed three times with 0.1 M NaOH, 1 mM Na2-EDTA and once in 6xSSC, dried and processed for autoradiography or liquid scintillation photometry. The results were visualized by autoradiography (inset) and quantitated by scintillation counting of the autoradiographic spots (graph).
increasing concentrations of 8-MOC in the incubation mixture with Drosophila topoisomerase II (Fig. 3, lanes 3 - 8 ) resulted in a stimulation of double stranded cleavage at the two sites recognized in the absence of drug, but new sites were not generated. Even the highest concentration of 8-MOC (1 mM) did not result in any change in the sequence specificity of topoisomerase II and cleavage was not inhibited although 8-MOC, at the concentrations used, unwinds D N A [22]. The conserved sequence specificity of Drosophila topoisomerase II in the presence of 8-MOC is in contrast to results obtained with mAMSA, where new sites beyond
32
linlI I
",~ AA
~
1 234567
I [inl7 A A
!
8
the two shown here, turned up at concentrations as low as 10 tzM (data not shown). The stimulation of cleavable complex formation by 8-MOC is expected to affect the strand passing activity of Drosophila topoisomerase II. To monitor the strand passing activity we employed the decatenation assay [23] u s i n g k i n e t o p l a s t D N A . A s s h o w n in Fig. 3, t h e d e c a t e n a t i n g a c t i v i t y o f Drosophila t o p o i s o m e r a s e II w a s s t r o n g l y i n h i b i t e d by 8 - M O C c o n c e n t r a t i o n s s t i m u l a t i n g t o p o i s o m e r a s e II c l e a v a b l e c o m p l e x f o r m a t i o n . 5 0 % i n h i b i t i o n w a s o b s e r v e d at 5 0 0 / z M . I n s p i t e o f t h e i n t e r c a l a t i v e m o d e o f 8 - M O C b i n d i n g t o D N A [22],
8-MOC, mM
C
0
0,25
0.5
1
kDNA
)
Minicircles
100
e., Q
y /,
50
I Fig. 2. Effect of 8-MOC on the sequence specificity of topoisomerase II. Map of the central spacer of the palindromic rDNA molecule from Tetrahymena: thin line, central spacer region of rDNA; thick line, coding region; arrow, palindrome symmetry axis; arrow heads, sites of DNA topoisomerase II cleavage. The HinfI rDNA fragment was labelled at the 5' ends by T4-polynucleotide kinase (New England Biolab) and [y-32p]ATP [19]. The 5' end labelled restriction fragment was incubated with topoisomerase I1 and 8-MOC, and cleavage was revealed as described in Fig. l. Cleaved DNA molecules were recovered as covalent DNA-protein complexes from interphasc after phenol extraction [18]. Following ethanol precipitation and proteinase K digestion (300 /zg/ml, 60 rain at 37°C), 1 volume of loading buffer containing 0.05% bromophenol blue, 0.03% xylene cyanole, 5 mM Na2-EDTA (pH 8.5) and 30% glycerol was added. Partial digestions with limiting amount of Xbal and Sphl (New England Biolab) were used as markers. Reaction products were analyzed by electrophoresis on a 6% native polyaerilamide gel and cleavage was visualized by autoradiography: lane 1, partial digestion of the fragment with Xbal; lane 2, partial digestion of the fragment with Sphl; lane 3, DNA topoisomerase II mediated cleavage in the absence of 8-MOC; lanes 4--8, DNA topoisomerase II mediated cleavage in the presence of 0.1 ,ttM; 1 /xM; 10 #M; 100 #M and 1 mM 8-MOC.
0
0
!
!
0,5
1
inhibitor, n~M Fig. 3. Effect of 8-MOC on the decatenating activity of DNA topoisomerase II. The strand-passing activity of DNA topoisomerase II was monitored by the decatenation of kinetoplast DNA [23]. A total assay volume of 20 p~l contained 50 mM Tris-HCl (pH 7.5). 120 mM KCI, 10 mM MgCI 2, 0.5 mM DTT, 0.5 mM Na2-EDTA, 1 mM ATP, 200 # g / m l kinetoplast-DNA (a generous gift of Dr. J.H..I. Hoeijmakers) and two units of DNA topoisomerase II. After incubation at 30°C for 30 min, reactions were quenched by addition of 5 ~zl of dye mixture (50% glycerol, 0.5% SDS, 0.025% bromophenol blue). The samples were electrophoresed at room temperature in a 1% agarose gel slab, in TPE-buffer (0.08 M Tris-phosphate, 0.008 M Na2-EDTA) for 3 h at 2 V/cm. Gels, stained with ethidium bromide (1 /zg/ml), were photographed on Polaroid type 55 films. Negatives of gel photographs were scanned by Beckman DU-8 gel scanner. Under the conditions employed, the intensity of the negative was directly proportional to the DNA present. Lane 1, DNA topoisomerase 11, no drug: Lane 2, 3, 4, 5, enzyme and increasing concentration of 8-MOC.
33 relaxation of supercoiled plasmid DNA by topoisomerase I purified from Drosophila was totally unaffected by 8-MOC, suggesting that intercalation per se was unable to cause the observed decrease in decatenation activity (data not shown). Consistent with the studies conducted in nuclei, we have found that 8-MOC stimulates cleavable complex formation between topoisomerase II and DNA. Treatment of the drug-affected complex by SDS results in DNA cleavage, where the enzyme becomes covalently linked to the broken DNA. 8-MOC stimulates the formation of both double and single stranded cleavage by topoisomerase II, where the cleaved DNA in both cases has protein covalently linked. Since the effect of 8-MOC on the cleavable complex formation parallels the inhibitory effect of the drug on the strand-passing activity of topoisomerase II, we suggest that 8-MOC, like the epipodophyllotoxins [24] and the intercalating antitumor drugs mAMSA and ellipticine [21], interferes with the cleavage/religation reaction of topoisomerase II by stabilizing the cleavable complex. In addition, the intercalative type of DNA-drug interaction, analogous to most antitumor intercalators inhibitors of DNA topoisomerase II, favours the possibility of a similar mode of action. Since inhibition of the strandpassing activity of DNA topoisomerase II has been observed not only with 8-MOC but also with caffeine [25,26], it is likely that induction of the cleavable complex might be a general property of methylated oxypurines. In previous experiments, where the effect of 8-MOC has been studied using low resolution gels, 8-MOC stimulation of mouse DNA topoisomerase II cleavable complexes was not visible [15]. The discrepancy between the two results may possibly derive from different properties of the two systems (sensibility of the enzymes or enzyme context), or from a sequence specific effect of 8-MOC inhibition. In this work we have used a highly specific recognition sequence [19] and, by high resolution gel analysis, we have observed that 8-MOC stimulation of the cleavable complex does not interfere with the sequence specificity of the enzyme. Although this property is not unique to this drug, since it has been described also for the trypanosomidal Ro 15-0216 [27]. it is not shared by the antineoplastic DNA topoisomerase II inhibitors that instead modify the sequence selectivity of DNA breakage by DNA topoisomerase II [28]. Comparison between DNA topoisomerase II inhibitors of different chemical classes has shown no correlation between drug cytotoxicity, the level of protein-associated DNA breaks and sequence selectivity. These results have been attributed to the fact that drugs from different chemical classes may produce cleavable complexes with different properties [11]. Therefore, the different stringency of the topoiso-
merase II nucleotide specificity between antineoplastic drugs and 8-MOC might be an important observation for the understanding of the different cellular responses of these two classes of inhibitors (e.g., DNA synthesis, G2-delay inhibition and cytotoxicity). It is possible that 8-MOC stimulated cleavable complexes are formed in the cell at a subset of DNA topoisomerase II sites, as for example those present at the nuclear matrix attachment sites [8], which do not trigger the lethal cellular responses generally observed with the antineoplastic drugs but only modify the normal pattern of DNA replication and stimulate chromatid exchanges. However, before the activities of methylated oxypurines on cell physiology can be fully understood, the mechanism by which these drugs determine the DNA topoisomerase II genomic sites of action must be delineated.
Acknowledgements We wish to thank Dr. B.A. Kihlman and Dr. O. Westergaard for support and collaboration during the course of this work. This work was supported by P.F. Ingegneria Genetica, C.N.R.
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