Introduction

Top Summary Introduction Experimental Procedures Results and Discussion References

Current platinum-based drugs are limited in their use by their severe side effects, narrow spectrum of anticancer activity, and problems due to drug resistance. We are investigating the design of novel platinum(II) aminophosphine complexes as potential anticancer agents.

The aminophosphine complexes of platinum(II), cis-[PtCl₂(Me₂N(CH₂)₃PPh₂-P)₂] (Com1) and cis-[PtCl(C₆H₁₁NH(CH₂)₂PPh₂-N,P)(C₆H₁₁NH(CH₂)₂PPh₂-P)] (Com2; Fig. 1) contain the possibility of cis amine ligands, which are a feature found in many active platinum anticancer agents (Reedijk, 1996), together with cis phosphine ligands. Certain diphosphines have also been shown to exhibit anticancer activity, especially 1,2-diphenylphosphine (dppe) complexes of Cu(I), Ag(I), and Au(I) (Berners-Price and Sadler, 1988). Furthermore, lipophilic cations such as [Au(dppe)]⁺ can disrupt the mitochondrial membrane potentials (Berners-Price et al., 1997), and thereby act via a different mode of action to platinum am(m)ine antitumor complexes for which DNA is the target (Johnson et al., 1989).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Structures of bis(aminophosphine) platinum(II) complexes: 1, cis-[PtCl2(Me2N(CH2)3PPh2-P)2], 2, cis-[PtCl(C6H11NH(CH2)2PPh2-N, P)(C6H11NH(CH2)2PPh2-P)], 3, cis-[PtCl(Me2N(CH2)2PPh2-N, P)(Me2N(CH2)2PPh2-P)].

Metal aminophosphine complexes have been shown to be cytotoxic toward cancer cells with a potency approaching that of cis-diamminedichloroplatinum(II) {cis-[PtCl₂(NH₃)₂]} (cisplatin) and, moreover, are active against some cisplatin-resistant cell lines (Habtemariam and Sadler, 1996; Papathanasiou et al., 1997). These complexes can exist in chelate ring-opened and ring-closed forms in aqueous solution (Fig. 2). The equilibrium can be controlled under conditions of biological relevance by variation of pH and chloride concentration (Habtemariam and Sadler, 1996). The ring-closed form (Fig. 2A), being a lipophilic cation, could thus act as an antimitochondrial agent and the ring-opened forms (Fig. 2, B and C), which contain labile chloride ligands, offer potential binding sites to DNA.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Schematic representation of chelate ring opening in bis(aminophosphine) platinum(II) complexes.

Recently we have shown that chelate ring-opening platinum(II) aminophosphine complexes can bind rapidly and reversibly to the DNA bases guanine (Habtemariam and Sadler, 1996) and thymine as well as to the RNA base uracil (Margiotta et al., 1997), under physiologically relevant conditions, in contrast to platinum am(m)ine anticancer complexes (Reedijk, 1996). The binding was observed to the bases contained in monomeric nucleotides or in a short oligonucleotide duplex [8 base pairs (bp)]. In this work the modification of natural DNA and synthetic single- or double-stranded polydeoxyribonucleotides in cell-free media was studied by using various biomedical and biophysical methods. Com1 was chosen for these studies as a representative of the bifunctional aminophosphine complexes with leaving chloride ligands in cis position to compare its effects on double-helical DNA with those of cisplatin, whereas Com2, which has one chloride-leaving group, was used as a model.

	Experimental Procedures

Top Summary Introduction Experimental Procedures Results and Discussion References

Materials. Cisplatin and chlorodiethylenetriamineplatinum(II) chloride {[PtCl(H₂NCH₂CH₂NHCH₂CH₂NH₂)]Cl} ([PtCl(dien)]Cl) were synthesized and characterized in Lachema (Brno, Czech Republic). Com1 and Com2 (Fig. 1) were prepared and characterized as reported elsewhere (A.H., R. Palmer, P. Potter, and P.J.S., submitted). Calf thymus (CT) DNA (42% guanine + cytosine, mean molecular mass ca. 2 × 10⁷) was prepared and characterized as described previously (Brabec and Paleek, 1970). Denatured CT DNA was prepared by heating at 100°C for 10 min and subsequent rapid cooling on an ice bath. Plasmids pSP73 (2464 bp) or pSP73KB (2455 bp) were isolated according to standard procedures and banded twice in CsCl/ethidium bromide (EtBr) equilibrium density gradients. Synthetic single-stranded homopolydeoxyribonucleotides poly(dA), poly(dC), poly(dG), poly(dT), and double-helical alternating polydeoxyribonucleotides poly(dA-dT) and poly(dG-dC) were purchased from Boehringer-Mannheim Biochemica (Mannheim, Germany) [all references in the text to poly(dA-dT) and poly(dG-dC) refer to duplex molecules] (the concentrations of synthetic polynucleotides are related to their phosphorus content). The oligodeoxyribonucleotide duplex containing 40 bp, 5'-CCCGGATTATACGGCTTAAACCAAATTGCTTAAATTGGCC)/5'-GGCCAATTTAAGCAATTTGGTTTAAGCCGTATAATCCGGG was obtained from East Port (Prague, Czech Republic); the single-stranded oligonucleotides constituting this duplex were purified by strong anion exchange chromatography (Pharmacia MonoQ column) on a Pharmacia fast protein liquid chromatography system with 10 mM NaOH, 0.2 to 0.8 M NaCl gradient. The duplex was formed by heating the mixture of the complementary single-stranded oligonucleotides at equal concentrations (related to the mononucleotide content) at 90°C for 5 min followed by incubation at 25°C for 4 h. Restriction endonucleases and Thermal Cycle Dideoxy DNA Sequencing Kit with Vent_R(exo^-) or Vent_R(exo⁺) DNA polymerases were purchased from New England Biolabs (Beverly, MA). A primer 5'-d(GATTTAGGTGACACTATAG) was obtained from BioVendor (Brno, Czech Republic). T4 polynucleotide kinase and Klenow fragment of DNA polymerase I were also obtained from Boehringer-Mannheim Biochemica (Mannheim, Germany). DNase I from bovine pancreas, nuclease P1 from Penicillium citrinum, and alkaline phosphatase from calf intestine were purchased from Sigma-Aldrich (Prague, Czech Republic). EtBr, acrylamide, (bis)acrylamide, urea, and agarose were obtained from Merck KgaA (Darmstadt, Germany). The radioactive products were purchased from Amersham (Arlington Heights, IL).

Platination Reactions. DNAs were modified by platinum complexes in 10 mM NaClO₄ (pH 7.0) at 37°C in the dark for 48 h unless stated otherwise. In these samples, the number of the molecules of the platinum complex fixed per nucleotide residue (r_b) was determined by flameless atomic absorption spectrophotometry (FAAS) or by differential pulse polarography (DPP; Kim et al., 1990).

HPLC Analyses. These analyses were performed using a Hitachi Series 4 liquid chromatograph equipped with a LCI-100 computing integrator and a Waters µBondapack C18 column. If not stated otherwise, the products were separated by reversed phase (RP)-HPLC (isocratic elution with 0.1 M ammonium acetate, pH 5.0 in 4% CH₃CN at 1 ml/min flow rate). The following enzymatic digestion protocol was used to characterize the platinated deoxyribooligonucleotides. The samples (50 µg of the oligonucleotide) were incubated with 72 U DNase I at 37°C. After 4 h nuclease P1 (40 µg) was added, and the reaction was allowed to continue at 37°C for 18 h. Finally, alkaline phosphatase (39 U) was added and the incubation continued for additional 4 h at 37°C. The digested samples containing constituent nucleosides were then heated for 2 min at 80°C, centrifuged, and the supernatant was analyzed by RP-HPLC. Each analysis was performed four times and the data varied on average ± 1% from their mean.

Sequence Specificity of DNA Adducts. The (HpaI/NdeI) restriction fragment of pSP73KB DNA (212 bp) was obtained as described previously (Brabec and Leng, 1993). Ten micrograms of pSP73KB were treated with NdeI to obtain linear plasmid followed by treatment with alkaline phosphatase. The linear fragment was then 5'-end labeled by treatment with T4 polynucleotide kinase in the presence of [³²P]-ATP. The 212-bp fragment was obtained by subsequent treatment with HpaI and isolated by electrophoresis through a preparative 1% agarose gel. The modification of this fragment by cisplatin, Com1, or Com2 was carried out in 10 mM NaClO₄ (pH 7) for 48 h at 37°C to obtain r_b = 0.01. CircumVent Thermal Cycle Dideoxy DNA Sequencing Kit with Vent_R(exo^-) or (exo⁺) DNA polymerases was used with the protocol for thermal cycle DNA sequencing with 5' end-labeled primer recommended by the manufacturer with small modifications (Nováková et al., 1995).

EtBr Fluorescence. These measurements were performed with the aid of a Shimadzu RF 40 spectrofluorophotometer using a 1-cm quartz cell. Fluorescence measurements of DNA modified by platinum in the presence of EtBr were performed using the excitation wavelength of 546 nm and the emitted fluorescence was measured at 590 nm. The fluorescence was measured at 25°C in 0.4 M NaCl to avoid the second fixation site of EtBr to DNA (Butour and Macquet, 1977). The concentrations were 0.01 mg/ml for DNA and 0.04 mg/ml for EtBr, which corresponded to the saturation of all intercalation sites of EtBr in DNA (Butour and Macquet, 1977).

Unwinding of Negatively Supercoiled DNA. Unwinding of closed circular supercoiled pSP73 plasmid DNA was assayed by an agarose gel mobility shift assay (Keck and Lippard, 1992). The unwinding angle , induced per platinum-DNA adduct was calculated upon the determination of the r_b value at which the complete transformation of the supercoiled to relaxed form of the plasmid was attained. Samples of pSP73 plasmid were incubated with Com1 or Com2 in 10 mM NaClO₄, pH 7.0 at 37°C in the dark for 48 h. All samples were precipitated by ethanol and redissolved in TAE or the TBE buffer (0.04 M Tris-acetate + 1 mM EDTA, pH 7.0 or 0.09 M Tris-borate + 1 mM EDTA, pH 9.0, respectively). An aliquot of the precipitated sample was subjected to electrophoresis on 1% agarose gels running at 25°C in the dark with TAE or TBE buffer with voltage set at 30 V. The gels were then stained with EtBr, followed by photography on Polaroid 667 film with transilluminator. The other aliquot was used for the determination of r_b values by FAAS.

Interstrand Cross-Link (ICL) Assay. If not stated otherwise, Com1 or Com2 at varying concentrations were incubated with 2 µg of pSP73 DNA linearized by EcoRI. The platinated samples were precipitated by ethanol and analyzed for DNA ICLs in the same way as described in several recent papers (Farrell et al., 1990; Brabec and Leng, 1993). The linear duplexes were first 3'-end labeled by means of Klenow fragment of DNA polymerase I and [-³²P]dATP. The samples were deproteinized by phenol, precipitated by ethanol, and the pellet was dissolved in 18 µl of a solution containing 30 mM NaOH, 1 mM EDTA, 6.6% sucrose, and 0.04% bromophenol blue. The number of ICLs was analyzed by electrophoresis under denaturing conditions on alkaline agarose gel (1%). After the electrophoresis was completed, the intensities of the bands corresponding to single strands of DNA and ICL duplex were quantified by means of a Molecular Dynamics PhosphorImager (Storm 860 System with ImageQuant software; Sunnyvale, CA). As shown below, some platinated DNA samples were also analyzed for the ICLs using milder conditions, when the DNA samples were treated with formamide or dimethyl sulfoxide at 40 or 55°C and then analyzed in native agarose gel. The details of these milder assays can be found in the papers previously published (Konopa, 1983; Cullinane and Phillips, 1994).

Circular Dichroism (CD). If not stated otherwise, CD spectra of DNA modified by the platinum complexes were recorded at 25°C in 10 mM NaClO₄ (pH 7.0) on a JASCO spectropolarimeter, Model J720 (Tokyo, Japan).

Immunochemical Analysis. Polyclonal antibodies that bind selectively to adducts formed on linear double-helical DNA by cisplatin at r_b = 0.08 (Ab_cis) were elicited against double-helical calf-thymus DNA modified by cisplatin at r_b = 0.08 in 10 mM NaClO₄ for 48 h at 37°C. They were purified and characterized as described in previously published papers (Sundquist et al., 1987; Vrána et al., 1992). The procedures for their immunoenzymatic analysis and enzyme-linked immunosorbent assay have been also described (Sundquist et al., 1987; Vrána et al., 1992).

	Results and Discussion

Top Summary Introduction Experimental Procedures Results and Discussion References

DNA Binding. Solutions of double-helical CT DNA at a concentration of 32 µg/ml were incubated with Com1 or Com2 at the molar ratio of free platinum complex to nucleotide-phosphates at the onset of incubation with DNA (r_i) values of 0.08 in 10 mM NaClO₄ (pH 7.0) at 37°C. At various time intervals an aliquot of the reaction mixture was withdrawn and assayed by DPP for the amount of platinum bound to DNA (r_b) (Kim et al., 1990). Figure 3 shows a plot of r_b against the time of DNA incubation with Com1 or Com2. The amount of platinum coordinated to DNA increased with time. After 48 h, approximately 100% or 80% of the complex Com1 or Com2, respectively, present in the reaction mixtures was coordinated to DNA [exhaustive dialysis of the samples of DNA treated with Com1 or Com2 against platinum-free background solution (10 mM NaClO₄) did not affect the amount of the platinum bound to DNA]. In these binding reactions, the time at which the binding reached 50% (T_50%) was ca. 3 h for both Com1 and Com2. The value of T_50% for the reaction of cisplatin or monofunctional [PtCl(dien)]Cl with DNA under conditions identical with those specified in Fig. 3 were ~4 h, respectively (Bancroft et al., 1990). This comparison indicates that the presence of the aminophosphine groups as nonleaving ligands enhances the rate of the coordination of monofunctional chloro or bifunctional dichloro platinum(II) complexes to natural double-helical DNA. When the same binding experiment was carried out with thermally denatured CT DNA, the binding of Com2 remained unchanged whereas the binding of Com1 was somewhat faster (T_50% was ~2 h; Table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Formation of DNA adducts by bis(aminophosphine) platinum(II) complexes as a function of incubation time. CT DNA at the concentration of 32 µg/ml was mixed with Com1 () or Com2 () at ri = 0.08 in 10 mM NaClO4 and at 37°C in the dark. At various time intervals the aliquots were withdrawn and platinum coordinated to DNA was estimated by DPP assay. Data points measured in triplicate varied on average ± 2% from their mean.

4

Characterization of DNA Adducts by HPLC Analysis of Enzymatically Digested DNA. To further characterize the coordination mode of the two aminophosphine platinum(II) complexes, a 40-bp deoxyribooligonucleotide duplex (with a random nucleotide sequence, see Experimental Procedures) modified by Com1 or Com2 (in 0.1 M NaClO₄, pH 7) was enzymatically digested to mononucleosides and analyzed by RP-HPLC. RP-HPLC analysis of enzymatic digests of Com1- or Com2-modified oligonucleotide duplex was performed by recording optical density at 260 nm. The profile in Fig. 4 (curve 1) shows well resolved mononucleoside peaks that reflect the proper proportions of the single mononucleosides in unplatinated duplex when integrated and normalized by their extinction coefficients. Digestion of the platinated samples (Fig. 4, curves 2 and 3) resulted in the decrease of the integrated area of the deoxyriboguanosine peak and, in the case of the digestion of the duplex modified by Com1, also in the decrease of deoxyriboadenosine peak. The peaks of deoxyribocytidine and thymidine were not affected. It was verified by FAAS that no product containing platinum coeluted with the peaks corresponding to unplatinated deoxyribonucleosides. The platinated products were not retained by the column under the conditions used, so they could not be identified and quantified.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   RP C18 HPLC separation of the products of enzymatic digest of the 40-bp oligonucleotide duplex nonmodified (curve 1) or modified by Com1 at rb = 0.09 (curve 2) or by Com2 at rb = 0.05 (curve 3). For other details, see the text.

Mapping of DNA Adducts. Recent work has shown that the in vitro DNA synthesis by DNA polymerases on DNA templates containing several types of bidentate adducts of platinum complexes can be prematurely terminated at the level or in the proximity of adducts (Comess et al., 1992; Murray et al., 1992; Vrána et al., 1996). Importantly, the efficiency of monofunctional DNA adducts of several platinum(II) complexes to terminate DNA synthesis is considerably smaller (Comess et al., 1992).

View larger version (39K): [in this window] [in a new window]   Fig. 5.   A, autoradiogram of 6% polyacrylamide/8 M urea sequencing gel showing inhibition of DNA synthesis by VentR DNA polymerase on the HpaI/NdeI restriction fragment of pSP73KB plasmid DNA modified by platinum complexes. The gel contained the linear amplification products of DNA treated with Com1, Com2, and cisplatin. Lanes: control, unmodified template; cisDDP, DNA modified by cisplatin at rb = 0.01; 1, DNA modified by Com1 at rb = 0.01; 2, DNA modified by Com2 at rb = 0.05; C, G, T, A, chain-terminated marker DNAs (note that these dideoxy-sequencing lanes give the sequence complementary to the template strand). The numbers correspond to the nucleotide sequence numbering of 5B. B, schematic diagram showing a portion of the sequence used to monitor inhibition of DNA synthesis on the template containing adducts of the platinum complexes. * indicates the 5'-end labeling of the primer. The dashed arrow indicates the start site of the DNA polymerase and the direction of the synthesis. , stop signals from A, lane 1. Nucleotides 1 and 18 correspond to 2539 and 1 on the pSP73KB nucleotide sequence map.

Characterization of DNA Adducts by EtBr Fluorescence. EtBr as a fluorescent probe can be used to distinguish between perturbations induced in DNA by monofunctional and bifunctional adducts of platinum(II) compounds (Butour and Macquet, 1977; aludová et al., 1997b). Binding of EtBr to DNA by intercalation is blocked in a stoichiometric manner by formation of the bifunctional adducts of a series of platinum complexes including cisplatin and transplatin, which results in a loss of fluorescence intensity (Butour and Macquet, 1977; ákovská et al., 1998). On the other hand, modification of DNA by monodentate platinum(II) complexes (having only one leaving ligand) results in only a slight decrease of EtBr fluorescence intensity as compared with nonplatinated DNA-EtBr complex.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Dependences of the EtBr fluorescence on rb for DNA modified by various platinum complexes in 10 mM NaClO4 at 37°C for 48 h. (+), cisplatin; (), [PtCl(dien)]Cl; (), Com1; and (), Com2. Data points measured in triplicate varied on average ± 2% from their mean.

CD. CD spectroscopy has already been used to obtain information about the global changes in DNA conformation induced by platinum complexes. It has been shown (Vrána et al., 1986; Brabec et al., 1990) that the intensity of the positive CD band yielded by B-DNA at ~275 nm is increased as a consequence of DNA modification by the complexes containing the cis-[PtCl₂(amine)₂] unit (Fig. 7, B and C); at higher levels of the modification (r_b > ~0.5) the intensity of this CD band begins to decrease (Fig. 7, B and C). On the other hand, the modification of DNA by clinically ineffective transplatin or dienPt only slightly decreases this positive band (Vrána et al., 1986; Brabec et al., 1990). It has been suggested (Vrána et al., 1986; Brabec et al., 1990) that the enhancement of the CD band at ~275 nm due to the modification by the complexes containing cis-[PtCl₂(amine)₂] unit reflects distortions in DNA of a nondenaturational nature. The slight reduction of this CD band induced by the binding of platinum complexes is consistent with the occurrence of short segments containing unpaired bases (denatured regions).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   CD spectroscopy of CT DNA modified by Com1 and cisplatin. The spectra were recorded for DNA in 10 mM NaClO4, pH 7.0. A, CD spectra of DNA modified by Com1. Curves: 1, control (nonmodified) DNA; 2, rb = 0.04; 3, rb = 0.065; 4, rb = 0.09; 5, rb = 0.13; 6, rb = 0.18. B, CD spectra of DNA modified by cisplatin. Curves: 1, control (nonmodified) DNA; 2, rb = 0.02; 3, rb = 0.04; 4, rb = 0.065; 5, rb = 0.09; 6, rb = 0.13; 7, rb = 0.18. C, dependence of the maximum ellipticity of the positive CD band at around 280 nm on rb. (×), cisplatin; (), Com1. Data points measured in duplicate varied on average ± 1% from their mean.

Unwinding Induced in DNA by Platinum Coordination. The results described above suggest that Com1 forms bifunctional adducts on DNA although to a smaller extent than its bifunctional analog, cisplatin. It has been shown that DNA adducts of cisplatin (and its direct analogs) unwind DNA (e.g., Bellon et al., 1991; Keck and Lippard, 1992; Huang et al., 1995; Gelasco and Lippard, 1998). CD spectra of DNA modified by Com1 (Fig. 7A) suggest that this modification also results in an overall overwinding of the DNA molecule as a whole if the platinated DNA is dissolved in neutral media, whereas no changes in CD spectra indicating overwinding occur if DNA is modified under identical conditions by cisplatin or if the pH of the sample of DNA modified by Com1 is adjusted to 9.

View larger version (49K): [in this window] [in a new window]   Fig. 8.   Unwinding of supercoiled pSP73 plasmid DNA by Com1. DNA was modified in 10 mM NaClO4, pH 7, precipitated by ethanol, dissolved in TBE buffer, pH 9.0 (A) or TAE buffer, pH 7.0 (B), and analyzed by gel electrophoresis using the same buffer in which DNA was dissolved. The top bands correspond to the form of nicked plasmid and the bottom bands to the closed, negatively supercoiled plasmid. A, lanes: 1 and 10, rb = 0; 2, rb = 0.008; 3, rb = 0.01; 4, rb = 0.02; 5, rb = 0.04; 6, rb = 0.06; 7, rb = 0.08; 8, rb = 0.09; 9, rb = 0.11. B, lanes: 1 and 14, rb = 0; 2, rb = 0.0008; 3, rb = 0.002; 4, rb = 0.004; 5, rb = 0.0064; 6, rb = 0.008; 7, rb = 0.02; 8, rb = 0.04; 9, rb = 0.064; 10, rb = 0.08; 11, rb = 0.2; 12, rb = 0.4; 13, rb = 0.64.

Interstrand Cross-Linking. The amounts of ICLs formed by Com1 or Com2 in linear DNA were measured in pSP73 plasmid (2464 bp) that was first linearized by EcoRI (EcoRI cuts only once within pSP73 plasmid) and subsequently modified by Com1 or Com2 at various r_b. The samples were analyzed for the ICLs by agarose gel electrophoresis under denaturing conditions.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   The formation of ICLs by Com1 in the pSP73 plasmid linearized by EcoRI. A, autoradiogram of a denaturing 1% agarose gel of 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). DNA was incubated with Com1 for 48 h at 37°C. rb values: lane 1, 0; lane 2, 0.0007; lane 3, 0.0016; lane 4, 0.0032. B, dependence on rb of the number of ICLs per adduct (%ICL/Pt). The ratio of ICLs to total platinum bound was calculated as described previously (Farrell et al., 1990; Brabec and Leng, 1993); %ICL/Pt was then calculated by multiplying this ratio by 100. Data points measured in triplicate varied on average ±3% from their mean.

Immunochemical Analysis. We prepared Ab_cis, which recognizes two neighboring purine residues of the same strand of DNA cis coordinated to the platinum atom of cis-[Pt(amine)₂]²⁺ (Sundquist et al., 1987; Vrána et al., 1992). They do not recognize monofunctional platinum adducts and the adducts of transplatin and its analogs.

BZ Transition. The effect of various platinum compounds on the salt-induced BZ transition in poly(dG-dC) has already been described in several papers (for instance, Ushay et al., 1982; Pérez-Martin et al., 1993; aludová et al., 1997a). In the present work, DNA modifications by Com1 or Com2 are compared with those by cisplatin or [PtCl(dien)]Cl, respectively. Cisplatin was found to facilitate the BZ transition, but the resulting Z form was distorted and the transition cooperativity was considerably reduced. [PtCl(dien)]Cl has been shown to stabilize the Z-form of DNA; for [PtCl(dien)]Cl-treated poly(dG-dC), the Z conformation is observed at a considerably lower salt concentration than for the nontreated polymer, and the transition cooperativity is considerably reduced.

View larger version (21K): [in this window] [in a new window]   Fig. 10.   CD spectroscopy of poly(dG-dC) nonmodified (A) or treated with Com1 (B) or Com2 (C) at rb = 0.1. The polymer was modified when it was in B-form (in 10 mM NaClO4) and was subsequently transferred into the media containing 10 mM NaClO4 with 1 mM phosphate buffer, pH 7.5, plus 10 mM EDTA and various concentrations of NaCl. A, control, nonmodified polymer; curves: 1, 0.19 M NaCl; 2, 1.53 M NaCl; 3, 2.17 M NaCl; 4, 2.29 M NaCl; 5, 4.18 M NaCl. B, polymer modified by Com1; curves: 1, no NaCl added; 2, 1.01 M NaCl; 3, 1.51 M NaCl; 4, 2.00 M NaCl; 5, 3.14 M NaCl. C, polymer modified by Com2; curves: 1, no NaCl added; 2, 0.97 M NaCl; 3, 1.44 M NaCl; 4, 1.89 M NaCl; 5, 2.82 NaCl. D, plot of the molar ellipticity at 290 nm (290 nm) as a function of Na+ concentration for poly(dG-dC) nonmodified (×) or treated with Com1 (), Com2 (), and cisplatin at rb = 0.1 (). Data points measured in duplicate varied on average ±1% from their mean. Other details are described in the text.

Conclusions. In broad terms, we have demonstrated that the DNA binding mode, and very likely also the mechanism of antitumor activity of the new class of platinum(II) aminophosphine complexes, is different from that of cisplatin. This difference reflects the distinct nature of nonleaving ligands. The replacement of amine ligands in cis-dichloroplatinum(II) complexes by phosphine groups has been shown to influence considerably the selectivity for the adducts containing adenine and guanine residues and for monodentate and bidentate lesions. In addition, conformational alterations induced in DNA by Com1 are different from those induced by cisplatin. The different conformational changes and the increased bulk of the lesions are consistent with a role of these factors in the processing of DNA platinated by Com1. In particular, Com1 is more efficient than cisplatin in blocking DNA synthesis. A specific role of aminophosphine ligands in the modification of DNA by Com1 is also evident from the observation that DNA platinated by Com1 is not recognized by the antibodies elicited against DNA modified by cisplatin despite an equally good specificity of these antibodies for DNA modified by several analogs of cisplatin having varied nonleaving amine groups. A further processing of platinum(II) adducts by cellular components has been suggested to play an important role in the mechanism underlying antitumor activity of platinum compounds. The different cellular processing of DNA modified by cis-dichloroplatinum(II) complexes containing on the one hand nonleaving amine groups and aminophosphine ligands on the other might be relevant to the different biological activity of these two classes of platinum compounds. Structural studies of site-specific DNA adducts with aminophosphine platinum(II) complexes should provide a basis for analyzing and re-evaluating the structure-pharmacological activity relationships of platinum compounds. Studies toward this end are in progress, from which may ultimately arise a rational basis for design of a novel class of platinum antitumor drugs.