Effects of a Piperidine Ligand on DNA Modification by Antitumor

ACS2GO © 2018. ← → → ←. loading. To add this web app to the home screen open the browser option menu and tap on Add to homescreen...
0 downloads 0 Views 156KB Size
Download PDF
1424

Chem. Res. Toxicol. 2003, 16, 1424-1432

Effects of a Piperidine Ligand on DNA Modification by Antitumor Cisplatin Analogues Jana Kasparkova,*,† Olga Novakova,† Yousef Najajreh,‡ Dan Gibson,‡,§ Jose-Manuel Perez,| and Viktor Brabec† Institute of Biophysics, Academy of Sciences of the Czech Republic, CZ-61265 Brno, Czech Republic, Department of Medicinal Chemistry and Natural Products, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel, and Departamento de Quimica Inorganica, Facultad de Ciencias, Universidad Autonoma de Madrid, 28049-Madrid, Spain Received June 25, 2003

Replacement of the ammine group in antitumor cisplatin by a heterocyclic ligand (piperidine, piperazine, or 4-picoline) results in reduction of cytotoxicity in human ovarian cancer cells. DNA is generally believed to be a major pharmacological target of antitumor platinum complexes. Therefore, we examined conformation of oligodeoxyribonucleotide duplexes containing a cross-link of cis-[PtCl2(NH3)(piperidine)], their recognition by high mobility group proteins, and nucleotide excision repair; that is, some of the processes that may mediate antitumor effects of platinum drugs. The replacement does not affect the DNA binding mode including conformational alterations and excision of the cross-links. The results suggest that in certain cancer cells the lower cytotoxicity of cis-[PtCl2(NH3)(piperidine)] might be partially associated with reduced affinity of the high mobility group proteins to the major intrastrand cross-links of this analogue relative to the same adducts of cisplatin. Besides this and a number of other biochemical factors, the reduced intracellular accumulation with subsequent effects on the level of DNA platination in the cells may also contribute to the reduced cytotoxicity of cis[PtCl2(NH3)(piperidine)]. The results support the view that the concept based on the design of the complexes structurally derived from cisplatin that do not present an altered DNA binding mode may be less effective in the search for new platinum drugs that would overcome cisplatin resistance.

Introduction Since the discovery of its anticancer activity, several new analogues of cisplatin1 have been synthesized and tested for biological activity. Some of these compounds are now considered potent anticancer drugs (1). Although the precise mechanism underlying antitumor action of platinum drugs is not completely understood, they are known to bind DNA primarily by forming bifunctional adducts (2). The anticancer activity displayed by cisplatin and its analogues is usually attributed to a unique type of 1,2-GG intrastrand CL with platinum cross-linking N7 atoms of neighboring guanine residues of DNA. It has also been shown that nonleaving amine ligands of cisplatin analogues appear to modulate the antitumor properties of this class of compounds. On the other hand, * To whom correspondence should be addressed. Tel: 420-541517174. Fax: 420-541240499. E-mail: [email protected]. † Academy of Sciences of the Czech Republic. ‡ The Hebrew University of Jerusalem. § Affiliated with the David R. Bloom Center for Pharmacy at The Hebrew University of Jerusalem, Israel. | Universidad Autonoma de Madrid. 1 Abbreviations: bp, base pair; CFE, cell-free extract; cisplatin, cisdiamminedichloroplatinum(II); CL, cross-link; DEPC, diethylpyrocarbonate; DMS, dimethyl sulfate; FPLC, fast protein liquid chromatography; HMGB1a, HMGB1 domain A; HMGB1b, HMGB1 domain B; HMG, high mobility group; IC50, the concentration of the compound that afforded 50% cell killing; NER, nucleotide excision repair; PAA, polyacrylamide; pip, piperidine; rb, the number of molecules of the platinum compound bound per nucleotide residue; transplatin, transdiamminedichloroplatinum(II); TXRF, reflection X-ray fluorescence.

the trans isomer of cisplatin (transplatin) is clinically ineffective (3). We have shown in our recent papers (4-6) that replacement of one of the ammine ligands of cisplatin by a heterocyclic ligand, such as pip, piperazine, or 4-picoline, results in reduction of cytotoxicity in human ovarian cancer cell lines, both sensitive and resistant to cisplatin. This observation was somewhat surprising since the analogues of transplatin in which one ammine ligand was replaced by the heterocyclic ligand were radically more potent than the parent compound and even more interestingly the analogues of transplatin were in most cases considerably more potent than their cis congeners (6). Although the mechanism underlying antitumor activity of platinum compounds is a complex process involving a number of factors, it is generally accepted that DNA is an important and major pharmacological target of platinum compounds (2). Therefore, we have also attempted in our previous work (6) to obtain some initial data on the modification of calf thymus and plasmid DNA by these analogues of cisplatin in cell-free media. These data showed that the replacement of one ammine ligand in cisplatin by pip, piperazine, or 4-picoline did not alter the overall properties of calf thymus or plasmid DNA relative to cisplatin-modified DNA. Hence, these initial data might be interpreted to mean that the reduced activity of these analogues of cisplatin may be associated with some features of the damaged DNA and/or its

10.1021/tx034128g CCC: $25.00 © 2003 American Chemical Society Published on Web 09/16/2003

DNA Modified by Cisplatin Analogues

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1425

DNA (in particular, bending and unwinding) and how these adducts are recognized by HMGB1 domain proteins and removed from DNA during in vitro NER reactions. In addition, antitumor activity of platinum compounds depends on the factors that do not operate directly at the level of the DNA adducts, such as the amount of platinum complex that can reach DNA (by affecting either the cell accumulation of the complexes or the levels of intracellular platinophiles). Therefore, we also studied the cellular uptake of cisplatin and its pip analogue and determined the amount of platinum bound to DNA in the cells treated with these platinum compounds.

Materials and Methods Figure 1. Structures of platinum complexes and sequences of the synthetic oligodeoxyribonucleotides used in the present study with their abbreviations. (A) Structures; (B) sequences. The top and bottom strands of each pair in panel B are designated top and bottom, respectively, in the text. The bold letters in the top strands of the duplexes TGGT, TGGT(21), TGGT(NER), or TGTGT(NER) indicate the platinated residues of the intrastrand CLs. The bold letters in the top strands of the duplexes CGC and TGC indicate the platinated site of the monofunctional adduct that was allowed to evolve in the interstrand CL (see the text).

cellular processing not perceptible by the methods used in the previous work or to an event that is not related to DNA binding. The overall properties of DNA, such as thermal stability or DNA conformation determined by circular dichroism spectroscopy used in our precedent work, represent a sum of the contributions from all platinum adducts making it difficult to deduce the features of the individual types of adducts. Structural details of individual DNA adducts formed by the cisplatin analogues containing pip, piperazine, or 4-picoline are not yet available. It is not clear how these adducts affect DNA conformation and how these alterations are further processed in the cells. Damaged DNA binding proteins, such as those containing for instance the HMG domain, bind with different affinities to DNA adducts of different platinum complexes (3, 7, 8). The binding of these proteins has been postulated to mediate the antitumor properties of the platinum drugs in a number of cancer cells (7). Importantly, it has been demonstrated for the 1,2-GG intrastrand CLs of several analogues of cisplatin that their affinity to HMG box proteins is changed if the ammine ligand in cisplatin is replaced by various, mostly cyclic nonleaving ligands (9, 10). It has also been demonstrated (11, 12) that DNA adducts of several platinum drugs are removed from DNA during NER reactions and that NER is also an important mechanism contributing to resistance to platinum drugs. We examined in the present work in detail short oligodeoxyribonucleotide duplexes containing a single, site specific adduct of the analogue containing the nonplanar pip ligand (Figure 1). cis-[PtCl2(NH3)(pip)] was chosen for these studies as the representative of the new platinum compounds synthesized by us recently (4-6). This choice was further sustained by the observation that among the cisplatin analogues containing the heterocyclic ligand tested in our recent work (6) the most pronounced changes in the activity in cancer cell lines were observed just for the pip analogue. We investigated how the adducts of this analogue affect the local conformation of

Chemicals. cis-[PtCl2(NH3)(pip)] (Figure 1A) was prepared by the methods described in detail previously (5). Cisplatin was obtained from Sigma (Prague, Czech Republic). 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. The synthetic oligodeoxyribonucleotides (Figure 1B) were synthesized and purified as described previously (13). Plasmid pSP73KB [2455 bps (14)] was isolated according to standard procedures and banded twice in CsCl/EtBr equilibrium density gradients. Expression and purification of the rat HMGB1a (residues 1-84) and HMGB1b (residues 85-180) were carried out as described (15). Restriction endonucleases, T4 DNA ligase, and T4 polynucleotide kinase were purchased from New England Biolabs (Beverly, MA). Acrylamide, bis(acrylamide), urea, thiourea, and NaCN were from Merck KgaA (Darmstadt, Germany). DMS, KMnO4, DEPC, KBr, and KHSO5 were from Sigma. [γ-32P]ATP was from Amersham (Arlington Heights, IL). Proteinase K, RNaseA, and ATP were from Boehringer (Mannheim, Germany). Sequence Specificity of DNA Adducts. To characterize specific nucleotide preference in the DNA template, RNA transcription mapping was employed. Transcription of the NdeI/ HpaI restriction fragment of pSP73KB DNA with T7 RNA polymerase and electrophoretic analysis of transcripts was performed according to the protocols recommended by Promega [Promega Protocols and Applications, 43-46 (1989/1990)] and previously described in detail (14). Platinations of Oligonucleotides. The duplexes containing single CL of cisplatin or cis-[PtCl2(NH3)(pip)] [1,2-GG, 1,3-GTG intrastrand CL, or interstrand CL (in the duplexes shown in Figure 1B)] were prepared as described (14, 16-18). The unmodified or CLs containing duplexes used in the studies of recognition by HMGB1 domain proteins were purified by electrophoresis on native 15% PAA gel [mono:bis(acrylamide) ratio ) 29:1]. Other details have been described previously (13, 14, 19). Hydroxyl Radical Footprinting of Interstrand CLs. Platinated (or unplatinated) oligodeoxyribonucleotide duplexes that had either the top or the bottom strand 32P-labeled at the 5′-end were dissolved in the medium of 50 mM NaCl and 10 mM Tris-HCl, pH 7.5, so that their concentration was 6 nM. The cleavage of the phosphodiesteric bonds was performed by incubating the duplexes in 0.04 mM Fe(NH4)2(SO4)2, 0.08 mM EDTA, 0.03% H2O2, and 2 mM sodium ascorbate for 5 min at 20 °C. The reaction was stopped by adding 15 mM thiourea, 3 mM EDTA, 0.3 M sodium acetate, and 0.3 mg tRNA/mL. After precipitation, the samples were loaded onto a 24% denaturing PAA/8 M urea gel. Maxam-Gilbert sequencing reactions were run in parallel. Chemical Modifications. The modification by KMnO4, DEPC, and KBr/KHSO5 was performed as described previously (19). The strands of the duplexes were 5′-end-labeled with [γ-32P]ATP. In the case of the platinated oligonucleotides, the platinum complex was removed after reaction of the DNA with the probe by incubation with 0.2 M NaCN (pH 11) at 45 °C for 10 h in the dark.

1426 Chem. Res. Toxicol., Vol. 16, No. 11, 2003 Ligation and Electrophoresis of Oligonucleotides. Details of these experiments were as described in previously published papers (20, 21). Gel Mobility Shift Assay. Reactions with HMG Domain Proteins. The 5′-end-labeled 20 bp oligonucleotide duplexes (TGGT shown in Figure 1B) either unplatinated (controls) or containing the central platinum CL in their top strands were used, and their reaction with HMG domain proteins was performed and analyzed as described previously (22). Nucleotide Excision Assay. The 148 bp substrates containing single, central intrastrand, or interstrand CL were assembled from three oligonucleotide duplexes as described previously (18, 23, 24). Oligonucleotide excision reactions were performed in CFEs prepared from the HeLa S3 and CHO AA8 cell lines as described (12). These extracts were kindly provided by J. T. Reardon and A. Sancar from the University of North Carolina (Chapel Hill, NC). In vitro repair was measured with the excision assay using these CFEs and 148 bp linear DNA substrates (vide supra) in the same way as described previously (12). Measurements of Platinum Accumulation in CH1cisR Cells. CH1cisR cells [human ovarian tumor cells primarily resistant to cisplatin through enhanced DNA repair/tolerance (25)] were routinely cultured in DMEM medium (Dulbecco Modified Eagle’s Medium). P100 cultures plates containing exponentially growing CH1cisR cells (cell density ) 2 × 105 cells/ mL) in 10 mL of DMEM were exposed for several periods of time to 25 µM platinum drugs dissolved in DMEM medium. This drug concentration is close to the IC50 of cisplatin for 24 h of incubation in the CH1cisR line. Cells were washed with icecold PBS, scraped, and resuspended in 0.7 mL of lysis buffer containing 20 mM TrisHCl (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 for 15 min. Afterward, supernatants were treated for 3 h at 37 °C with 20 µg of proteinase K/mL. The platinum content in the samples was determined by total TXRF as described below. Experiments were carried out as four independent experiments with duplicate cultures. Determination of Platinum Binding to DNA in Culture Cells. Culture plates containing exponentially growing CH1cisR cells in DMEM medium (cell density ) 2 × 105 cells/mL) were exposed to 25 µM of the platinum drugs dissolved in DMEM. The plates were incubated for several periods of time under the conditions described above. Following drug incubation, culture medium was removed from the plates and the cell plates were washed with PBS. Subsequently, the cells were lysed with 0.7 mL of a buffer solution containing 0.15 M TrisHCl (pH 8.0), 0.1 M EDTA, and 0.1 M NaCl, incubated for 15 min at 4 °C, and centrifuged at 12 000 rpm for 15 min. Supernatants were treated for 3 h at 37 °C with 20 µg of proteinase K/mL. Afterward, supernatants were incubated for 16 h at 37 °C with 4 µL of RNase A at the concentration of 0.1 mg/mL. Finally, DNA was extracted with a volume of phenol-chloroform-isoamyl alcohol (50 + 49 + 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 in a Shimadzu UV-240 spectrophotometer and platinum bound to DNA was determined by TXRF. The data were obtained from four independent experiments with duplicate cultures. Total TXRF Measurements. The analysis by TXRF of the platinum content in biological samples was performed using a Seifert Extra-II spectrometer (Seifert, Ahrensburg, Germany). TXRF determinations were carried out according to a procedure previously reported (26, 27). The analytical sensitivity of the TXRF measurements was 0.3-22.4 ng of Pt in a solution volume of 0.1 mL, with repeatability between 2 and 8% (n ) 8, four independent experiments with duplicate cultures).

Kasparkova et al.

Results We have shown (6) that several features of the binding of cis-[PtCl2(NH3)(pip)] and cisplatin in a cell-free medium to calf thymus and plasmid DNAs, such as the rate of binding, the frequency of interstrand CLs, conformational alterations evaluated by circular dichroism spectroscopy, DNA melting curves, and unwinding of supercoiled plasmid DNA, are similar. In the present work, we expanded these analyses by studying sequence specificity of the DNA binding of cis-[PtCl2(NH3)(pip)], local conformational alterations by its major adduct, recognition of this adduct by HMG domain proteins, and its removal from DNA by NER system, i.e., the important components of the mechanism underlying antitumor activity of cisplatin that operate directly at the level of DNA. Sequence Specificity. In vitro RNA synthesis by RNA polymerases on DNA templates containing several types of bifunctional adducts of platinum complexes can be prematurely terminated at the level or in the proximity of adducts (14). Importantly, monofunctional DNA adducts of several platinum complexes are unable to terminate RNA synthesis. Cutting of pSP73KB DNA (14) by NdeI and HpaI restriction endonucleases yielded a 212 bp fragment (a substantial part of its nucleotide sequence is shown in Figure 2B). This fragment contained T7 RNA polymerase promoter [in the upper strand close to its 3′-end (Figure 2B)]. The experiments were carried out using this linear DNA fragment, modified by cisplatin and cis-[PtCl2(NH3)(pip)] at rb ) 0.005, for RNA synthesis by T7 RNA polymerase (Figure 2, lanes cisPt and cis-pip). RNA synthesis on the template modified by the platinum complexes yielded fragments of defined sizes, which indicates that RNA synthesis on these templates was prematurely terminated. The sequence analysis revealed that the major bands resulting from termination of RNA synthesis by the adducts of cisplatin and cis-[PtCl2(NH3)(pip)] were identical and appeared at guanine (G) sites and to a considerably lesser extent at adenine (A) sites. These G and A sites were mostly contained in GG or AG sites, which are preferential DNA binding sites for cisplatin. Taken together, the results of the transcription mapping experiments suggest that base sequence selectivity of cisplatin and its pip analogue is similar and that also the major adducts formed on DNA by cis-[PtCl2(NH3)(pip)] and cisplatin are identical, presumably 1,2GG or AG intrastrand CLs. Interstrand Cross-Linking. Cisplatin also forms in DNA a small amount of interstrand CLs. Despite their low abundance, these lesions are also considered relevant to the antitumor effects of cisplatin (28). These CLs are formed by cisplatin preferentially between G residues in the 5′-GC/5′-GC sequences (28). We have carried out hydroxyl radical (29) and DMS footprinting experiments (30) (Figure 3) to unambiguously identify the nature of the interstrand CLs formed by cis-[PtCl2(NH3)(pip)]. Dichloroplatinum(II) complexes react with DNA in a two step process (31). Monofunctional adducts are formed preferentially at N7 atoms of G residues. These lesions subsequently close to bifunctional CLs (intrastrand and/ or interstrand). Bearing this in mind, we designed for the present study synthetic oligodeoxyribonucleotide duplexes CGC and TGC whose sequences are shown in Figure 1B. The pyrimidine-rich top strands of these

DNA Modified by Cisplatin Analogues

Figure 2. Inhibition of RNA synthesis by T7 RNA polymerase on the NdeI/HpaI fragment of pSP73KB plasmid containing adducts of cisplatin or cis-[PtCl2(NH3)(pip)]. (A) Autoradiograms of a 6% PAA/8 M urea sequencing gel. Lanes: control, nonplatinated template; cisPt and cis-pip, DNA modified by cisplatin and cis-[PtCl2(NH3)(pip)], respectively at rb ) 0.005; A, C, G, and U, chain-terminated marker RNAs. The numbers correspond to the nucleotide sequence numbering of panel B. (B) Schematic diagram showing the portion of the sequence used to monitor inhibition of RNA synthesis by the platinum complexes. The arrow indicates the start of T7 RNA polymerase, which used as template the upper strand of NdeI/HpaI fragment of pSP73KB DNA. Full circles indicate the sites corresponding stop signals from panel A, lane cis-pip. The numbers correspond to the nucleotide numbering in the sequence map of pSP73KB plasmid.

duplexes contain a unique G residue at which the monofunctional adduct of cisplatin or its pip analogue was formed. Thus, the choice of this nucleotide allowed for a cross-linking study under competitive conditions, i.e., interstrand CLs were in principle possible: in the CGC duplex between the central G in the top strand and either complementary C or adjacent 5′ or 3′ guanine bases on the opposite strand and in the case of the TGC duplex between the central G in the top strand and complementary C or adjacent 5′ G in the bottom strand. The top strands of the duplexes containing the monofunctional adduct of cisplatin or cis-[PtCl2(NH3)(pip)] were hybridized with their complementary (bottom), 5′end 32P-labeled strands. The mixtures were incubated at 37 °C for 48 h in 0.1 M NaClO4 and finally subjected to FPLC or gel electrophoresis under denaturing (strand separating) conditions to purify interstrand cross-linked duplexes. These incubations afforded single products as revealed by FPLC and gel electrophoresis. From the observation that all fragments corresponding to the cleavage by hydroxyl radicals from the 5′-end up to the interstrand CL were detected (shown for the duplex CGC in Figure 3A) or from the systematic absence of the Maxam-Gilbert (DMS) sequencing fragments due to platination of guanine-N7, separated according to size

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1427

Figure 3. Hydroxyl radical and Maxam-Gilbert footprinting of interstrand CLs. Autoradiograms of denaturing 24% PAA/8 M urea gels of the products of the reaction between hydroxyl radicals (A) or DMS (G-specific reactions) (B) and the duplex CGC either nonmodified or containing an interstrand CL of cisplatin or cis-[PtCl2(NH3)(pip)]. The bottom strands of the duplexes were 5′-end labeled. Lanes: NoPt, unplatinated duplex; cisPt, the duplex containing interstrand CL of cisplatin; cisPt/ CN-, same as lane cisPt but with platinum removed by NaCN after methylation; cis-pip, the duplex containing interstrand CL of cis-[PtCl2(NH3)(pip)]; cis-pip/CN-, same as lane cis-pip but with platinum removed by NaCN after methylation; G, a Maxam-Gilbert specific reaction for the unplatinated duplex that had only bottom strand end labeled. For other details, see the text.

on a PAA gel (shown for the duplex CGC in Figure 3B), the exact locations of the bases involved in the interstrand CL were deduced (14, 29, 30). The interstrand adducts formed by either cisplatin or cis-[PtCl2(NH3)(pip)] in the duplexes CGC (Figure 3) and TGC (not shown) were unambiguously identified as 1,2 GG interstrand adducts involving the central G site in the top strand and G in the bottom strand adjacent 5′ to the C complementary to platinated G in the top strand. Thus, substitution of NH3 by a nonplanar pip produces “cisplatin-like” interstrand CLs. Taken together, the results of the interstrand crosslinking assay and transcription mapping experiments are consistent with the idea that the replacement of one NH3 nonleaving ligand in cisplatin by the pip has not significantly altered base sequence selectivity of the parent platinum drug or the spectrum of its DNA adducts.

1428 Chem. Res. Toxicol., Vol. 16, No. 11, 2003

Recognition by HMGB1 Proteins. An important feature of the mechanism of action of cisplatin is that the major adducts of this platinum drug (1,2-GG intrastrand CLs) are recognized by proteins containing HMG domains (3). Importantly, DNA modified by transplatin or monodentate platinum(II) compounds, such as chlorodiethylenetriamineplatinum(II) chloride or [PtCl(NH3)3]Cl, is not recognized by these cellular proteins. It has also been shown (3) that the binding of these proteins to cisplatinated DNA may mediate the antitumor effects of this platinum drug. In addition, it has also been shown (9) that the replacement of one or both ammine groups in cisplatin by various, mostly cyclic nonleaving ligands lowered affinity of HMG box proteins to the 1,2-GG intrastrand CLs. Hence, it was of great interest to examine also whether the replacement of the ammine group in cisplatin by pip could also affect affinity of HMG box proteins to this major adduct. The interactions of the HMGB1a and HMGB1b proteins with the 1,2-GG intrastrand CLs of cis-[PtCl2(NH3)(pip)] were investigated using gel mobility shift assay. In these experiments, the 20 bp duplex TGGT (Figure 1B) was modified so that it contained a single, site specific 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)] or cisplatin. As cis-[PtCl2(NH3)(pip)] contains two different nonleaving ligands, two 5′ and 3′ orientational isomers could be formed as in the case of cis-[PtCl2(NH3)(cyclohexylamine)] (32). The reactions of all oligonucleotides containing the central GG sequences (the top strands in the duplexes TGGT in Figure 1B) with cis[PtCl2(NH3)(pip)] gave single products as revealed by ion exchange FPLC and 24% PAA/8 M urea gel electrophoresis. It is possible that one orientational isomer was formed exclusively in these reactions. Alternatively, two orientational isomers were formed, but they could not be resolved by FPLC or gel electrophoresis. The binding of the HMGB1a and HMGB1b to the DNA probes containing the 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)] was detected by retardation of the migration of the radiolabeled 20 bp probes through the gel (22) (Figure 4A,B). HMGB1a and HMGB1b exhibited negligible binding to the unmodified 20 bp duplexes. As indicated by the presence of a shifted band whose intensity increases with increasing protein concentration, both HMGB1a and HMGB1b recognize the duplex containing the 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)] (Figure 4A,B). These data indicate that HMGB1a binds the probe containing the 1,2-GG intrastrand CL of cis[PtCl2(NH3)(pip)] with a relatively high affinity, although somewhat lower than to the probe containing the same adduct of cisplatin (Figure 4C). Similarly, the titration of the 20 bp duplex containing the 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)] with HMGB1b has revealed (Figure 4B) that this protein also binds to the 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)] with a lower affinity than to the probe containing 1,2-GG intrastrand CL of cisplatin (Figure 4D). The affinity of HMGB1b to these platinum lesions was, however, considerably smaller (∼50 times) than that of HMGB1a, as expected (9, 10). NER. NER is a pathway used by human cells for the removal of damaged nucleotides from DNA (33). In mammalian cells, this repair pathway is an important mechanism for the removal of bulky, helix-distorting DNA adducts, such as those generated by various

Kasparkova et al.

Figure 4. Analysis of the binding affinity of platinated 20 bp DNA probes containing 1,2-GG intrastrand CL of cisplatin or cis-[PtCl2(NH3)(pip)] to HMGB1 domain proteins in a 6% PAA gel. (A) HMGB1a; (B) HMGB1b. The oligonucleotide duplex TGGT (20 nM) was either nonplatinated (lanes 1-5) or contained 1,2-GG intrastrand CL of cisplatin (lanes 6-10) or cis[PtCl2(NH3)(pip)] (lanes 10-15). Lanes in A: 1, 6, 11, no protein; 2, 7, 12, 5 ng of HMGB1a; 3, 8, 13, 10 ng of HMGB1a; 4, 9, 14, 20 ng of HMGB1a; 5, 10, 15, 30 ng of HMGB1a. Lanes in B: 1, 6, 11, no protein; 2, 7, 12, 25 ng of HMGB1b; 3, 8, 13, 50 ng of HMGB1b; 4, 9, 14, 100 ng of HMGB1b; 5, 10, 15, 200 ng of HMGB1b. (C,D) Plots of Θ values (ratio of protein-bound duplex to total duplex × 100) obtained from electrophoretic mobility shift assays (A,B). Each data point is the mean of four binding reactions. Open and shadowed bars represent the Θ values for cisplatin and cis-[PtCl2(NH3)(pip)], respectively.

chemotherapeutics including cisplatin (34). Efficient repair of 1,2-GG or 1,3 GNG intrastrand CL of cisplatin has been reported by various NER systems including human and rodent excinucleases (11, 12). The results presented in Figure 5A, lanes 4 and 6, are consistent with these reports. The major excision fragment contains 28 nucleotides, and other primary excision fragments are 23-27 nucleotides in length, although 22-31 nucleotide long fragments are also observed (10). This range of product sizes reflects variability at both the 3′ and 5′ incision sites (12); smaller excision products are due to degradation of the primary excision products by exonucleases present in the extracts (12). Importantly, the 1,2GG intrastrand CL of cis-[PtCl2(NH3)(pip)] was also excised with a similar efficiency by both human and rodent excinucleases (shown in Figure 5A, lane 8, and 5B for the adduct repaired by rodent excinuclease). Bending, Unwinding, and Chemical Probes of DNA Conformation. Important structural motifs that attract HMG domain and NER proteins to 1,2-intrastrand CLs of cisplatin are the bending and unwinding of the helix axis (3, 23). For DNA adducts of cisplatin, the structural details responsible for bending and unwinding and subsequent protein recognition have recently been elucidated (7). Given the recent advances in our understanding of the structural basis for the bending of DNA caused by cisplatin CLs, it is also of considerable interest to examine how 1,2-GG intrastrand CLs of its pip analogue affect conformational properties of DNA such as bending and unwinding. In this work, we further performed studies on the bending and unwinding induced by single, site specific 1,2-GG intrastrand CL of

DNA Modified by Cisplatin Analogues

Figure 5. Excision of the 1,3-GTG intrastrand CL of cisplatin and 1,2-GG intrastrand CLs of cisplatin or cis-[PtCl2(NH3)(pip)] by rodent excinuclease. (A) The substrates were incubated with CHO AA8 CFE and subsequently treated overnight with NaCN prior to analysis in 10% PAA/8 M urea denaturing gel. Lanes: 1 and 2, control, unplatinated substrate; 3 and 4, the substrate containing the 1,3-GTG intrastrand CL of cisplatin; 5 and 6, the substrate containing the 1,2-GG intrastrand CL of cisplatin; 7 and 8, the substrate containing the 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)]; 1, 3, 5, and 7, no extract added; 2, 4, 6, and 8, the substrates were incubated with CHO AA8 CFE for 40 min at 30 °C. Lane 9, the 20 and 30 nucleotides markers. (B) Quantitative analysis of removal of the adducts. The columns marked as cisPt and cis-pip are for 1,2-GG intrastrand CL of cisplatin and cis-[PtCl2(NH3)(pip)], respectively. The radioactivity associated with the fragments excised from the duplex containing the adduct of cisplatin was taken as 100%. Data are the average of two independent experiments done under the same conditions; bars indicate range of excision.

cis-[PtCl2(NH3)(pip)] using electrophoretic retardation as a quantitative measure of the extent of planar curvature. The oligodeoxyribonucleotide duplexes TGGT(19-22) [19-22 bp long, whose sequences were identical (21 bp) or similar to that of the duplex TGGT(21) shown in Figure 1B; the 19 and 20 bp duplexes had one or two marginal C‚G pair deleted, respectively, while one additional T‚A pair was added to one end in the 22 bp duplex] were used for the bending and unwinding studies of the present work. Experimental details of these studies are given in our recent papers (16, 17). The DNA unwinding due to one intrastrand adduct of cis-[PtCl2(NH3)(pip)] has been found to be 13 ( 1°. Moreover, the 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)] bends the DNA by about 34° toward the major groove. Thus, the bending and local unwinding induced by 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)] are very similar to those afforded by the 1,2-GG interstrand CL of cisplatin using the same experimental procedure [32-34 and 13°, respectively (20, 35)]. Further studies of the present work focused on analysis of the distortion induced by the 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)] by chemical probes of DNA conformation. The duplex TGGT (Figure 1B) containing the single, site specific adduct was treated with several chemical agents that are used as tools for monitoring the existence of conformations other than canonical B-DNA. These agents included KMnO4, DEPC, and bromine. They react preferentially with single-stranded DNA and distorted double-stranded DNA (19). We used for this analysis exactly the same methodology described in detail in our recent papers aimed at DNA adducts of various antitumor platinum drugs (36) so that these experiments

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1429

Figure 6. CH1cisR cell uptake of platinum complexes and platinum accumulation on DNA isolated from CH1cisR cells treated with platinum complexes. (A) Uptake; each data point is the mean ( SD of four determinations from two independent experiments; cisplatin, 4; cis-[PtCl2(NH3)(pip)], O. (B) Accumulation; cisplatin, 4; cis-[PtCl2(NH3)(pip)], O. The absence of error bars indicates that the size of the calculated error bar is smaller than the symbol. For other details, see the text.

are not described in more detail in the present work. The results (not shown) indicate that this adduct induces in DNA the distortion that extends over at least 5 bp and is localized mainly at the platinated bps and those on its 5′ side. Hence, the extent of the distortion was the same as that induced by 1,2-GG intrastrand CL of cisplatin (36). Platinum Complex Uptake and Platinum Binding to DNA in Culture Cells. A possible explanation for the difference in cytotoxicity between cisplatin and cis[PtCl2(NH3)(pip)] may be associated with differential cellular uptake of the compounds. Different platinum complexes may have different cellular uptake that may affect their cytotoxicity. Results in CH1cisR cells [this cancer cell line was among those used for testing activity of the novel cisplatin analogues in which cis-[PtCl2(NH3)(pip)] exhibited a reduced activity in comparison with cisplatin (6)] showed that platinum associated with the cells treated with cis-[PtCl2(NH3)(pip)] was lower than that associated with the cells treated with cisplatin (Figure 6A). Given that significantly less cis-[PtCl2(NH3)(pip)] appears to be accumulated in cells, the question arises as to how the pip ligand affects cisplatin binding to DNA in cells (treated with this drug). Results in the Figure 6B demonstrate that cis-[PtCl2(NH3)(pip)] binds to DNA in CH1cisR cells more slowly than cisplatin. However, it has been shown (6) that in a cell-free medium cisplatin and cis-[PtCl2(NH3)(pip)] bind to DNA with approximately the same rate. Hence, it is reasonable to suggest that the slower binding of cis-[PtCl2(NH3)(pip)] to DNA in culture cells is mainly a consequence of its slower intracellular uptake.

Discussion Cisplatin damage throughout the genome leads to cell cycle arrest and cell death through apoptotic or necrotic

1430 Chem. Res. Toxicol., Vol. 16, No. 11, 2003

pathways (37). Hence, the mechanism that underlies antitumor effects of platinum compounds or resistance to these drugs is a complex process, in which modification of DNA by these compounds and its processing by cellular components play undoubtedly a very important role. Nonetheless, details of these mechanisms are not entirely understood despite intensive research in this area. Adducts formed by cisplatin distort DNA conformation but are also removed from DNA mainly by NER and recognized by a number of proteins (3), which could block DNA adducts of cisplatin from damage recognition needed for repair. In this so-called “shielding” mechanism, these adducts may thus persist, which potentiates their cytotoxicity (7, 38). The other hypothesis of the mechanism of antitumor activity of cisplatin (so-called “hijacking” mechanism) may be effective in the other type of cells in which intracellular concentration of the proteins that specifically recognize platinated DNA is relatively low. A number of proteins that exhibit an enhanced affinity to DNA modified by cisplatin, such as transcription factors (which have key roles in the cells) may be recruited by cisplatin-DNA adducts from their native binding sites, thereby disrupting fundamental cellular processes. Experimental support for the mechanisms suggested for antitumor activity of cisplatin has been recently reviewed thoroughly (see, for instance, refs 7, 39). The studies of the modification of DNA by cisplatin and its analogues in cell-free media performed to date show that the replacement of one ammine ligand in cisplatin by pip and other heterocyclic ligands such as piperazine or 4-picoline does not affect radically its binding to DNA including resulting conformational alterations. On the other hand, we demonstrate in the present work that HMG box proteins HMGB1a and HMGB1b have slightly but significantly reduced affinity, relative to the 1,2-GG intrastrand CL of cisplatin, for the same adducts of cis[PtCl2(NH3)(pip)] (Figure 4). It is reasonable to expect that the full-length HMGB1 protein, which is an abundant chromosomal protein in mammalian cells and the candidate protein for participation in the repair shielding mechanism, has a reduced affinity to the 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)] as well since the interaction between these CLs of cisplatin and full-length protein is dominated by its domain A (9, 22). In addition, we also show in the present work that in in vitro assay the 1,2-GG intrastrand CLs of cisplatin and cis-[PtCl2(NH3)(pip)] are removed from DNA by the components of mammalian NER system present in CFEs with the same efficiency (Figure 5). However, the presence of higher concentrations of HMG domain proteins, such as HMGB1 in nuclei of a certain type of tumor cells, may shield the adducts of cisplatin and its analogues and thus reduce their removal from DNA. Consistent with this conclusion is the finding that addition of HMGB1 to an in vitro NER assay inhibited the repair of DNA damage induced by cisplatin and its analogue containing 2,3diaminobutane ligand (10, 11). In this way, because of the lower affinity of HMG domain proteins to the 1,2intrastrand CLs of cis-[PtCl2(NH3)(pip)], these adducts can be shielded less efficiently than the same adducts of cisplatin and consequently removed from DNA and repaired more efficiently. In other words, in the cell lines in which the repair “shielding” mechanism is effective, the excision repair of the 1,2-intrastrand CL of

Kasparkova et al.

cis-[PtCl2(NH3)(pip)] will be more efficient. Alternatively, in the other type of cells where the intracellular levels of HMG domain proteins, such as HMGB1, are low or in which these proteins are engaged in tight interactions with other targets, the other considerably less abundant proteins, such as transcription factors of vital importance for cells, may specifically bind 1,2-intrastrand CLs of cis[PtCl2(NH3)(pip)] with a smaller affinity than those of cisplatin. Consequently, the 1,2-intrastrand CLs of cis[PtCl2(NH3)(pip)] would recruit these factors from their native binding sites in a smaller extent than those of cisplatin, thereby diminishing the sensitivity of cells to the platinum compounds of this class. Hence, the results of the present work are consistent with the idea and support the hypothesis that at least in a certain type of cancer cells, in which for instance the shielding or hijacking mechanism involving HMG domain proteins is effective, the reduced affinity of the HMG domain proteins to the 1,2-GG intrastrand CLs of cis-[PtCl2(NH3)(pip)] may be partially responsible for its lower activity in cancer cell lines. The reasons for the reduced affinity of the HMG domain proteins to the 1,2-GG intrastrand CLs of the analogues of cisplatin containing heterocyclic ligands, such as pip, are unclear. As shown for the 1,2-GG intrastrand CL of cisplatin (7), HMGB1a binds to DNA around the site of this adduct in the minor groove while the ammine ligands coordinated to platinum reside in the major groove. It is therefore unlikely that the reduced affinity of HMGB1a to the 1,2-GG intrastrand CL of cis[PtCl2(NH3)(pip)] is due to direct interactions of the pip nonleaving ligand with the protein that would perturb the protein-DNA interaction. On the other hand, when HMGB1 proteins bind to the 1,2-GG intrastrand CL of cisplatin, the protein induces further bending of the DNA toward the major groove (7). Hence, it is also possible that the bulky pip group in the 1,2-GG intrastrand CL of cis-[PtCl2(NH3)(pip)] that resides in the major groove of DNA restricts the additional DNA bending toward the major groove, required for HMGB1a binding. Interestingly, the replacement of one ammine group in cisplatin by several cyclic ligands other than pip also reduced affinity of the 1,2-GG intrastrand CLs of these analogues to HMGB1a protein (9). Hence, the lowered capability of major DNA adducts of cisplatin analogues, in which the NH3 group is replaced by a bulkier cyclic ligand, to attract HMG domain proteins may be a common feature of this class of platinum compounds. The therapeutic profiles of platinum compounds do not correlate merely with DNA platination properties as can be noted from the different therapeutic profiles of cisplatin and carboplatin that form the identical DNA adducts. Among the factors influencing the pharmacological behavior of the drugs are those that affect the amount of platinum complex that ends up on the DNA in cancer cells. This can be done by affecting the cellular accumulation of the complexes and/or by inactivating them intracellularly. The results reported here (Figure 6) show that the activity of cis-[PtCl2(NH3)(pip)] in some cancer cell lines in comparison with cisplatin may be affected by the reduced accumulation of this drug with subsequent effects on the level of DNA platination in the cells. It is therefore reasonable to suggest that besides a number of biochemical factors including reduced affinity of HMG domain proteins to the major adduct of cis-[PtCl2(NH3)(pip)], the lowered intracellular accumula-

DNA Modified by Cisplatin Analogues

tion of this cisplatin analogue may also contribute to its smaller potency in tumor cell lines observed earlier (6). The differences in the affinities of HMG domain proteins to the major adducts of cis-[PtCl2(NH3)(pip)] and cisplatin and in the intracellular accumulation of these compounds are, however, not so pronounced to explain entirely the differences seen in the respective toxicities of the two compounds reported previously (6). Thus, it cannot be excluded that not only the biochemical factors associated with DNA modification examined in the present work contribute to the reduced potency of cis[PtCl2(NH3)(pip)] in comparison with cisplatin. DNA is considered a major pharmacological target of platinum antitumor drugs, and this is why we examined in the present work mainly several aspects of the mechanism of antitumor efficiency of cisplatin and its analogue that are associated with DNA. The cells represent complex systems, and a small fraction of cell-associated platinum is only covalently bound to DNA of the cells treated with platinum compounds whereas an overwhelming fraction of cell-associated platinum is bound to non-DNA cell components (6). Thus, differences in the reactions of cisplatin and its analogues with other non-DNA cellular components may also contribute to the reduced potency of the analogues of cisplatin in which the ammine group was replaced by the heterocyclic ligand. Further studies are needed to test this hypothesis. In conclusion, the results show that replacement of the NH3 group in cisplatin by pip does not modulate its DNA binding mode but reveals that the effects of two other factors (the reduced affinity of HMG domain proteins to the major adduct of cis-[PtCl2(NH3)(piperidine)] and the reduced accumulation of this drug with subsequent effects on the level of DNA platination in the cells) may partially contribute to the reduced activity of the cisplatin analogue. There are many biochemical factors affecting activity of platinum compounds in tumor cells. Further studies are, therefore, warranted to reveal a relative contribution of all potential factors contributing to the reduced potency of analogues of cisplatin containing heterocyclic ligands in various types of cancer cells. The results are also consistent with the idea that the concept based on the design of the complexes structurally derived from cisplatin that do not present an altered DNA binding mode may be less effective in the search for new platinum drugs that would overcome cisplatin resistance. In other words, a rational “molecular” approach to design new platinum antitumor drugs should also be focused on the platinum compounds structurally distinct from cisplatin that would also present a markedly different DNA binding mode.

Acknowledgment. This research was supported by the Grant Agency of the Czech Republic (Grant 305/02/ 1552) and the Grant Agency of the Academy of Sciences of the Czech Republic (Grants B5004301 and S5004009). J.K. is the international research scholar of the Howard Hughes Medical Institute. The research of J.K. and V.B. was also supported in part by the Wellcome Trust (U.K.). We acknowledge that our participation in the EC COST Chemistry Action D20 enabled us to exchange regularly the most recent ideas in the field of platinum anticancer drugs with several European colleagues. Y.N. thanks the David R. Bloom Center for Drug Design and for partial support. D.G. acknowledges partial support from the Project Grant from the Israel Cancer Research.

Chem. Res. Toxicol., Vol. 16, No. 11, 2003 1431

References (1) Kelland, L. R. (2000) New platinum drugs. The pathway to oral therapy. In Platinum-Based Drugs in Cancer Therapy (Kelland, L. R., and Farrell, N. P., Eds.) pp 299-319, Humana Press Inc., Totowa, NJ. (2) 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. (3) Brabec, V. (2002) DNA modifications by antitumor platinum and ruthenium compounds: their recognition and repair. In Progress in Nucleic Acid Research and Molecular Biology (Moldave, K., Ed.) Vol. 71, pp 1-68, Academic Press Inc., San Diego, CA. (4) Khazanov, E., Barenholz, Y., Gibson, D., and Najajreh, Y. (2002) Novel apoptosis-inducing trans-platinum piperidine derivatives: Synthesis and biological characterization. J. Med. Chem. 45, 5196-5204. (5) Najajreh, Y., Perez, J. M., Navarro-Ranninger, C., and Gibson, D. (2002) Novel soluble cationic trans-diamminedichloroplatinum(II) complexes that are active against cisplatin resistant ovarian cancer cell lines. J. Med. Chem. 45, 5189-5195. (6) Kasparkova, J., Marini, V., Najajreh, Y., Gibson, D., and Brabec, V. (2003) DNA binding mode of the cis and trans geometries of new antitumor nonclassical platinum complexes containing piperidine, piperazine or 4-picoline ligand in cell-free media. Relations to their activity in cancer cell lines. Biochemistry 42, 63216332. (7) 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 73-110, VHCA, WILEY-VCH, Zu¨rich, Weinheim. (8) Kasparkova, J., Delalande, O., Stros, M., Elizondo-Riojas, M. A., Vojtiskova, M., Kozelka, J., and Brabec, V. (2003) Recognition of DNA interstrand cross-link of antitumor cisplatin by HMGB1 protein. Biochemistry 42, 1234-1244. (9) 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 TATA-binding proteins. J. Biol. Chem. 276, 38774-38780. (10) Malina, J., Kasparkova, J., Natile, G., and Brabec, V. (2002) Recognition of major DNA adducts of enantiomeric cisplatin analogues by HMG box proteins and nucleotide excision repair of these adducts. Chem. Biol. 9, 629-638. (11) Zamble, D. B., Mu, D., Reardon, J. T., Sancar, A., and Lippard, S. J. (1996) Repair of cisplatin-DNA adducts by the mammalian excision nuclease. Biochemistry 35, 10004-10013. (12) Reardon, J. T., Vaisman, A., Chaney, S. G., and Sancar, A. (1999) Efficient nucleotide excision repair of cisplatin, oxaliplatin, and bis-aceto-ammine-dichloro-cyclohexylamine-platinum(IV) (JM216) platinum intrastrand DNA diadducts. Cancer Res. 59, 3968-3971. (13) Brabec, V., Reedijk, J., and Leng, M. (1992) Sequence-dependent distortions induced in DNA by monofunctional platinum(II) binding. Biochemistry 31, 12397-12402. (14) Brabec, V., and Leng, M. (1993) DNA interstrand cross-links of trans-diamminedichloroplatinum(II) are preferentially formed between guanine and complementary cytosine residues. Proc. Natl. Acad. Sci. U.S.A. 90, 5345-5349. (15) Stros, M. (2001) Two mutations of basic residues within the N-terminus of HMG-1 B domain with different effects on DNA supercoiling and binding to bent DNA. Biochemistry 40, 47694779. (16) 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. (17) 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. (18) 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. (19) 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. (20) Bellon, S. F., and Lippard, S. J. (1990) Bending studies of DNA site-specifically modified by cisplatin, trans-diamminedichloroplatinum(II) and cis-Pt(NH3)2(N3-cytosine)Cl+. Biophys. Chem. 35, 179-188. (21) Kasparkova, J., Novakova, O., Vrana, O., Farrell, N., and Brabec, V. (1999) Effect of geometric isomerism in dinuclear platinum

1432 Chem. Res. Toxicol., Vol. 16, No. 11, 2003

(22) (23)

(24)

(25)

(26) (27)

(28)

(29)

antitumor complexes on DNA interstrand cross-linking. Biochemistry 38, 10997-11005. He, Q., Ohndorf, U.-A., and Lippard, S. J. (2000) Intercalating residues determine the mode of HMG1 domains A and B binding to cisplatin-modified DNA. Biochemistry 39, 14426-14435. Missura, M., Buterin, T., Hindges, R., Hubscher, U., Kasparkova, J., Brabec, V., and Naegeli, H. (2001) Double-check probing of DNA bending and unwinding by XPA-RPA: an architectural function in DNA repair. EMBO J. 20, 3554-3564. 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. Kelland, L. R., Mistry, P., Abel, G., Loh, S. Y., O’Neill, C. F., Murrer, B. A., and Harrap, K. R. (1992) Mechanism-related circumvention of acquired cis-diamminedichloroplatinum(II) resistance using 2 pairs of human ovarian carcinoma cell lines by ammine/amine platinum(IV) dicarboxylates. Cancer Res. 52, 3857-3864. Ferna´ndez-Ruı´z, R., Tornero, J. D., Gonza´lez, V. M., and Alonso, C. (1999) Quantification of Pt bound to DNA using total-reflection X-ray fluorescente (TXRF). Analyst 124, 583-585. Pe´rez, J. M., Montero, E. I., Quiroga, A. G., Fuertes, M. A., Alonso, C., and Navarro-Ranniger, C. (2001) Cellular uptake, DNA binding and apoptosis induction of cytotoxic trans-[PtCl2(N,Ndimethylamine)(isopropylamine)] in A2780cisR ovarian tumor cells. Metal-Based Drugs 8, 29-37. Brabec, V. (2000) Chemistry and structural biology of 1,2interstrand adducts of cisplatin. In Platinum-Based Drugs in Cancer Therapy (Kelland, L. R., and Farrell, N. P., Eds.) pp 3761, Humana Press Inc., Totowa, NJ. Leng, M., Locker, D., Giraud-Panis, M. J., Schwartz, A., Intini, F. P., Natile, G., Pisano, C., Boccarelli, A., Giordano, D., and Coluccia, M. (2000) Replacement of an NH3 by an iminoether in transplatin makes an antitumor drug from an inactive compound. Mol. Pharmacol. 58, 1525-1535.

Kasparkova et al. (30) Brabec, V., Neplechova, K., Kasparkova, J., and Farrell, N. (2000) Steric control of DNA interstrand cross-link sites of trans platinum complexes: specificity can be dictated by planar nonleaving groups. J. Biol. Inorg. Chem. 5, 364-368. (31) Bancroft, D. P., Lepre, C. A., and Lippard, S. J. (1990) Pt-195 NMR kinetic and mechanistic studies of cis-diamminedichloroplatinum and trans-diamminedichloroplatinum(II) binding to DNA. J. Am. Chem. Soc. 112, 6860-6871. (32) 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. (33) Sancar, A. (1996) DNA excision repair. Annu. Rev. Biochem. 65, 43-81. (34) Reardon, J. T., and Sancar, A. (1998) Molecular mechanism of nucleotide excision repair in mammalian cells. In Advances in DNA Damage and Repair (Dizdaroglu, M., and Karakaya, A., Eds.) pp 377-393, Plenum Publishing Corp., New York. (35) 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. (36) Malina, J., Hofr, C., Maresca, L., Natile, G., and Brabec, V. (2000) DNA interactions of antitumor cisplatin analogues containing enantiomeric amine ligands. Biophys. J. 78, 2008-2021. (37) Fuertes, M. A., Castilla, J., Alonso, C., and Perez, J. M. (2003) Cisplatin biochemical mechanism of action: From cytotoxicity to induction of cell death through interconnections between apoptotic and necrotic pathways. Curr. Med. Chem. 10, 257-266. (38) Kartalou, M., and Essigmann, J. M. (2001) Recognition of cisplatin adducts by cellular proteins. Mutat. Res. 478, 1-21. (39) Kelland, L. R. (2000) Preclinical perspectives on platinum resistance. Drugs 59, 1-8.

TX034128G