The Formation of DNA Interstrand Cross-Links by a Novel Bis-[Pt2Cl4(diminazene aceturate)2]Cl4z4H2O Complex Inhibits the B to Z Transition

We present data demonstrating that the cytotoxic compound [Pt2Cl4(diminazene aceturate)2]Cl4z4H2O (Pt-berenil) circumvents cisplatin resistance in ovarian carcinoma cells. The analysis of the interaction of Pt-berenil with linear and supercoiled DNA indicates that this compound induces the formation of a large number of covalent interstrand cross-links on DNA and that this number is significantly higher than that produced by cis-diamminedichloroplatinum(II) (cis-DDP). Renaturation experiments, interstrand cross-link assays, and electron microscopy indicate that the kinetics of DNA interstrand cross-link formation caused by Pt-berenil binding is faster than that caused by cis-DDP at similar levels of platinum bound to DNA. Furthermore, the number of DNA interstrand cross-links in Ptberenil-DNA complexes is influenced by supercoiling. Circular dichroism experiments show that Pt-berenil strongly inhibits the B-DNA-to-Z-DNA transition of poly(dG-m dC)zpoly(dGmdC) at salt concentrations (3 mM MgCl2) at which the native methylated polynucleotide readily adopts the Z-DNA conformation, which suggests that the induction of interstrand crosslinks by Pt-berenil inhibits the Z-DNA transition. On the basis of these results, we propose that bis(platinum) compounds with structure similar to Pt-berenil may act as blockers of DNA conformational changes and may also display activity in cisplatin-resistant cells. It has been postulated that the DNA interstrand cross-link adducts formed by platinum drugs may contribute to the cytotoxicity of these compounds (Leng and Brabec, 1994). In fact, the interstrand cross-links formed in DNA by cis-diamminedichloroplatinum(II) (cis-DDP) between guanine residues in opposite strands preferentially inhibit DNA or RNA polymerases in vitro (Lemaire et al., 1991; Vrána et al., 1996). Moreover, the binding of platinum drugs on DNA may induce conformational changes of the double helix structure that may be responsible for the drug activity (Johnson et al., 1989; Leng and Brabec, 1994; Pil and Lippard, 1997). The secondary structure most commonly adopted by DNA in vivo is the B-form, which is right-handed; however, DNA may also adopt other conformations (Cantor, 1981). Thus, a new conformational state of the DNA, namely Z-DNA, characterized by a left-handed conformation of the helix, has been described (Pohl and Jovin, 1972; Herbert and Rich, 1996). It is now well established that DNA sequences that are in Z conformation or having potential to adopt the Z-DNA form can be found in biological systems (Nordheim et al., 1981; Nordheim and Rich, 1983; Lancilloti et al., 1987; Casasnovas et al., 1989; Wittig et al., 1991). Moreover, a possible role for Z-DNA in gene regulation has also been proposed (JiménezRuı́z et al., 1991). Pohl and Jovin showed that at high salt concentration, poly(dG-dC)zpoly(dG-dC) undergoes a cooperative, conformational transition from the B-DNA form toward the Z-DNA form (Pohl and Jovin, 1972). Later on, Behe and Felsenfeld noticed that the methylated polymer poly(dG-m dC)z poly(dG-mdC) also undergoes a transition from the B form to the Z form, but it does so at lower salt concentrations (Behe and Felsenfeld, 1981). Malinge and Leng (1984) found that binding of the antitumor drug cis-DDP to poly(dGmdC)zpoly(dG-mdC) in B conformation induces a conformational change toward a distorted Z-like form and that binding of cis-DDP to the polynucleotide in Z conformation stabilizes its Z-DNA structure. Moreover, it has been also observed that a family of trans-bis(platinum) complexes provokes the Bto-Z transition in poly(dG-mdC)zpoly(dG-mdC) (Johnson et This work was supported by Comisión Interministerial de Ciencia y Tecnologica Grants BIO-096–0405, SAF-96–0041 and CAM 160/92. The institutional support of Fundación Ramón Areces is also acknowledged. ABBREVIATIONS: cis-DDP, cis-diamminedichloroplatinum(II); berenil, diminazene aceturate; CD, circular dichroism; Pt-berenil, [Pt2Cl4(diminazene aceturate)2]Cl4z4H2O; ri, input molar ratio of platinum to nucleotides; rb, molar ratio of platinum bound to nucleotides; TXRF, total reflection X-ray fluorescence; DMEM, Dulbecco’s modified Eagle’s medium; kb, kilobase pair(s). 0026-895X/99/040770-08$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 55:770–777 (1999). 770 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from al., 1992). In contrast, the clinically ineffective trans-isomer of cis-DDP inhibits B-to-Z transition in DNA (Peticolas and Thomas, 1985; Rahmouni et al., 1985; Zaludová et al., 1997). We have previously reported that the cytotoxic bis(platinum) complex [Pt2Cl4(diminazene aceturate)2]Cl4z4H2O (Ptberenil) (Fig. 1) induces drastic changes on DNA secondary structure, leading to a compaction of the double helix, and that the diminazene aceturate (berenil) ligand directs the DNA binding of the Pt-berenil drug (González et al., 1996, 1997). We show herein that the interaction between the Pt-berenil compound and DNA results in the formation of a large number of covalent interstrand cross-links and that this number is significantly higher than that caused by cisDDP-DNA interaction. Our data indicate, moreover, that the kinetics of DNA interstrand cross-link formation by Pt-berenil binding is faster than that of cis-DDP-DNA interaction and that the number of DNA interstrand cross-links in Ptberenil-DNA complexes is influenced by supercoiling. To determine whether the large number of DNA interstrand crosslinks formed by the drug affect the conformational changes of the double helix, we have analyzed the effect of Pt-berenil binding on the MgCl2-induced B-DNA-to-Z-DNA transition of poly(dG-mdC)zpoly(dG-mdC). We have chosen this methylated polymer because the dinucleotide sequence “mdC-dG” appears so frequently in eukaryotic DNA [it may account for more than half of all “d(CpG)” sequences (Razin and Riggs, 1980)]. Moreover, we have chosen MgCl2 as the Z-DNA inducer because it allows work at Cl concentrations close to those found intracellularly (Rich et al., 1984). The results show that Pt-berenil strongly inhibits the salt-induced BDNA-to-Z-DNA transition of poly(dG-m dC)zpoly(dG-mdC). We think that these results are interesting in view of the fact that the currently known platinum drugs, in contrast with Pt-berenil, induce the B-DNA-to-Z-DNA transition (Malinge and Leng, 1984; Johnson et al., 1992; Zaludová et al., 1997). Because we also present data here demonstrating that Ptberenil is active in cis-DDP-resistant cells, we propose that other bis(platinum) compounds with structures similar to Pt-berenil might be designed in the search for new cytotoxic platinum compounds capable of circumventing cisplatin resistance. Experimental Procedures Materials. The stock solutions of the cis-DDP, berenil, and Ptberenil compounds were prepared to a final concentration of 1 mg/ml. Pt-berenil was synthesized as described previously (González et al., 1996). Cis-DDP was kindly supplied by Bristol-Myers S.A. (Madrid, Spain). The Pt-berenil and cis-DDP compounds were dissolved in 10 mM NaClO4. The drug solutions were freshly prepared before use. Calf thymus DNA was purchased from Sigma Chemical Co. (St. Louis, MO). The pF18 plasmid DNA (2927 base pairs in length) containing a Z-DNA forming sequence (Jiménez-Ruı́z et al., 1989) was isolated from the Escherichia coli JM83 strain by a modification of the alkaline lysis method (Maniatis et al., 1989). The BamHI restriction enzyme and the Klenow fragment of E coli DNA polymerase I were obtained from Boehringer Mannheim (Madrid, Spain). [a-P]dCTP (10 mCi/ml) was purchased from Amersham International (Madrid, Spain). Cytotoxicity Assays. The ovarian carcinoma cell lines A2780, A2780cisR, CH1, CH1cisR, SKOV-3 and SKOV-3cisR were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of newborn calf serum together with 2 mM glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37°C in an atmosphere of 95% air/5% CO2. A2780cisR, CH1cisR, and SKOV3cisR are a sublines of their respective parent lines that have acquired cisplatin resistance (Kelland et al., 1993). Cell survival in compound-treated cultures was analyzed by using a system based on the tetrazolium compound 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, which is reduced by living cells to yield a soluble formazan product that can be assayed colorimetrically (Alley et al., 1988). Cells were plated in 96-well sterile plates, at a density of 10 cells/well in 100 ml of medium and were incubated for 3–4 h. Compounds dissolved in DMEM were added to final concentrations from 0.1 to 100 mM, in a volume of 100 ml/well. Ninety-six hours later, 50 ml of a freshly diluted 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide solution (1/5 in culture medium) was Fig. 1. Chemical structure of Pt-berenil (A) and berenil (B). Inhibition of the B-to-Z-DNA Transition 771 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from added at a concentration of 1 mg/ml into each well and the plates were incubated for 5 h at 37°C in a humidified 5% CO2 atmosphere. Cell survival was evaluated by measuring the absorbance at 520 nm, using a Whittaker Microplate Reader 2001. All experiments were made in quadruplicate. Platination Reactions. Calf thymus DNA, pF18 DNA, and poly(dG-m dC)zpoly(dG-mdC) at concentrations of 200 mg/ml were incubated with the platinum drugs at ri 5 0.1 in 10 mM NaClO4 at 37°C. At various time periods (10 min, 30 min, 1 h, 2 h, 3 h, 18 h, and 24 h), aliquots of the reaction mixture were withdrawn and assayed by total X-ray fluorescence (TXRF) for platinum not bound to DNA (Niemann et al., 1990; Wobrauschek, 1994; González et al., 1996). The molar ratio of platinum bound to nucleotides (rb) was calculated by subtracting the amount of free (unbound) platinum present in the reaction mixture. In agreement with previously reported data (González et al., 1996), it was observed that the kinetics of DNA platination by both Pt-berenil and cis-DDP were similar and that after 24 h of incubation, DNA platination reached a plateau because . 95% of the cis-Pt(II) centers from both Pt-berenil and cisDDP were bound to DNA. DNA Renaturation Experiments. Sonicated calf thymus DNA (average length of 500 base pairs) was dissolved in 0.02 3 standard saline citrate (150 mM NaCl, 15 mM sodium citrate, pH 7.0) at a concentration of 20 mg/ml. Aliquots of 1 ml of the calf thymus DNA solution were modified by the drugs at rb of 0.05. The DNA was melted at 45°C to 95°C. After 2 min at 95°C, control DNA was allowed to reanneal by decreasing the temperature from 95°C to 12°C above the melting temperature of native DNA (57 6 0.5°C) at a 1°C/min rate. DNA modified by Pt-berenil or cis-DDP were exposed to comparable renaturation conditions. Thus, Pt-berenil-modified DNA or cis-DDP-modified DNA were allowed to reanneal by decreasing the temperature at a 1°C/min rate from 95°C to 12°C above the melting temperature of Pt-berenil-DNA (62 60.7°C) or cis-DDP-DNA complexes (55 6 0.3), respectively. The decrease in chromicity upon renaturation was measured in a Beckman Acta CIII spectrophotometer attached to a temperature programmer. The percentage of DNA renaturation was calculated from the hypochromicity value at 95°C. Interstrand Cross-Link Assay. To linearize the pF18 plasmid, the DNA was digested in 150 mM NaCl with 10 units/mg DNA of BamHI (unique restriction site in pF18) at 37°C for 4 h. The plasmid DNA was 39-end labeled by incubation with 2.5 mCi/mg DNA of [a-P]dCTP and 1.25 units/mg DNA of the Klenow fragment of E coli DNA polymerase I for 30 min at room temperature. The reaction was stopped by heating at 70°C for 5 min. The unincorporated radioactivity was removed by passing the labeling reaction through a Sephadex G-50 column. The labeled DNA was precipitated with 0.1 volumes of sodium acetate and 2 volumes of cold ethanol. Sonicated DNA was added to the eluted solution of the labeled pF18 DNA to a final DNA concentration of 180 mg/ml. Afterwards, the DNA, at a concentration of 90 ng/ml, was incubated with the Pt-berenil and cis-DDP drugs in 10 mM NaClO4 at ri of 0.1 for different periods of incubation. Then, 10-ml aliquots were removed and the reaction was ended by addition of an equal volume of the loading dye (90% formamide, 10 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue). At each period of incubation, the Pt bound to DNA (rb) was monitored by TXRF. The DNA was melted for 10 min at 90°C and chilled on ice. Agarose gel (1.5%) electrophoresis in denaturing conditions was carried out at 20 V for 16 h (Maniatis et al., 1989). Interstrand cross-link formation was also detected in covalently closed circular pF18 plasmid DNA (density of supercoiling, s 5 20.067). Supercoiled pF18 plasmid was incubated with Pt-berenil or cis-DDP under the conditions indicated above and subsequently linearized with BamHI endonuclease, 39-end labeled with [a-P]dCTP, denatured by heat, and subjected to agarose gel electrophoresis in denaturing conditions. The gels were dried and autoradiographed. Band quantification was made using a Molecular Dynamics model 300A densitometer (Sunnyvale, CA). Determination of the Cross-Linking Rate. The number of cross-links was estimated assuming a Poisson distribution [(XL/fg 5 2ln(1 2 FDS)] (Vos and Hanawalt, 1987). XL/fg represents the number of cross-links per fragment. FDS results from dividing the peak of the integrated area of the double-stranded band by the sum of the integrated areas of the double-stranded and single-stranded bands. The (XL/fg)/fragment size ratio gives the number of cross-links per kilobase pair (kb). Electron Microscopy of Pt-berenil-pF18 DNA Complexes. Supercoiled (s 5 20.067) or linear pF18 plasmid DNA (2 mg) were incubated at 37°C in 10 mM NaClO4 with Pt-berenil at ri 5 0.1 until an rb 5 0.05 was achieved. Then, the DNA was precipitated with 2.5 volumes of cold ethanol and 0.1 volume of 3 M sodium acetate, pH 4.8. Afterward, the precipitate was washed with 100 ml of 75% ethanol. Sample aliquots of the Pt-berenil-pF18 DNA complexes (DNA concentration 5 0.1 mg/ml) were denatured and extended as a monolayer on an aqueous hypophase according to the method of Sogo and Thoma (1989). The DNA interstrand cross-links were visualized in electron micrographs of the denatured supercoiled and linear DNA samples. Determination of Platinum Bound to DNA In Vivo. Culture plates containing 10 ml of HeLa cells (human cervix carcinoma line) in DMEM (cell density, 2 3 10 cells/ml) were preincubated at 37°C for 24 h in an atmosphere of 5% CO2. Then, 500 ml of culture medium containing the desired amount of drug to reach a final drug concentration of 35 mM (the IC50 value of Pt-berenil in HeLa cells) was added to the plates. Subsequently, the plates were incubated for 48 h at 37°C and 5% CO2. Afterward, the cells were centrifuged at 1500 rpm for 5 min, washed with phosphate-buffered saline, resuspended in 400 ml of buffer containing 20 mM TriszHCl, pH 7.5, 2 mM EDTA, and 0.4% Triton X-100, incubated at 4°C for 15 min, and centrifuged at 12,000 rpm during 15 min in a Microfuge (Hettich Microfuge, Madrid, Spain). The supernatants were then treated for 3 h at 37°C with 20 mg/ml of proteinase K in a buffer containing 150 mM NaCl, 40 mM TriszHCl, pH 8.0, 40 mM EDTA, and 1% SDS. Finally, the DNA was extracted with phenol/chloroform/isoamyl alcohol (25:24: 1), precipitated with 2.5 volumes of cold ethanol and 0.1 volumes of 3 M sodium acetate, washed with 75% ethanol, dried, and resuspended in 1 ml of water. The DNA content in each sample was measured by UV spectrophotometry at 260 nm. The platinum bound to DNA was determined by TXRF (Niemann et al., 1990; Wobrauschek, 1994). Experiments were carried out in triplicate. In Vivo Detection of DNA Modifications Induced by Binding of Pt-Berenil. The HeLa cells cultivated and treated with 35 mM concentrations of the Pt-berenil drug for 24 h were harvested with trypsin/EDTA and centrifuged. The pellets were treated as described in the previous section to isolate the DNA. Aliquots (250 ml) of genomic DNA (60 ng/ml of DNA) were digested to completion with EcoRI. Subsequently, 20-ml aliquots were subjected to 1% agarose gel electrophoresis during 16 h at 1.5 V/cm and transferred to a nylon membrane (Amersham). Southern blot detection of genomic DNA was performed using a [P]-labeled 18 S ribosomal DNA probe (10 mCi/ml). CD Spectroscopy. CD spectra were recorded in a JASCO J-600 spectropolarimeter interfaced to a 486 PC. Measurements were performed at 37°C using 1-cm path-length cells. Each spectrum represents the mean of three scans. Aliquots containing 15 mg/ml of poly(dG-m dC)zpoly(dG-mdC) were prepared from the stock solution. CD spectra were run in a range of wavelength from 220 to 320 nm and at a speed of 50 nm/min. Scans were recorded at 0.4 nm