(JM118) and Cisplatin - American Chemical Society

Oct 12, 2010 - metabolite of the first orally administered PtIV drug satraplatin. In an effort to design improved platinum antitumor agents, it is imp...
0 downloads 0 Views 3MB Size
Download PDF
Chem. Res. Toxicol. 2010, 23, 1833–1842

1833

Different Features of the DNA Binding Mode of Antitumor cis-Amminedichlorido(cyclohexylamine)platinum(II) (JM118) and Cisplatin in Vitro Hana Kostrhunova,† Oldrich Vrana,† Tereza Suchankova,‡ Dan Gibson,§ Jana Kasparkova,† and Viktor Brabec*,† Institute of Biophysics, Academy of Sciences of the Czech Republic, V.V.i., CZ-61265 Brno, Czech Republic, Department of Experimental Physics, Faculty of Sciences, Palacky UniVersity, 17. listopadu 12, 77146 Olomouc, Czech Republic, and Department of Medicinal Chemistry and Natural Products, School of Pharmacy, The Hebrew UniVersity of Jerusalem, Jerusalem 91120, Israel ReceiVed August 27, 2010

cis-Amminedichlorido(cyclohexylamine)platinum(II) (JM118) is an antitumor PtII analogue of cisplatin exhibiting considerably higher activity than cisplatin in human tumor cells. JM118 is also the major metabolite of the first orally administered PtIV drug satraplatin. In an effort to design improved platinum antitumor agents, it is important to elucidate the biochemical factors that affect the cytotoxic properties of existing platinum drugs. Since DNA is considered the major pharmacological target of platinum drugs, the objective in the present work was to understand more fully the DNA binding mode of antitumor JM118. We examined the rate of aquation of the first chloride of bifunctional JM118 and found that it was considerably lower than that of cisplatin; consequently, the rate of the reaction of JM118 with DNA was lower compared to cisplatin. The influence of global modification by JM118 and its major sitespecific adducts on DNA conformation by biochemical methods was investigated as well. While examination of the global modification revealed in several cases no substantial differences in the lesions induced by JM118 and cisplatin, DNA bending due to the 1,2-GG intrastrand adduct of JM118 was lower than that of cisplatin. The bending angles afforded by the adducts of JM118 were only slightly affected by the orientation of the cyclohexylamine ligand toward the 3′ or 5′ direction of the duplex. We also used in vitro assays that make it possible to monitor DNA repair synthesis by cell-free extracts and DNA-protein cross-linking to probe properties of DNA adducts of JM118. These results showed a higher DNA-protein cross-linking efficiency of JM118 and a less efficient removal from DNA of the adducts of JM118 in comparison with cisplatin. Thus, the results of the present work provide additional evidence that DNA binding of JM118 is in several aspects different from that of conventional cisplatin. Introduction In spite of the widespread success of platinum anticancer drugs already used in the clinic [cisplatin (Figure 1), carboplatin, nedaplatin, and oxaliplatin], the search for new platinum antitumor drugs continues. It has been motivated by the desire to overcome drawbacks associated with the use of cisplatin to treat malignancies. In this search various strategies have been explored. The target for platinum antitumor compounds is genomic DNA, to which they bind efficiently forming a variety of adducts (1, 2). Thus, one strategy is based on testing the hypothesis that there is a correlation between the antitumor activity of platinum compounds and their capability to induce in DNA a certain sort of conformational change induced in DNA by their binding (3, 4). Since the discovery of antitumor activity of cisplatin, several active compounds that bind to DNA in a way different from that of cisplatin have been identified, including platinum compounds with a trans stereochemistry (5), monofunctional PtII complexes (6-8), polynuclear platinum complexes (9, 10), platinum(IV) complexes (11), photoactivat* To whom correspondence should be addressed. Tel: +420-541517148. Fax: +420-541240499. E-mail: [email protected]. † Academy of Sciences of the Czech Republic. ‡ Palacky University. § The Hebrew University of Jerusalem.

Figure 1. Structures of platinum complexes.

able platinum complexes (12), and various direct mononuclear and bifunctional analogues of cisplatin (13, 14). JM118 (cis-amminedichlorido(cyclohexylamine)platinum(II), Figure 1) is a bifunctional mononuclear PtII analogue of cisplatin which is itself an active antitumor complex (15). Importantly, JM118 was considerably more active than cisplatin in a number of both cisplatin sensitive and resistant human tumor cells (16-18). In addition, JM118 is the major metabolite of satraplatin [bis-acetatoamminedichloro(cyclohexylamine)platinum(IV), JM216, Figure 1]; the latter PtIV complex is the first orally administered platinum drug, which showed promise in patients with prostate cancer (19). Importantly, it is the PtII complex which confers the cytotoxic mechanism of action of satraplatin (20). During their reaction with DNA, dichlorido leaving groups of bifunctional PtII drugs are displaced, but the nonleaving amine groups remain intact. Compared with platinum drugs already used in the clinic, JM118 or satraplatin

10.1021/tx1002904  2010 American Chemical Society Published on Web 10/12/2010

1834

Chem. Res. Toxicol., Vol. 23, No. 11, 2010

are unique in that their nonleaving ligands are asymmetrical (an NH3 and a cyclohexamine, compared with the two NH3 groups of cisplatin, carboplatin, or nedaplatin or the diaminocyclohexane group of oxaliplatin). These differences may affect some intracellular processes on the level of a major pharmacological target of JM118 or satraplatin, which is DNA (17, 21-25), contributing to their unique properties. It is generally accepted that JM118 binds to DNA in a similar fashion as does cisplatin (26), although some differences in the DNA binding mode of JM118 and cisplatin have been reported (27). Our primary objective in the present work was to understand more fully the DNA binding mode of JM118 in a cell free medium. We examined the influence of global modification by JM118 on the conformation of natural DNA. In addition, we constructed a site-specifically platinated 20-23 bp duplexes containing a centrally located 1,2-GG intrastrand cross-link (CL) of JM118 and determined local bending and unwinding induced in DNA by this major DNA adduct of JM118. Comparisons of these results with those obtained for conventional cisplatin yielded new information that the DNA binding mode of JM118 is in several aspects different from the DNA binding mode of cisplatin. In order to further demonstrate that some features of the DNA adducts of JM118 and cisplatin are different, we also probed these adducts using the assay that makes it possible to monitor DNA repair synthesis in vitro.

Experimental Procedures Material and Reagents. JM118 was prepared as described (28, 29). The purity of JM118 was higher than 95% as established by combustion analysis carried out with a Hewlett-Packard 185 C, H, and N analyzer. Cisplatin was obtained from Sigma (Prague, Czech Republic) (the purity of cisplatin was ∼99.9% based on elemental and ICP trace analysis). If not stated otherwise, the stock solutions of platinum compounds were prepared at the concentration of 5 × 10-4 M in 10 mM NaClO4 and stored at 4 °C in the dark. Calf thymus (CT) DNA (42% G + C, mean molecular mass ca. 20 000 kDa) was prepared and characterized as described previously (30, 31). Plasmids, pUC19 [2686 base pairs (bp)], pBR322 (4361 bp), and pSP73 (2464 bp) were isolated according to standard procedures. T4 DNA ligase, the Klenow fragment from DNA polymerase I (exonuclease minus, mutated to remove the 3′-5′ proofreading domain) (KF-), restriction endonucleases, T4 polynucleotide kinase, Circum VentTM Thermal Cycle Sequencing Kit with Vent(exo-) DNA polymerase, and bovine serum albumin (BSA) were purchased from New England Biolabs (Beverly, MA). Deoxyribonucleoside 5′-triphosphates (dNTPs) were from Roche Diagnostics, GmbH (Mannheim, Germany). Agarose was from FMC BioProducts (Rockland, ME). Acrylamide, bis(acrylamide), ethidium bromide (EtBr), urea, dithiothreitol (DTT), and NaCN were from Merck KgaA (Darmstadt, Germany). Proteinase K and ATP were from Boehringer (Mannheim, Germany). Dimethyl sulfate (DMS) was from Sigma (Prague, Czech Republic). Sodium dodecyl sulfate (SDS) was from Serva (Heidelberg, Germany). NFκB protein (p50 homodimer) was kindly provided by Professor Vasak (University of Zurich, Switzerland). Radioactive products were from Amersham (Arlington Heights, IL, USA). A cell-free extract (CFE) was prepared from the repair proficient HeLa S3 cell line as described (32, 33). Quantitative Evaluation of Binding of JM118 and Cisplatin to Mammalian DNA in a Cell-Free Medium. Solutions of doublehelical CT DNA at a concentration of 0.024 mg mL-1 (7.5 × 10-5 M related to the phosphorus content) were incubated with JM118 (1.5 µM) at a value of ri ) 0.02 in 0.1 mM NaCl at 37 °C (ri is defined as the molar ratio of free platinum complex to nucleotide phosphates at the onset of incubation with DNA). Two different stock solutions of JM118 (1.5 mM) or cisplatin (1.5 mM) were prepared. One contained the PtII complex incubated for 7 days in

KostrhunoVa et al. unbuffered NaCl (0.1 M, pH 6) at 37 °C in the dark, whereas the PtII complexes in the other type of the stock solutions were incubated for 7 days in double distilled water at 37 °C in the dark. Five microliters of the PtII complex aged in NaCl (0.1 M) or in water were quickly mixed with 4995 µL of DNA dissolved in NaClO4 (10 mM), and the reaction mixture was kept at 37 °C. In the experiments in which the PtII complex aged in water was used, the final reaction mixture was still supplemented at the onset of the reaction with NaCl so that the resulting concentration of NaCl in the reaction mixtures was always 0.1 mM. At various time intervals, an aliquot of the reaction mixture was withdrawn and assayed by differential pulse polarography (DPP) for platinum not bound to DNA (34). Platination Reactions in Cell-Free Media. If not stated otherwise, CT or plasmid DNAs were incubated with the platinum complex in NaClO4 (10 mM) at 37 °C in the dark. After 24 h, the samples were exhaustively dialyzed against the medium required for subsequent biochemical or biophysical analysis. An aliquot of these samples was used to determine the value of rb (the number of molecules of the Pt complex bound per nucleotide residue) by flameless atomic absorption spectrophotometry (FAAS) or by DPP (34). The duplexes containing single, central 1,2-GG intrastrand CL of JM118 or cisplatin in the pyrimidine-rich top strands were prepared as described (35, 36). The platinated oligonucleotides were purified by ion-exchange high-pressure liquid chromatography (HPLC), which made it possible to separate two orientational isomers of 1,2-GG intrastrand CL of JM118 (27). It was verified by platinum FAAS and by the measurements of the optical density that the modified oligonucleotides contained one platinum atom. It was also verified using DMS footprinting of platinum on DNA (36, 37) that one JM118 or cisplatin molecule was coordinated to the N7 atoms of the two Gs in the top strands of each duplex. Other details have been described previously (35, 37). Sequence Preference of DNA Adducts. The primer extension footprinting assay was used to evaluate the sequence selectivity of DNA modification by JM118 in comparison with that of cisplatin. The fragment of pSP73 DNA linearized by NdeI (2464 bp) was incubated with the platinum complexes in NaClO4 (10 mM) for 24 h at 37 °C to obtain rb ) 0.005. DNA Interstrand Cross-Linking. Platinum complexes at varying concentrations were incubated for 24 h with 0.5 µg of a linear 1663-bp fragment of pSP73 DNA linearized by NdeI/PvuI. The linear fragment was first 3′-end labeled by means of the Klenow fragment of DNA polymerase I in the presence of [R-32P]dATP. The platinated samples were analyzed for DNA interstrand CLs by previously published procedures (37, 38). The number of interstrand CLs was analyzed by electrophoresis under denaturing conditions on alkaline agarose gel (1%). After the electrophoresis had been completed, the intensities of the bands corresponding to single strands of DNA and interstrand cross-linked duplex were quantified. The frequency of interstrand CLs was calculated as % ICL/Pt ) XL/3326·rb (the DNA fragment contained 3326 nucleotide residues), where % ICL/Pt is the number of interstrand CLs per adduct, and XL is the number of interstrand CLs per molecule of the linearized DNA duplex and was calculated assuming a Poisson distribution of the interstrand CLs as XL ) -ln A, where A is the fraction of molecules running as a band corresponding to the noncross-linked DNA. Fluorescence Measurements. These measurements were performed on a Varian Cary fluorescence spectrophotometer using a 0.5 cm quartz cell. Fluorescence measurements were performed at an excitation wavelength of 546 nm, and the emitted fluorescence was analyzed at 590 nm. The fluorescence intensity was measured at 25 °C in 0.4 M NaCl to avoid secondary binding of EtBr to DNA (39, 40). The concentrations were 0.01 mg mL-1 for DNA and 0.04 mg mL-1 for EtBr, which corresponded to the saturation of all intercalation sites of EtBr in DNA (39). Unwinding of Negatively Supercoiled DNA. Unwinding of closed circular supercoiled pSP73 plasmid DNA was assayed by an agarose gel mobility shift assay (41). The unwinding angle Φ, induced per one DNA adduct of the platinum complex, was

DNA Binding Mode of JM118

Figure 2. Sequence preference of DNA adducts. Replication mapping of platinum-DNA adducts. (A) Autoradiogram of 6% polyacrylamide/8 M urea sequencing gel showing the inhibition of DNA synthesis by VentR DNA polymerase on the pSP73 plasmid DNA linearized by the NdeI restriction enzyme and subsequently modified by platinum complexes. The gel contained the linear amplification products of the control, unplatinated DNA, and DNA treated with JM118 or cisplatin. Lanes: NoPt, unplatinated template; C, G, T, A, chain-terminated marker DNAs (note that these dideoxy sequencing lanes give the sequence complementary to the template strand); cisPt and JM118, DNA modified by cisplatin and JM118 at rb ) 0.005, respectively. The numbers on the right side of the gel correspond to the nucleotide sequence numbering in panel B. (B) Schematic diagram showing a portion of the sequence used to monitor the inhibition of DNA synthesis on the template containing adducts of platinum complexes. The arrow indicates the direction of the synthesis. *, major stop signals from panel A, lane JM118.

calculated upon the determination of the rb value at which the complete transformation of the supercoiled to relaxed form of the plasmid was attained. Samples of plasmid DNA were incubated with the platinum complex at 37 °C in the dark for 24 h. The samples were subsequently subjected to electrophoresis on 1% native agarose gel running at 25 °C in the dark with TAE (Trisaceate/Na2H2EDTA) buffer and the voltage set at 18 V. The gels were then stained with EtBr, followed by photography with transilluminator. DNA Melting. The melting curves of CT DNAs were recorded by measuring the absorbance at 260 nm. The melting curves were recorded in a medium containing 0.1 M Na+ with Tris·HCl (1 mM, pH 7.4) plus Na2H2EDTA (0.1 mM). The value of the melting temperature (tm) was determined as the temperature corresponding to a maximum on the first derivative profile of the melting curves. Ligation and Electrophoresis of Oligonucleotides. Unplatinated 20-23-mer single strands (bottom strands of the duplexes shown in Figure 2A) were 5′-end-labeled with [γ-32P]ATP by using T4 polynucleotide kinase. Then they were annealed with their phosphorylated complementary strands containing a single GG platinated site (unplatinated or containing the platinum CL). The duplexes were allowed to react with T4 DNA ligase. The resulting samples along with ligated unplatinated duplexes were subsequently examined on 8% native polyacrylamide (PAA) [mono/bis(acrylamide) ratio ) 29:1] electrophoresis gels. Other details of these experiments were as described in previous papers (42-44). The oligonucleotides

Chem. Res. Toxicol., Vol. 23, No. 11, 2010 1835 containing either 3′- or 5′-orientational isomers of the 1,2-GG intrastrand CLs of JM118 were separated, purified, and identified by a reversed phase C18 HPLC column under the same conditions as those described previously (27). DNA Repair Synthesis by Human Cell Extract. DNA repair DNA synthesis of CFEs was assayed using nonmodified pBR322 and nonmodified or platinated pUC19 plasmids along with CFE from the HeLa S3 cells in the same way as that described in our recently published paper (45). Briefly, the measurement of DNA excision repair activity in vitro, originally developed by Wood et al. (46), utilizes protein extract obtained from mammalian cells by the method of Manley et al. (47). The repair assay relies on the incorporation of radiolabeled deoxyribonucleotide during the repair synthesis step. In order to observe repair-DNA synthesis, supercoiled plasmid DNA damaged by the platinum complex is incubated with whole cell extract in a reaction mixture including the four dNTPs, one of them (dATP) radiolabeled, and an ATP-regenerating system. Undamaged plasmid DNA of different size is added to the reaction mixture as an internal control. DNA repair synthesis is determined after the recovery of plasmid DNA from the mixture, linearization with restriction enzyme, agarose-gel electrophorersis autoradiography, and measurement of the radioactivity incorporated into each plasmid (46). Repair activity is expressed as specific repair synthesis (incorporation in the damaged plasmid minus background incorporation in the control plasmid). The plasmid DNA recovery in each reaction sample is normalized by densitometry of the EtBrstained gel. Formation of the Ternary DNA-Platinum-Protein Complexes. Platinated DNA (40- or 21-bp oligodeoxyribonucleotide duplexes) at the concentration of 10 nM were incubated with the proteins (KF- or NF-κB) at the concentration of 100 nM overnight at room temperature in the appropriate buffer: Tris·HCl (10 mM, pH 8), Na2H2EDTA (10 mM), BSA (0.1 mM), glycerol (0.8%), and MgSO4 (2 mM) (KF-); HEPES (42 mM), KCl (42 mM), MgCl2 (1 mM), Na2H2EDTA (0.02 mM), DTT (210 mM), glycerol (2.5%), and Ficoll (2%) (NF-κB). The protein samples used in this study were prepared in the following way: the final composition of the storage buffers, KF-, Tris·HCl (10 mM, pH 8.0), Na2H2EDTA (0.5 mM), BSA (100 mg mL-1), glycerol (50%), and MgSO4 (10 mM); NF-κB protein (p50 dimer), Tris·HCl (25 mM, pH 8.0) and NaCl (50 mM). The commercially available sample of KF- was in the manufacturer’s storage buffer containing DTT; the manufacturer’s storage buffer was exchanged for that specified earlier using microcon concentrators. The ability to form CLs by JM118 or cisplatin between the oligonucleotide duplex and proteins was assessed by 10% SDS/PAA gel electrophoresis after mixing the samples with the loading buffer (Tris-HCl (50 mM, pH 6.8), DTT (100 mM), SDS (2%), bromophenol blue (0.1%), and glycerol (10%)) and denaturing by heat at 90 °C for 5 min. Gels were electrophoresed for 1-2 h at 140 V, dried, and visualized by using the bioimaging analyzer. Other Physical Methods. Absorption spectra were measured with a Beckmann DU-7400 spectrophotometer. FAAS measurements were carried out with a Varian AA240Z Zeeman atomic absorption spectrometer equipped with a GTA 120 graphite tube atomizer. For FAAS analysis, DNA was precipitated with ethanol and dissolved in 0.1 M HCl. DPP was performed with an EG&G Princeton Applied Research Corporation Model 384B Polarographic Analyzer. Circular dichroism spectra of CT DNA modified by PtII complexes were recorded at 25 °C in NaClO4 (10 mM) using a JASCO J-720 spectropolarimeter. The cell path length was 1 cm. Spectra were recorded in the range 230-500 nm in 0.5-nm increments with an averaging time of 1 s. The gels were visualized by using the BAS 2500 FUJIFILM bioimaging analyzer, and the radioactivities associated with bands were quantitated with the AIDA image analyzer software (Raytest, Germany).

Results Global Modification of Natural Double-Helical DNA by JM118. One of the important early phases of the mechanism by which platinum compounds exert their anticancer activity is

1836

Chem. Res. Toxicol., Vol. 23, No. 11, 2010

KostrhunoVa et al.

Table 1. Binding of JM118 and Cisplatin to Calf Thymus DNAa t50%b (min) JM118 aged in H2O cisplatin aged in H2O JM118 aged in 0.1 M NaCl cisplatin aged in 0.1 M NaCl

112 ( 3 43 ( 2 193 ( 4 128 ( 3

a Solutions of double-helical DNA at a concentration of 0.024 mg mL-1 (7.5 × 10-5 M related to the phosphorus content) were incubated with JM118 or cisplatin (1.5 µM) at a value of ri of 0.02. For other details, see the text. Medium: NaCl (0.1 mM, pH 6) at 37 °C. b The time at which the binding reached 50%. Values shown in the table are the means ((SEM) of three separate experiments.

the formation of adducts on nuclear DNA by these agents (14, 22, 48). Hence, data on the DNA binding mode of platinum complexes are of interest. In the present study, we have applied some methodologies previously developed for cisplatin and its analogues to investigate the reaction products of JM118 with DNA in cell-free media. In order to determine the character of DNA adducts of JM118, we examined the DNA binding properties of this complex and compared these binding properties with those of the parent cisplatin. The first experiments were aimed at quantifying the binding of JM118 to mammalian double-helical DNA in a cellfree medium. The amount of the platinum compound bound to DNA increased with time. The times at which the binding reached 50% (t50%) are summarized in Table 1. Importantly, after 48 h cisplatin and JM118 aged in water, and cisplatin aged in 0.1 M NaCl or in water were quantitatively bound, whereas only 91% JM118 aged in 0.1 M NaCl was bound. As expected, the PtII complexes preincubated in water reacted with DNA significantly more rapidly than those preincubated in NaCl (0.1 M), but the rate of the reaction of cisplatin was considerably higher than that of JM118. Further experiments were aimed at the determination of preferential DNA binding sites (Figure 2). In vitro DNA synthesis by DNA polymerases on DNA templates containing several types of adducts of platinum complexes can be prematurely terminated at the level or in the proximity of adducts (49, 50). The procedure involved the extension by VentR(exo-) DNA polymerase at the 3′-end of the primer up to the metal adduct on the template strand of pSP73 DNA. The products of the synthesis were then examined on DNA sequencing gels, and the sequence specificity of the platinum adduct formation was determined to the exact base pair. In vitro DNA synthesis on DNA templates containing adducts of JM118 generated a population of DNA fragments, indicating that the adducts of this complex effectively terminated DNA synthesis (Figure 2A, lane JM118). Sequence analysis of the termination sites produced by adducts of JM118 shows that the major stop sites were similar to those produced by cisplatin (Figure 2A, lane cisPt) and were mainly at GG or AG sites. We further characterized DNA lesions induced by global modification by JM118 or cisplatin within natural double-helical DNA using the methods of molecular biophysics (Figures 3-7). DNA Interstrand Cross-Linking. Bifunctional platinum compounds, which coordinate base residues in DNA, form various types of interstrand and intrastrand CLs. Considerable evidence suggests that the antitumor efficacy of bifunctional platinum compounds is the result of the formation of these lesions, but their relative efficacy remains unknown. Therefore, we have decided to quantitate the interstrand cross-linking efficiency of JM118 in the linear 1663 bp fragment (NdeI/PvuI restriction fragment of pUC19 plasmid), radioactively labeled

Figure 3. Formation of interstrand cross-links. Interstrand cross-linking by JM118 and cisplatin in the linear 1663 bp fragment of the pSP73 plasmid. Autoradiogram of denaturing 1% agarose gels of linearized DNA which was 3′-end labeled. The interstrand cross-linked DNA appears as the top bands migrating on the gel more slowly than the single-stranded DNA (contained in the bottom bands). Lanes: 1, control, unplatinated DNA; 2-4, DNA modified by cisplatin; 5-7, DNA modified by JM118. rb values: 0.0003 (lanes 2 and 5); 0.0007 (lanes 3 and 6); 0.0015 (lanes 4 and 7).

Figure 4. Ethidium bromide fluorescence. Dependences of the EtBr fluorescence on rb for CT DNA modified by platinum complexes in 10 mM NaClO4 at 37 °C for 24 h. (0), JM118; (+), cisplatin.

and modified by JM118 at various rb values. The samples were analyzed for the interstrand CLs by agarose gel electrophoresis under denaturing conditions (37). Upon electrophoresis, 3′-endlabeled strands of the linear fragment containing no interstrand CLs migrate as a 1663-base single strand, whereas the interstrand cross-linked strands migrate more slowly as a higher molecular mass species (Figure 3). The bands corresponding to more slowly migrating interstrand cross-linked fragments were seen for rb values as low as 3 × 10-4. The intensity of the more slowly migrating band increased with the growing level of the modification. The radioactivity associated with the individual bands in each lane was measured to obtain estimates of the fraction of noncross-linked or cross-linked DNA under each condition. The frequency of interstrand CLs was calculated using the Poisson distribution from the fraction of interstrandcross-linked DNA in combination with the rb values and the fragment size (38). The interstrand cross-linking efficiency of JM118 (5.4 ( 0.4% Figure 3) was similar to that of cisplatin (6% (37)). Characterization of DNA Adducts by Ethidium Bromide Fluorescence. The fluorescent probe ethidium bromide (EtBr) can be used to distinguish between perturbations induced in DNA by monofunctional and bifunctional adducts of platinum compounds (39, 40, 51-53). Binding of EtBr to DNA by intercalation is blocked in a stoichiometric manner by the formation of the bifunctional adducts of a series of platinum complexes including cisplatin and transplatin, which results in a loss of fluorescence intensity. Double helical DNA was first modified by the PtII complex (JM118 or cisplatin) for 24 h. The levels of the modification corresponded to the values of rb in the range between 0 and 0.125. Modification of DNA by both platinum complexes resulted in a decrease of EtBr fluorescence (Figure 4). The formation of adducts of JM118 resulted in a decrease of EtBr fluorescence intensity, which was similar to that of cisplatin. This result suggests that the conformational distortion induced in DNA by the adducts of JM118 is delocalized and extends over the same number of base pairs

DNA Binding Mode of JM118

Chem. Res. Toxicol., Vol. 23, No. 11, 2010 1837

Figure 5. Unwinding of supercoiled pSP73 plasmid DNA by JM118. The top bands correspond to the form of nicked plasmid and the bottom bands to closed, negatively supercoiled plasmid. The plasmid was incubated with each complex with the following rb values: lanes 1 and 12, control, nonmodified DNA; 2, 0.002; 3, 0.005; 4, 0.018; 5, 0.03; 6, 0.038; 7, 0.046; 8, 0.05; 9, 0.06; 10, 0.07; 11, 0.08.

Figure 7. Circular dichroism (CD) spectra of calf thymus DNA modified by JM118 (A) and cisplatin (B). Curves: 1, control (unplatinated) DNA; 2, rb ) 0.02; 3, rb ) 0.05; 4, rb ) 0.08; 5, rb ) 0.1. (C) Changes in the CD spectra of DNA at λmax around 280 nm (at the wavelength at which the maximum of the positive CD band around 280 nm occurred) induced by the binding of JM118 (0) or cisplatin (+).

Figure 6. ∆tm of calf thymus DNA modified by JM118 (0) or cisplatin (+) versus rb measured in NaCl (0.1M) plus Tris·HCl (10 mM, pH 7.4) with Na2H2EDTA (0.1 mM) (with ∆tm defined as the difference between the tm values of platinated and unplatinated DNAs).

around the platination sites as in the case of the adducts of cisplatin. Thus, characterization of DNA adducts of JM118 by EtBr fluorescence supports the view that the DNA binding modes of this PtII complex is similar to that of cisplatin. Unwinding of Negatively Supercoiled DNA. Electrophoresis in native agarose gel was used to quantify the unwinding induced in pSP73 plasmid by the platinum complexes by monitoring the degree of supercoiling (Figure 5). A compound that unwinds the DNA duplex reduces the number of supercoils so that the superhelical density of closed circular DNA decreases. This decrease upon the binding of unwinding agents causes a decrease of the rate of migration through the agarose gel, which makes it possible to observe and quantify the mean value of unwinding per one adduct. Figure 5 shows an electrophoresis gel in which increasing amounts of JM118 were bound to a mixture of relaxed and supercoiled pSP73 DNA. The unwinding angle is given by Φ ) -18σ/rb(c) where σ is the superhelical density, and rb(c) is the value of rb at which the supercoiled and relaxed forms comigrate (41). Under the present experimental conditions, σ was calculated to be -0.028 on the basis of the data of cisplatin for which the rb(c) was determined in this study and Φ ) 13° was assumed (41). By using this approach, we determined the DNA unwinding angle of 13° for JM118 (the comigration point of the modified supercoiled and nicked DNA, rb(c), was reached at rb ) 0.038). Hence, the efficiency of JM118 adducts to unwind the DNA double helix is similar to that of cisplatin adducts. DNA Thermal Melting. CT DNA was modified in NaClO4 (10 mM) by JM118 or cisplatin to the value of rb ) 0-0.06. After modification, the salt concentration was further adjusted by the addition of NaCl to 0.1 M, and the samples were further supplemented by Tris-HCl (1 mM) and Na2H2EDTA (0.1 mM). Thus, the melting curves for DNA modified by the platinum compounds were measured at a relatively high salt concentration (Figure 6). We find that the effect of DNA platination by JM118 on the melting temperature of DNA (tm) is slightly more pronounced than that of cisplatin.

Table 2. Summary of the Biophysical and Biochemical Properties of DNA Globally Modified by JM118 and Cisplatina preferential DNA binding sitesb reduction of EtBr fluorescence plasmid DNA unwinding angle/adduct % interstrand cross-links/adduct CD band at 278 nm melting temperature a See also Figures 2-7. 75. d Ref 41. e Ref 37.

b

JM118

cisplatin

GG, AG medium 13° 6(1 increase decrease

GG, AGc medium 13°d 6e increase decrease

Determined by replication mapping.

c

Ref

Circular Dichroism Spectroscopy. CD spectral characteristics were compared for CT DNA modified by JM118 and cisplatin to values of rb in the range 0.02-0.1 (Figure 7). Upon binding of these compounds to CT DNA, the conservative CD spectrum normally found for DNA in the canonical B-conformation considerably transforms at wavelengths below 300 nm. There was a significant increase in the intensity of the positive band around 280 nm if DNA was modified by JM118. At higher levels of the modification (rb > 0.08), the intensity of this CD band leveled off (Figure 7C). This behavior was similar to that observed for DNA modified by cisplatin under identical conditions. On the basis of the analogy with the changes in the CD spectra of DNA modified by cisplatin and clinically ineffective transplatin (54), it might be suggested that the binding of JM118 in particular at low levels of modification results in the conformational alterations in double-helical DNA that do not involve denaturation (hydrogen-bond breakage) similar to those induced in DNA by cisplatin. The results of these experiments aimed at the characterization of DNA lesions induced by global modification by JM118 or cisplatin within natural double-helical DNA are summarized in the Table 2. These results support the thesis that the preferential DNA binding sites of JM118 are similar to those of cisplatin and that several features of conformational alterations induced in DNA by adducts of JM118, which could be revealed by the biophysical and biochemical methods used, are not substantially different from those induced in DNA by adducts of cisplatin. Decomposition of JM118 Involving Displacement of the Chloride Ligand. Studies providing insights into the stability of dichlorido forms of cisplatin analogues are of interest since differences between the activities of the cisplatin analogues may

1838

Chem. Res. Toxicol., Vol. 23, No. 11, 2010

Figure 8. Time dependence of the decomposition of dichlorido forms of JM118 (50 µM) (0) and cisplatin (50 µM) (+) involving the displacement of the chloride ligand examined in 5 mM NaCl at 37 °C by differential pulse polarography (34). See the text for details.

arise from differences in the reactivities of their monofunctional DNA adducts (55). In the present investigation, the method for the assay of JM118 and cisplatin stability based on the polarographic activity of these drugs (56) has been applied to solutions of JM118 and cisplatin. The stock solutions containing the PtII complex (1.0 mM) were incubated for 7 days in unbuffered NaCl (0.1 M, pH 6) at 37 °C in the dark. The PtII complex (50 µL) was quickly mixed with double distilled water (950 µL), and the reaction mixture was kept at 37 °C in the dark. Thus, the resulting concentrations of NaCl and the PtII complex in these solutions were 5 mM and 50 µM, respectively. At various time intervals, an aliquot of the reaction mixture was withdrawn and assayed by DPP for the intact (dichlorido) form of the PtII complex (56) (Figure 8). The amount of the intact PtII complex decreased with time, and the rate of the aquation of the first chloride ion of cisplatin was considerably higher than that of JM118 (the time at which this aquation reached 50% was 170 ( 8 min for JM118 or 80 ( 5 min for cisplatin; these values represent the means ((SEM) of three separate experiments). Bending and Unwinding Produced in Double-Helical DNA by the Site-Specific 1,2-GG Intrastrand Cross-Link. Local duplex bending and unwinding are important determinants in the molecular mechanism of antitumor effects of cisplatin and its analogues (57). In this work, we further performed studies on the bending and unwinding induced by single, sitespecific intrastrand CLs of JM118 formed in oligodeoxyribonucleotide duplexes between neighboring guanine residues. When JM118 forms 1,2-GG intrastrand CLs, two orientational isomers are formed as a result of the asymmetry in the platinum coordination sphere. These isomers differ with respect to the positioning of the cyclohexyl group toward either the 3′ or the 5′ direction of the phosphodiester linkage (3′ or 5′ isomer, respectively) (27). Thus, site-specifically modified duplexes allow for the evaluation of the effect of the cyclohexylamine ligand, and its orientation toward the 3′ or 5′ direction of the duplex, on local bending and unwinding induced in DNA by the major adduct of JM118. As in the previous studies (58-60), we used electrophoretic retardation as a quantitative measure of the extent of planar curvature to analyze bending and unwinding induced by the single, site-specific orientational isomer of 1,2-GG intrastrand CL of JM118. This CL was formed in the sequence TGGT frequently used in previous studies of the same adducts formed by cisplatin and its analogues. The oligodeoxyribonucleotide duplexes TGGT(20-23) (for their sequence, see Figure 9A) were 20-23-bp long. The ligation products of these unplatinated duplexes or duplexes containing CL of either JM118 or cisplatin were analyzed on native PAA electrophoresis gel. Experimental details of these studies are given in our recent reports (58-60). A representative gel and its analysis showing the mobility of the ligation products of

KostrhunoVa et al.

20-23-bp duplexes containing single, site-specific 1,2-GG intrastrand CL of JM118 (or cisplatin for comparative purposes) at the central sequence TGGT in a PAA gel is demonstrated in Figure 9B. The DNA bending toward the major groove of 28 ( 1° and 26 ( 1° due to the single, site-specific 1,2-GG intrastrand CL of JM118 (3′ and 5′ orientational isomer, respectively) was significantly lower than that found in this experiment for the same adduct of cisplatin (34°; see also refs 43 and 61). The direction of the bend was determined using the 33-bp duplexes, which also contained, besides the single 1,2-GG intrastrand CL formed by JM118, the (A-T)5 tract located “in phase” from the CL (the cross-linked basepair and the center of the A tract were separated by 11 bp), in the same way as that in our recent articles (59, 62, 63). In contrast, DNA unwinding 11 ( 1° and 12 ( 1° due to the single, site-specific 1,2-GG intrastrand CL of JM118 (3′ and 5′ orientational isomer, respectively) was almost identical to that found in this experiment for the same adduct of cisplatin (13°; see also refs 61 and 64). Also produced in the ligations of monomers investigated in this work were separate bands arising from small DNA circles that migrate close to the top of the gel (see the bands marked by asterisks in Figure 9B, lanes cisPt and JM118-B for the 22mer). The occurrence of small DNA circles was even better evident if the PtII complex was removed from the products of the ligation reaction by NaCN (not shown). The tendency to yield DNA circles was observed only for the 22-bp intrastrand cross-linked multimers of the duplex containing the 3′ but not 5′ orientational isomer of the 1,2-GG intrastrand CL, confirming a close match between the 22-bp sequence repeat and the helix screw (65, 66). DNA Repair Synthesis by Human Cell Extract. Properties of DNA adducts of JM118 were also probed by using the in vitro assay that makes it possible to monitor DNA repair synthesis by the CFE of repair proficient HeLa cells. pUC19 plasmid (2686 bp) globally modified by JM118 or cisplatin at rb ) 0.03 was incubated with this CFE, and the amount of radiolabeled nucleotide incorporated into the platinated pUC19 plasmid was monitored as a measure of adduct-induced DNA repair synthesis. The incorporation of radioactive material was corrected for the relative DNA content in each band. As illustrated in Figure 10, adduct-induced DNA repair synthesis detected in the plasmid modified by JM118 was approximately 70% of that found for the cisplatin at the same level of modification. DNA-Protein Cross-Linking. The complex JM118 was also investigated for its ability to form ternary DNA-protein complexes covalently linked by the platinum moiety. The proteins were chosen for these studies that bind to DNA with a relatively high affinity. KF- was chosen as the representative of nonsequence specific DNA-binding protein with enzymatic function, whereas transcription factor NF-κB (p50 dimer) was chosen as the representative of a sequence-specific DNA-binding protein with a regulation function. The 40-bp duplex (with a random sequence shown in Figure 11A) 5′-end-labeled at its top strand was globally modified by JM118 or cisplatin for 24 h so that 1 platinum atom was bound per duplex on average (rb ) 0.0125). The duplex modified by JM118 or cisplatin (10 nM) was mixed with KF- (the molar ratio protein/duplex was 10). For the studies of the formation of ternary DNA-PtII-NF-κB complexes, the 21-bp duplex NF-κB (its nucleotide sequence shown in Figure 11A corresponds to the DNA consensus

DNA Binding Mode of JM118

Chem. Res. Toxicol., Vol. 23, No. 11, 2010 1839

Figure 9. Ligation and electrophoresis of oligonucleotides. (A) Sequences of the synthetic oligodeoxyribonucleotides used in this study with their abbreviations. The top and bottom strands of each pair are designated top and bottom, respectively, in the text. The bold letters in the top strand indicate the location of the intrastrand CL after the modification of the oligonucleotides by JM118 or cisplatin as described in Experimental Procedures. (B) Autoradiograms of the ligation products of double-stranded oligonucleotides TGGT (20-23) containing a unique 1,2-GG intrastrand CL of JM118 (3′ and 5′ orientational isomers) or cisplatin separated on an 8% polyacrylamide gel. Lanes: NoPt, unplatinated oligomers; cisPt, oligomers contacting CL of cisplatin; JM118-A, oligomers containing CL of JM118 (5′ orientational isomer); JM118-B, oligomers containing CL of JM118 (3′ orientational isomer). (C,D) Plots showing the relative mobility K (defined as the ratio of calculated to actual length) versus sequence length curves for the oligomers 20-23 bp long containing the CL of JM118, 3′ (C) or 5′ (D) orientation isomer. (o), 20-mer; (1), 21-mer; (2), 22-mer; (b), 23-mer. (E) The plot showing the relative mobility K versus interadduct distance in bp for the oligomers 20-23 bp long containing the CL of JM118, 5′ (O) or (3′) (b) orientational isomer with a total length of 130 bp. The experimental points represent the average of three independent electrophoresis experiments. The curves represent the best fit of these experimental points to the equation K ) ad2 + bd + c (64).

Figure 10. In vitro DNA repair synthesis assay. Repair synthesis of the extract prepared from the repair-proficient HeLa cell line used as substrates nonmodified pBR322 plasmid and pUC19 plasmid nonmodified or modified at rb ) 0.03 by JM118 or cisplatin. (A) Results of a typical experiment. Top panel, autoradiogram of the gel showing the incorporation of [R-32P]dCTP; bottom panel, a photograph of the EtBr stained gel. Lanes: 1, nonmodified pBR322 plus pUC19 plasmids; 2, nonmodified pBR322 plus pUC19 modified by cisplatin; 3, nonmodified pBR322 plus pUC19 modified by JM118. (B) Incorporation of dCTP into nonmodified or platinated pUC19 plasmid. For all quantifications representing the mean values of three separate experiments, incorporation of radioactive material is corrected for the relative DNA content in each band. The radioactivity associated with the incorporation of [R-32P]dCTP into DNA modified by cisplatin was taken as 100%. Values shown in the graph are the means ((SEM) of three separate experiments, each conducted with four replicates.

sequence of NF-κB) globally modified by JM118 or cisplatin for 24 h (rb ) 0.024) and 5′-end-labeled at its top strand was used. Ternary DNA-PtII-protein cross-linking efficiency was assessed by SDS/PAGE shift assay. Fractions were detected by SDS/PAGE with significantly retarded mobility (Figures 11B,C, lanes 5,6) compared with that of the free probes (in absence of proteins) (Figures 11B,C, lanes 1-3). These more slowly migrating fractions were eliminated after treatment with NaCN or proteinase K converting them to those of the unmodified probes (not shown). These results suggest that the species is a

Figure 11. Formation of ternary DNA-PtII-protein complexes of unmodified and platinated oligodeoxyribonucleotide duplexes of 40 bp (B) or 21 bp (C) (see Figure 11A for their nucleotide sequences) with KF- (B) or NF-κB (C) assessed by SDS/PAA gel electrophoresis, globally modified by JM118 or cisplatin. The 40 bp duplex was globally modified at rb ) 0.0125, whereas the 21 bp duplex was at rb ) 0.024. Lanes: 1-3, the duplex unplatinated or modified by JM118 or cisplatin, respectively, in the absence of the protein; 4-6, the duplex unplatinated or modified by JM118 or cisplatin, respectively, incubated with the protein for 24 h at 25 °C. See the text for other details.

protein-DNA CL tethered by platinum-DNA and platinumprotein coordination bonds. While the proteinase K and NaCN experiments clearly indicate that protein is the species crosslinked to DNA, the amino acids participating in the cross-linking reaction have not been determined. Importantly, the amount of radioactivity associated with the bands corresponding to DNA-PtII-protein CLs formed by JM118 was higher than that by cisplatin (cf. Figure 11B,C, lanes 5,6 and Table 3) demonstrating that JM118 exhibited a significantly higher efficiency (by ca. 70%) to form ternary DNA-PtII-protein CLs than cisplatin.

1840

Chem. Res. Toxicol., Vol. 23, No. 11, 2010

Table 3. Formation of the DNA-PtII-Protein Cross-Links of KF- or NF-KB with Platinated Oligodeoxyribonucleotide Duplexesa JM118 cisplatin

KF- (%)

NF-κB (%)

15.7 ( 0.4 9.1 ( 0.2

17.8 ( 0.3 10.6 ( 0.2

a The 40-bp duplex was used in the experiments with KF-, whereas the 21-bp duplex was used in the experiments with NF-κB (see Figure 11A for their nucleotide sequences). The duplexes were globally modified by cisplatin or JM118 (40-bp duplex, rb ) 0.0125; and 21-bp duplex, rb ) 0.024). Formation of CLs was assessed by SDS/PAA gel electrophoresis. Each value represents the average of four samples, and standard errors are indicated.

Discussion The rate-limiting step for the initial binding of cisplatin and its bifunctional mononuclear analogues to DNA is hydrolysis (aquation) of the first chloride ion, after which the complexes coordinate primarily to the N7 positions of purine bases to form monofunctional adducts (55). These monofunctional adducts subsequently react with a second nucleophile, forming primarily 1,2-intrastrand CLs between neighboring purine bases in vitro. We quantified in the present work the rate of the hydrolysis of the first chloride ion of JM118. JM118 and cisplatin in the dichlorido forms [aged in NaCl (0.1 M)] were quickly transferred into the medium in which the concentration of NaCl was radically reduced (5 mM), and the decomposition of these PtII complexes involving displacement of the chloride ligand was assayed by DPP (56). The rate of the aquation of the first chloride ion of JM118 (very likely trans to cyclohexylamine (67)) was considerably lower than that of cisplatin (the time at which this aquation reached 50% was 2.1-fold longer than that for cisplatin). Hence, it is possible that differences between some features of DNA binding modes of JM118 and cisplatin may also arise from differences in the susceptibility of these PtII analogues to replace the first chloride ion by the water molecule in the platinum coordination sphere, or in other words, from differences in the stability of their dichlorido forms. Consistent with the lower aquation of the first chloride ion of JM118 was the lower rate of the reaction of JM118 with double-helical DNA in comparison with that of cisplatin (Table 1). Results obtained earlier (68) or some results of the present work (Table 2) do not reveal substantial differences if the lesions were induced in DNA by its global modification by JM118 or cisplatin and were analyzed by the biophysical and biochemical techniques used in the present work (see also Figures 2-7). Yet, there are some indicia suggesting differences in the character of the lesions induced in DNA by these two bifunctional PtII complexes. For instance, some reports suggest that the affinity of HMGB1 protein, which is known to mediate antitumor effects of conventional cisplatin, to JM118-induced adducts is considerably reduced, compared with adducts formed by cisplatin (23, 24). Also, interestingly, in contrast to cisplatin, JM118-induced adducts are not recognized by DNA mismatch repair proteins (25), whereas these adducts are more effective at blocking translesion synthesis by several eukaryotic DNA polymerases (23). Not least, the hydrogen bonding between the amine ligand and the 5′-phosphate group, previously identified as being important in the major cisplatin adducts with DNA, can be disrupted by the replacement of the NH3 group in cisplatin by the cyclohexylamine ligand (27), which may be a factor responsible for the different character of DNA lesions induced by JM118 and cisplatin. Moreover, properties of DNA adducts of JM118 were also probed in the present work by using the assay that makes it possible to monitor DNA repair synthesis

KostrhunoVa et al.

in vitro. JM118 adducts induced a considerably lower level of repair synthesis than the adducts of cisplatin (by ∼30%, Figure 10B). Thus, this result is consistent with the thesis that some features of DNA adducts of JM118 are different in comparison with the adducts of cisplatin. Moreover, it has been shown (69) that removal of platinum adducts from leukocyte DNA of patients who received cisplatin or carboplatin was directly related to clinical resistance to the cisplatin/carboplatin treatment regimen. It is therefore possible that the factors responsible for a higher potency of JM118 compared with cisplatin in a number of cisplatin resistant human tumor cells (16-18) also involve enhanced resistance of DNA adducts of JM118 to DNA repair. Also, interestingly, we show in the present work that in cellfree media JM118 forms DNA-PtII-protein CLs more effectively than cisplatin (Figure 11 and Table 3). Hence, also this observation represents support for the thesis that the DNA binding mode of JM118 is different from that of cisplatin. Among the alterations of secondary and tertiary structures of DNA to which it may be subjected, the role of intrinsic bending and unwinding of DNA is increasingly recognized as being potentially important in regulating replication, transcription, and repair functions through specific DNA-protein interactions. For major 1,2-GG intrastrand CLs of cisplatin, the structural details responsible for bending and subsequent protein recognition have recently been elucidated (43, 64, 70). Given the recent advances in our understanding of the structural basis for the bending of DNA caused by cisplatin, it was of considerable interest to examine how the character and orientation (3′ or 5′) of a carrier amine in the major 1,2-GG intrastrand adduct of JM118 affects conformational properties of DNA, such as bending and unwinding. Interestingly, we find in the present work that the DNA bending toward the major groove of 26-28° due to the single, site-specific 1,2-GG intrastrand CLs of JM118 is lower than that found for the same adduct of cisplatin using the same electrophoresis gel shift methodology (61, 64). However, unwinding of 11-12° is almost identical. In addition, the results of these experiments also show that bending angles in DNA due to the major adduct of JM118 are slightly affected by the orientation of the cyclohexylamine ligand toward the 3′ or 5′ direction of the duplex (28° versus 26°, respectively). Interestingly, the crystal structure of the isomer of the 1,2-GG intrastrand CL of JM118 having the cyclohexylamine ligand directed toward the 3′-end of the platinated strand revealed a higher global bend angle (∼38°) (71). This difference suggests that the form of platinated-DNA in the gel differs from that in the crystal lattice. Also, interestingly, a band arising from small DNA circles that migrate close to the top of the gel can be observed only for 22-bp multimers of duplexes containing the 3′ but not the 5′ orientational isomer of the 1,2-GG intrastrand CL of JM118 (Figure 9B). It is because only the duplexes containing these adducts with the cyclohexylamine ligand in 3′ orientation of the cyclohexylamine ligand phase the platinum induced bends sufficiently well to allow the ends of the polymers to approach one another with the proper orientation to close covalently (64). Structural studies have indicated that a hydrogen bonding interaction occurs between the 5′-phosphate and the ammine ligand of cisplatin (72). In addition, the orientational isomer of the 1,2-GG intrastrand CL of JM118 having the cyclohexyl group directed toward the 3′ end of the platinated strand is less disruptive to the hydrogen bonding between the NH3 ligand and the 5′-phosphate group (27). It is possible that the different tendencies of the 3′ and 5′ orientational isomers of the 1,2-GG intrastrand CL of JM118 to form circles in the phasing assay

DNA Binding Mode of JM118

experiment (Figure 9B) arises from the different abilities of the two isomers to form hydrogen bonding interactions with the 5′-phosphate. The cytotoxicity of cisplatin and its analoguess can be potentiated by the binding of nuclear proteins to DNA adducts of these metallodrugs (73). For instance, HMG domain proteins play a role in sensitizing cells to cisplatin recognizing and binding to 1,2-intrastrand DNA CLs between purine residues (70, 73). It has been suggested that shielding cisplatin-DNA adducts from repair or titrating these proteins away from their transcriptional regulatory function (74) could be clues for how these proteins are involved in the antitumor activity. An important structural motif recognized by HMG domain proteins on DNA modified by cisplatin is a stable, directional bend of the helix axis (74). As it was shown previously (23, 24) the affinity of HMGB1 protein to the 1,2-GG intrastrand CL of JM118 is considerably lower than that to the same adduct of cisplatin. Hence, cellular sensitivity to JM118 is likely to be dependent on the HMG-domain and probably other structurespecific recognition proteins not involved in DNA repair much less than cellular sensitivity to cisplatin. The lower affinity of HMG-domain proteins to the CLs of JM118 is consistent with the view that the cyclohexylamine ligand could restrict the bending angle of the 1,2-GG intrastrand CL of JM118. In addition, when HMGB1 protein binds to the 1,2-GG intrastrand CL of cisplatin, the protein induces further bending of the DNA (70, 74). Thus, it is also possible that the bulky cyclohexylamine group in the 1,2-GG intrastrand CL of JM118 located in the major groove of DNA restricts the additional DNA bending toward the major groove, required for HMGB1 binding, more than in the CL of cisplatin. In conclusion, the results of the present work provide additional evidence that the DNA binding mode of JM118 is in several aspects different from the DNA binding mode of cisplatin. Hence, the results of the present work may expand the theoretical background needed to understand more fully why some cisplatin resistance may be overcome by JM118 and satraplatin. Acknowledgment. This research was supported by the Ministry of Education of the CR (MSMT LC06030, 6198959216, ME08017, ME10066, OC08003, and OC09018), the Academy of Sciences of the Czech Republic (Grants KAN200200651, M200040901, AV0Z50040507, and AV0Z50040702), the Grant Agency of the Academy of Sciences of the CR (IAA400040803), and the Grant Agency of the CR (P301/10/0598). J.K. is an international research scholar of the Howard Hughes Medical Institute. We also acknowledge that our participation in the EU COST Action D39 has enabled us to exchange regularly their most recent ideas in the field of anticancer metallodrugs with several European colleagues.

References (1) Johnson, N. P., Butour, J.-L., Villani, G., Wimmer, F. L., Defais, M., Pierson, V., and Brabec, V. (1989) Metal antitumor compounds: The mechanism of action of platinum complexes. Prog. Clin. Biochem. Med. 10, 1–24. (2) Jamieson, E. R., and Lippard, S. J. (1999) Structure, recognition, and processing of cisplatin-DNA adducts. Chem. ReV. 99, 2467–2498. (3) Vrana, O., Brabec, V., and Kleinwa¨chter, V. (1986) Polarographic studies on the conformation of some platinum complexes: relations to anti-tumour activity. Anti-Cancer Drug Des. 1, 95–109. (4) Farrell, N., Kelland, L. R., Roberts, J. D., and Van Beusichem, M. (1992) Activation of the trans geometry in platinum antitumor complexes: A Survey of the cytotoxicity of trans complexes containing planar ligands in murine-L1210 and human tumor panels and studies on their mechanism of action. Cancer Res. 52, 5065–5072.

Chem. Res. Toxicol., Vol. 23, No. 11, 2010 1841 (5) Aris, S. M., and Farrell, N. P. (2009) Towards antitumor active transplatinum compounds. Eur. J. Inorg. Chem. 2009, 1293–1302. (6) Hollis, L. S., Sundquist, W. I., Burstyn, J. N., Heiger-Bernays, W. J., Bellon, S. F., Ahmed, K. J., Amundsen, A. R., Stern, E. W., and Lippard, S. J. (1991) Mechanistic studies of a novel class of trisubstituted platinum(II) antitumor agents. Cancer Res. 51, 1866–1875. (7) Balcarova, Z., Kasparkova, J., Zakovska, A., Novakova, O., Sivo, M. F., Natile, G., and Brabec, V. (1998) DNA interactions of a novel platinum drug, cis-[PtCl(NH3)2(N7-acyclovir)]+. Mol. Pharmacol. 53, 846–855. (8) Lovejoy, K. S., Todd, R. C., Zhang, S. Z., McCormick, M. S., D’Aquino, J. A., Reardon, J. T., Sancar, A., Giacomini, K. M., and Lippard, S. J. (2008) cis-Diammine(pyridine)chloroplatinum(II), a monofunctional platinum(II) antitumor agent: Uptake, structure, function, and prospects. Proc. Natl. Acad. Sci. U.S.A. 105, 8902–8907. (9) Farrell, N. (2004) Polynuclear Platinum Drugs, in Metal Ions in Biological Systems (Sigel, A., and Sigel, H., Eds.) pp251-296, Marcel Dekker, Inc., New York. (10) Wheate, N. J., and Collins, J. G. (2005) Multi-nuclear platinum drugs: A new paradigm in chemotherapy. Curr. Med. Chem.-Anti-Cancer Agents 5, 267–279. (11) Hall, M. D., Mellor, H. R., Callaghan, R., and Hambley, T. W. (2007) Basis for design and development of platinum(IV) anticancer complexes. J. Med. Chem. 50, 3403–3411. (12) Bednarski, P. J., Mackay, F. S., and Sadler, P. (2007) Photoactivatable platinum complexes. Anti-Cancer Agents Med. Chem. 7, 75–93. (13) Wong, E., and Giandomenico, C. M. (1999) Current status of platinumbased antitumor drugs. Chem. ReV. 99, 2451–2466. (14) Brabec, V., and Kasparkova, J. (2005) DNA Interactions of Platinum Anticancer Drugs. Recent Advances and Mechanisms of Action, in Metal Compounds in Cancer Chemotherapy (Perez-Martin, J.-M., Fuertes, M. A., and Alonso, C., Eds.) pp 187-218, Research Signpost, Trivandrum, India. (15) Raynaud, F. I., Boxall, F. E., Goddard, P., Barnard, C. F., Murrer, B. A., and Kelland, L. R. (1996) Metabolism, protein binding and in vivo activity of the oral platinum drug JM216 and its biotransformation products. Anticancer Res. 16, 1857–1862. (16) O’Neill, C. F., Hunakova, L., and Kelland, L. R. (1999) Cellular pharmacology of cis and trans pairs of platinum complexes in cisplatinsensitive and -resistant human ovarian carcinoma cells. Chem.-Biol. Interact 123, 11–29. (17) Fokkema, E., Groen, H. J. M., Helder, M. N., deVries, E. G. E., and Meijer, C. (2002) JM216-, JM118-, and cisplatin-induced cytotoxicity in relation to platinum-DNA adduct formation, glutathione levels and p53 status in human tumour cell lines with different sensitivities to cisplatin. Biochem. Pharmacol. 63, 1989–1996. (18) Wosikowski, K., Lamphere, L., Unteregger, G., Jung, V., Kaplan, F., Xu, J., Rattel, B., and Caligiuri, M. (2007) Preclinical antitumor activity of the oral platinum analog satraplatin. Cancer Chemother. Pharmacol. 60, 589–600. (19) Kelland, L. (2007) Broadening the clinical use of platinum drug-based chemotherapy with new analogues: satraplatin and picoplatin. Expert Opin. InVest. Drugs 16, 1009–1021. (20) Hall, M. D., and Hambley, T. W. (2002) Platinum(IV) antitumour compounds: their bioinorganic chemistry. Coord. Chem. ReV. 232, 49–67. (21) McA’Nulty, M. M., and Lippard, S. J. (1996) The HMG-domain protein Ixr1 blocks excision repair of cisplatin-DNA adducts in yeast. Mutat. Res. 362, 75–86. (22) Jung, Y., and Lippard, S. J. (2007) Direct cellular responses to platinum-induced DNA damage. Chem. ReV. 107, 1387–1407. (23) Vaisman, A., Lim, S. E., Patrick, S. M., Copeland, W. C., Hinkle, D. C., Turchi, J. J., and Chaney, S. G. (1999) Effect of DNA polymerases and high mobility group protein 1 on the carrier ligand specificity for translesion synthesis past platinum-DNA adducts. Biochemistry 38, 11026–11039. (24) Wei, M., Cohen, S. M., Silverman, A. P., and Lippard, S. J. (2001) Effects of spectator ligands on the specific recognition of intrastrand platinum-DNA cross-links by high mobility group box and TATAbinding proteins. J. Biol. Chem. 276, 38774–38780. (25) Fink, D., Nebel, S., Aebi, S., Zheng, H., Cenni, B., Nehme, A., Christen, R. D., and Howell, S. B. (1996) The role of DNA mismatch repair in platinum drug resistance. Cancer Res. 56, 4881–4886. (26) Choy, H., Park, C., and Yao, M. (2008) Current status and future prospects for satraplatin, an oral platinum analogue. Clin. Cancer Res. 14, 1633–1638. (27) Hartwig, J. F., and Lippard, S. J. (1992) DNA binding properties of cis-Pt(NH3)(C6H11NH2)Cl2, a metabolite of an orally active platinum anticancer drug. J. Am. Chem. Soc. 114, 5646–5654. (28) Giandomenico, C. M., Abrams, M. J., Murrer, B. A., Vollano, J. F., Bernard, C. F. J., Harrap, K. R., Goddard, P. M., Kelland, L. R., and Morgan, S. E. (1991) Synthesis and Reactions of a New Class of Orally Active Pt(IV) Antitumor Complexes, in Platinum and Other Metal

1842

(29)

(30) (31) (32) (33)

(34) (35) (36)

(37)

(38)

(39) (40)

(41) (42) (43) (44)

(45)

(46) (47) (48) (49)

(50)

(51) (52)

Chem. Res. Toxicol., Vol. 23, No. 11, 2010

Coordination Compounds in Cancer Chemotherapy (Howell, S. B., Ed.) pp93-100, Plenum Press, New York. Giandomenico, C. M., Abrams, M. J., Murrer, B. A., Vollano, J. F., Rheinheimer, M. I., Wyer, S. B., Bossard, G. E., and Higgins, J. D. (1995) Carboxylation of kinetically inert platinum(IV) hydroxy complexes: An entree into orally-active platinum(IV) antitumor agents. Inorg. Chem. 34, 1015–1021. Brabec, V., and Palecek, E. (1970) The influence of salts and pH on polarographic currents produced by denatured DNA. Biophysik 6, 290–300. Brabec, V., and Palecek, E. (1976) Interaction of nucleic acids with electrically charged surfaces. II. Conformational changes in doublehelical polynucleotides. Biophys. Chem. 4, 76–92. Manley, J. L., Fire, A., Cano, A., Sharp, P. A., and Gefter, M. L. (1980) DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc. Natl. Acad. Sci. U.S.A. 77, 3855–3859. Reardon, J. T., Vaisman, A., Chaney, S. G., and Sancar, A. (1999) Efficient nucleotide excision repair of cisplatin, oxaliplatin, and bisaceto-ammine-dichloro-cyclohexylamine-platinum(IV) (JM216) platinum intrastrand DNA diadducts. Cancer Res. 59, 3968–3971. Kim, S. D., Vrana, O., Kleinwa¨chter, V., Niki, K., and Brabec, V. (1990) Polarographic determination of subnanogram quantities of free platinum in reaction mixture with DNA. Anal. Lett. 23, 1505–1518. Brabec, V., Sip, M., and Leng, M. (1993) DNA conformational distortion produced by site-specific interstrand cross-link of transdiamminedichloroplatinum(II). Biochemistry 32, 11676–11681. Kasparkova, J., Mellish, K. J., Qu, Y., Brabec, V., and Farrell, N. (1996) Site-specific d(GpG) intrastrand cross-links formed by dinuclear platinum complexes. Bending and NMR studies. Biochemistry 35, 16705–16713. Brabec, V., and Leng, M. (1993) DNA interstrand cross-links of transdiamminedichloroplatinum(II) are preferentially formed between guanine and complementary cytosine residues. Proc. Natl. Acad. Sci. U.S.A. 90, 5345–5349. Farrell, N., Qu, Y., Feng, L., and Van Houten, B. (1990) Comparison of chemical reactivity, cytotoxicity, interstrand cross-linking and DNA sequence specificity of bis(platinum) complexes containing monodentate or bidentate coordination spheres with their monomeric analogues. Biochemistry 29, 9522–9531. Butour, J. L., and Macquet, J. P. (1977) Differentiation of DNAplatinum complexes by fluorescence. The use of an intercalating dye as a probe. Eur. J. Biochem. 78, 455–463. Butour, J. L., Alvinerie, P., Souchard, J. P., Colson, P., Houssier, C., and Johnson, N. P. (1991) Effect of the amine nonleaving group on the structure and stability of DNA complexes with cis-[Pt(RNH2)2(NO3)2]. Eur. J. Biochem. 202, 975–980. Keck, M. V., and Lippard, S. J. (1992) Unwinding of supercoiled DNA by platinum ethidium and related complexes. J. Am. Chem. Soc. 114, 3386–3390. Koo, H. S., Wu, H. M., and Crothers, D. M. (1986) DNA bending at adenine.thymine tracts. Nature 320, 501–506. Bellon, S. F., and Lippard, S. J. (1990) Bending studies of DNA sitespecifically modified by cisplatin, trans-diamminedichloroplatinum(II) and cis-Pt(NH3)2(N3-cytosine)Cl+. Biophys. Chem. 35, 179–188. Kasparkova, J., Farrell, N., and Brabec, V. (2000) Sequence specificity, conformation, and recognition by HMG1 protein of major DNA interstrand cross-links of antitumor dinuclear platinum complexes. J. Biol. Chem. 275, 15789–15798. Zerzankova, L., Kostrhunova, H., Vojtiskova, M., Novakova, O., Suchankova, T., Lin, M., Guo, Z., Kasparkova, J., and Brabec, V. (2010) Mechanistic insights into antitumor effects of new dinuclear cis PtII complexes containing aromatic linkers. Biochem. Pharmacol. 80, 344–351. Wood, R. D., Robins, P., and Lindahl, T. (1988) Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts. Cell 53, 97–106. Manley, J. L., Fire, A., Samuels, M., and Sharp, P. A. (1983) In Vitro transcription: Whole-cell extract. Methods Enzymol. 101, 568–582. Siddik, Z. H. (2003) Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22, 7265–7279. Coluccia, M., Nassii, F., Loseto, F., Boccarelli, A., Marigio´, M. A., Giordano, D., Intini, F. P., Caputo, P., and Natile, G. (1993) A transplatinum complex showing higher antitumor activity than the cis congeners. J. Med. Chem. 36, 510–512. Zerzankova, L., Suchankova, T., Vrana, O., Farrell, N. P., Brabec, V., and Kasparkova, J. (2010) Conformation and recognition of DNA modified by a new antitumor dinuclear PtII complex resistant to decomposition by sulfur nucleophiles. Biochem. Pharmacol. 79, 112–121. Brabec, V., Vrana, O., Novakova, O., Kleinwachter, V., Intini, F. P., Coluccia, M., and Natile, G. (1996) DNA adducts of antitumor trans[PtCl2(E-imino ether)2]. Nucleic Acids Res. 24, 336–341. Zakovska, A., Novakova, O., Balcarova, Z., Bierbach, U., Farrell, N., and Brabec, V. (1998) DNA interactions of antitumor trans[PtCl2(NH3)(quinoline)]. Eur. J. Biochem. 254, 547–557.

KostrhunoVa et al. (53) Brabec, V., Kasparkova, J., Vrana, O., Novakova, O., Cox, J. W., Qu, Y., and Farrell, N. (1999) DNA modifications by a novel bifunctional trinuclear platinum Phase I anticancer agent. Biochemistry 38, 6781–6790. (54) Brabec, V., Kleinwa¨chter, V., Butour, J. L., and Johnson, N. P. (1990) Biophysical studies of the modification of DNA by antitumour platinum coordination complexes. Biophys. Chem. 35, 129–141. (55) Bancroft, D. P., Lepre, C. A., and Lippard, S. J. (1990) 195Pt NMR kinetic and mechanistic studies of cis-diamminedichloroplatinum and trans-diamminedichloroplatinum(II) binding to DNA. J. Am. Chem. Soc. 112, 6860–6871. (56) Vrana, O., Kleinwa¨chter, V., and Brabec, V. (1984) Polarographic activity of the antitumor drug cis-dichlorodiammineplatinum(II). The effect of hydrolysis and trans-isomerization of the drug. Experientia 40, 446–451. (57) Cohen, S. M., and Lippard, S. J. (2001) Cisplatin: From DNA damage to cancer chemotherapy. Prog. Nucleic Acid Res. Mol. Biol. 67, 93–130. (58) Malina, J., Novakova, O., Vojtiskova, M., Natile, G., and Brabec, V. (2007) Conformation of DNA GG intrastrand cross-link of antitumor oxaliplatin and its enantiomeric analog. Biophys. J. 93, 3950–3962. (59) Kostrhunova, H., and Brabec, V. (2000) Conformational analysis of site-specific DNA cross-links of cisplatin-distamycin conjugates. Biochemistry 39, 12639–12649. (60) Malina, J., Hofr, C., Maresca, L., Natile, G., and Brabec, V. (2000) DNA interactions of antitumor cisplatin analogs containing enantiomeric amine ligands. Biophys. J. 78, 2008–2021. (61) Stehlikova, K., Kostrhunova, H., Kasparkova, J., and Brabec, V. (2002) DNA bending and unwinding due to the major 1,2-GG intrastrand cross-link formed by antitumor cis-diamminedichloroplatinum(II) are flanking-base independent. Nucleic Acids Res. 30, 2894–2898. (62) Kasparkova, J., Zehnulova, J., Farrell, N., and Brabec, V. (2002) DNA interstrand cross-links of the novel antitumor trinuclear platinum complex BBR3464. Conformation, recognition by high mobility group domain proteins, and nucleotide excision repair. J. Biol. Chem. 277, 48076–48086. (63) Kasparkova, J., Novakova, O., Farrell, N., and Brabec, V. (2003) DNA binding by antitumor trans-[PtCl2(NH3)(thiazole)]. Protein recognition and nucleotide excision repair of monofunctional adducts. Biochemistry 42, 792–800. (64) Bellon, S. F., Coleman, J. H., and Lippard, S. J. (1991) DNA unwinding produced by site-specific intrastrand cross- links of the antitumor drug cis-diamminedichloroplatinum(II). Biochemistry 30, 8026–8035. (65) Rice, J. A., Crothers, D. M., Pinto, A. L., and Lippard, S. J. (1988) The major adduct of the antitumor drug cis-diamminedichloroplatinum(II) with DNA bends the duplex by ∼40° toward the major groove. Proc. Natl. Acad. Sci. U.S.A. 85, 4158–4161. (66) Ulanovsky, L., Bodner, M., Trifonov, E. N., and Choder, M. (1986) Curved DNA: Design, synthesis, and circularization. Proc. Natl. Acad. Sci. U.S.A. 83, 862–866. (67) Barton, S. J., Barnham, K. J., Frey, U., Habtemariam, A., Sue, R. E., and Sadler, P. J. (1999) 1H,15N N.M.R. kinetic studies of reactions of cis- and trans-PtCl2(15NH3))(c-(C6H1115NH2)) with guanosine 5′monophosphate. Aust. J. Chem. 52, 173–177. (68) Mellish, K. J., Barnard, C. F. J., Murrer, B. A., and Kelland, L. R. (1995) DNA-binding properties of novel cis- and trans platinum-based anticancer agents in 2 human ovarian carcinoma cell lines. Int. J. Cancer 62, 717–723. (69) Parker, R. J., Dimery, I. W., Dabholkar, M., Vionnet, J., and Reed, E. (1993) Platinum-DNA adduct in head and neck-cancer patients receiving cisplatin and carboplatin chemotherapy. Int. J. Oncol. 3, 331–335. (70) Ohndorf, U. M., Rould, M. A., He, Q., Pabo, C. O., and Lippard, S. J. (1999) Basis for recognition of cisplatin-modified DNA by highmobility-group proteins. Nature 399, 708–712. (71) Silverman, A. P., Bu, W. M., Cohen, S. M., and Lippard, S. J. (2002) 2.4-A crystal structure of the asymmetric platinum complex {Pt(ammine)(cyclohexylamine)}2+ bound to a dodecamer DNA complex. J. Biol. Chem. 277, 49743–49749. (72) Sherman, S. E., Gibson, D., Wang, A. H. J., and Lippard, S. J. (1988) Crystal and molecular structure of cis-[Pt(NH3)2{d(pGpG)}], the principal adduct formed by cis-diamminedichloroplatinum(II) with DNA. J. Am. Chem. Soc. 110, 7368–7380. (73) Kartalou, M., and Essigmann, J. M. (2001) Recognition of cisplatin adducts by cellular proteins. Mutat. Res. 478, 1–21. (74) Zamble, D. B., and Lippard, S. J. (1999) The Response of Cellular Proteins to Cisplatin-Damaged DNA, in Cisplatin. Chemistry and Biochemistry of a Leading Anticancer Drug (Lippert, B., Ed.) pp 73110, VHCA, WILEY-VCH, Zu¨rich, Switzerland. (75) Fichtinger-Schepman, A. M. J., Van der Veer, J. L., Den Hartog, J. H. J., Lohman, P. H. M., and Reedijk, J. (1985) Adducts of the antitumor drug cis-diamminedichloroplatinum(II) with DNA: Formation, identification, and quantitation. Biochemistry 24, 707–713.

TX1002904