Formation and repair of cisplatin-induced adducts to DNA in cultured normal and repair-deficient human fibroblasts.

The formation and repair of cisplatin [cis-PtCl2(NH3)2] adducts in the DNA of cultured normal and repair-deficient human fibroblasts are presented in relation to cell survival after cisplatin treatment. Directly after treatment with cisplatin, in normal (MB), Fanconi's anemia (FA), and xeroderma pigmentosum (XP) fibroblasts four platinated products are found. The major adduct is cisplatin bound to two neighboring guanines, Pt-GG (62-75%). A less abundant product is cisplatin bound to an AG sequence (Pt-AG). Binding to two guanines separated by one or more bases or to two guanines in opposite DNA strands (together measured as G-Pt-G) and cisplatin bound monofunctionally to guanine (Pt-G) are also found in small amounts. The distribution of the four products is similar to that found previously, in in vitro systems as well as in living cells. Directly after cisplatin treatment, the removal of cisplatin-DNA adducts is fast in normal and FA fibroblasts, whereas in XP fibroblasts adduct removal proceeds slowly throughout the repair period studied. Both FA and XP fibroblasts are extremely sensitive to cisplatin with regard to cell killing. For FA fibroblasts this sensitivity may be attributed to the fact that in these cells initially more DNA-adducts are formed than in normal fibroblasts, and/or to their known deficiency in the repair of DNA interstrand cross-links. For XP fibroblasts this sensitivity may be caused by their deficiency in the fast repair process, known as excision repair.

Influence of temperature on platinum binding to DNA, cell killing, and mutation induction in Escherichia coli K-12 cells treated with cis-diamminedichloroplatinum(II).

Cell killing and specificity of mutation induction by treatment of Escherichia coli K-12 cells with cis-Pt(NH3)2Cl2 at various temperatures have been studied. Survival experiments show that the cell killing by cis-Pt(NH3)2Cl2 is enhanced with increasing temperature. This effect is explained by an increase in the amount of platinum bound to the DNA. The binding is the same in repair-proficient and in repair-deficient cells. However, the mutation induction in the lacI gene is much more strongly enhanced than could be expected from the increased platinum binding. This phenomenon is attributed to a different distribution of the induced lesions by cis-Pt(NH3)2Cl2 at various temperatures and not to an aberrant excision repair. Analysis of the induced lacI mutants revealed an increase in the percentage of nonsense mutants at higher temperature. Among the nonsense mutations, base-pair substitutions at GAG and particularly at GCG sequences are enhanced by the increasing temperature. The results are in agreement with our hypothesis that local denaturation of DNA, known to be promoted at higher temperature, is necessary for the formation of intrastrand cross-links at two guanine bases separated by a third base.

The pH and redox-state dependence of the copper site in azurin from Pseudomonas aeruginosa as studied by EXAFS.

The EXAFS of the K-edge of copper in azurin from Pseudomonas aeruginosa has been measured in solutions of the oxidized and reduced protein, at both low and high pH. Model compounds of known molecular structure, exhibiting Cu-N and Cu-S bonds of varying length, were studied as well. The major shell of the high-pH oxidized azurin EXAFS contains contributions of two N(His) at 1.95 +/- 0.03 A, and one S(Cys) at 2.23 +/- 0.03 A. Some minor contributions from the carbon atoms of the histidine residues and the distal sulfur atom are observed in the 3-4 A region. Upon reduction a decrease is seen in amplitude of the main peak in the Fourier transform, due to a lengthening of one of the Cu-N(His) bonds (2.05 +/- 0.03 A), and a shortening of the other (1.89 +/- 0.03 A), both by approx. 0.1 A. Indications for a Cu-S(Met) bond are found in the reduced azurin data (2.70 +/- 0.05 A). However, in the oxidized protein, this bond could not be determined unambiguously, in line with results of a model compound featuring weak Cu-thioether coordination. The effect of pH is only slight for both the oxidized and the reduced protein, and no significant changes in bond lengths are found upon a change of pH from 4.1 to 9.1. The relevance of these findings for the interpretation of the existing data on the redox activity of the protein is discussed.

The interaction of the anti-cancer drug cisplatin with phospholipids is specific for negatively charged phospholipids and takes place at low chloride ion concentration.

The interaction of the anti-cancer drug cis-diamminedichloroplatinum(II) (cisPt) with model membranes was studied, with emphasis on the cisPt and phospholipid species involved. Binding studies using large unilamellar vesicles have revealed that: (i) Interaction involved negatively charged phospholipids only, and (ii) Interaction with negatively charged phospholipids was observed only in buffers with low Cl- concentration, indicating that aquated, positively charged cisPt is involved. Binding to all negatively charged phospholipids tested was highest at pH 6.0. At pH 7.4 a high and specific binding was observed with phosphatidic acid and phosphatidylserine. The consequences of cisPt binding on the organization of lipids was investigated with differential scanning calorimetry studies. These studies have indicated a higher ordering of dispersions of negatively charged phospholipids in the presence of divalent cationic cisPt. Summarizing, the interaction of positively charged cisPt species with negatively charged phospholipids is significant and should be considered in in vivo experiments.

Medicinal applications of heavy-metal compounds.

A brief summary of the key role for certain heavy-metal compounds in medicine is discussed, with a special focus on very recent findings in the following four topics: platinum anti-tumor compounds (novel mononuclear compounds, dinuclear compounds and trinuclear compounds with promising activity); ruthenium anti-tumor compounds (the first clinical trial for a Ru compound has begun); gadolinium NMR-imaging compounds (association with biomacromolecules is now possible); technetium compounds (the use of organometallic precursors opens a plethora of new species and enables the labeling of, for example, neurotransmitter molecules).

A phosphorus NMR study of the reaction products of cis-diamminedichloroplatinum (II) with a double-helical oligonucleotide and with DNA.

The structural distortion of oligonucleotides upon cis-PtCl2(NH3)2[d(T-C-T-C-G-G-T-C-T-C)-N7(5), N7(6)] reveals shifting of 4 phosphorus resonances due to platination. 3 Resonances could be assigned by selective 31P-irradiation, showing P(6) (P between the two Gs) to be shifted 1.5 ppm to low field. In the concomitant double strands P(6) is shifted 0.9 ppm to lower field. A similar peak has been observed in platinated salmon sperm DNA (37 degrees C), indicating that Pt-binding to GpG-fragments in DNA is similar to that found for the decanucleotide, so the distortion of DNA might be comparable.

Dinuclear alkyldiamine platinum antitumor compounds: a structure-activity relationship study.

Six related dinuclear trans-platinum complexes, with the formula [[trans-PtCl(2)(NH(3))(L)](2)(mu-H(2)N(CH(2))(n)NH(2))](2+) (L = pyridine, 2-picoline, 4-picoline; n = 4, 6) and chloride or nitrate anions, are compared with known cytotoxic dinuclear compounds (L = NH(3); n = 4, 6) that overcome cisplatin resistance. The cytotoxicity of the compounds was determined in L1210 murine leukemia and L1210/2, a cisplatin-resistant derivative. Unlike the L = NH(3) compounds, the substituted n = 4 compounds are more susceptible toward the resistance mechanisms in L1201/2. The n = 6 compounds, however, have comparable IC(50) values in both cell lines. In general, the substituted compounds are less cytotoxic than their NH(3) counterparts. After incubation with equimolar concentrations, the amount of platinum bound to cellular DNA was determined. The compounds show comparable binding, except for the sterically hindered 2-picoline compounds that bind significantly less. The amounts of platinum bound to DNA do not correlate with the cytotoxicity data. As DNA is considered to be the cellular target of platinum antitumor drugs, structural details of the DNA adducts probably account for the differences in cytotoxic activity.

Tuning the recovery of an Rh-containing catalyst with silica-based (poly) amine ion exchangers.

Highly efficient Rh-recovery from different adsorption media has been effected with silica-based (chelating) ion exchangers containing (poly) amine functionalities; recoveries have been found to correlate well with the stability of the metal-to-ligand complexes.

Structural effect of intra-strand cisplatin-crosslink on palindromic DNA sequences.

Three self-complementary DNA oligonucleotides, each having a single GpG site, have been reacted with the anticancer platinum compound cis-Pt(NH3)2Cl2 (cisplatin). Their covalent intra-strand didentate adducts have been purified and studied by NMR. In d(GAC-CATATG*G*TC), the two G*G* sites (G*G* denoting the cisplatin crosslinked lesion site) are separated by four base pairs and capped by two base pairs at the ends of the helix and the dodecamer forms a doubly-kinked duplex structure. Multi-stranded aggregate of the dodecamer was formed over time in the presence of chloride. This is due to the meta-stable property of the intra-strand Pt-G*pG* crosslink in dsDNA, similar to that first seen recently in another duplex (Yang et al., Biochemistry (1995) 34, 12912-12920). In d([c7A]CC[c7G][c7G]CCG*G*T), the CG*G*T segment of the decamer is essentially single-stranded with the G*8 in the syn conformation. In d([c7G]CC[c7G]CG*G*C), two possible structures, a full duplex and a staggered partial duplex, were formed. Therefore, the structural consequence of the incorporation of the G*G* lesion site into palindromic sequences is dependent on the location of the lesion sites in the sequence. The destabilizing effect of G*G* in dsDNA may facilitate the formation of a hairpin structure as shown recently (Iwamoto et al., J. Amer. Chem. Soc. (1994) 116, 6238-6244). Such alternative structural distortions may be relevant in understanding the protein recognition of the lesions induced by cisplatin.

cis-diamminedichloroplatinum(II) induced distortion of a single and double stranded deoxydecanucleosidenonaphosphate studied by nuclear magnetic resonance.

The structural distortion of a single- and a double-stranded decadeoxynucleotide upon binding of cis-PtCl2(NH3)2 was studied by 1H-NMR. After selective platination of d(T-C-T-C-G-G-T-C-T-C) (I) at the central d(-GpG-) site (resulting in I-Pt), several non-exchangeable base protons as well as H1', H2', H2" and H3' protons could be assigned by means of conventional NMR double-resonance techniques. Addition of the complementary decamer strand to I and I-Pt yielded the double-stranded III and III-Pt, respectively. All non-exchangeable base, H1', and most of the H2' and H2" protons in the two double stranded compounds could be assigned using 2D-chemical shift correlation (COSY) and nuclear Overhauser enhancement (NOESY) techniques. The double stranded compound III appears to adopt a B-DNA like structure. Comparison of NOEs and proton-proton coupling constants in the d(-GpG-).cisPt part in I-Pt and III-Pt reveals that their structure displays large similarity. Significant chemical shift changes (i.e. larger than 0.1 ppm) between III and III-Pt are restricted to the central four base pairs. It follows that the outer three base pairs, located on either side of the central four base pairs in III-Pt are likely to adopt a regular B-DNA type helix. The observed large upfield and downfield chemical shifts in the d(-CpGpG-) part of III with respect to III-Pt can be rationalized by describing the distortion of the double helix as a kink. A discussion of the observed physical effects upon platination of a double-stranded oligonucleotide is presented.

Reaction products from platinum(IV) amine compounds and 5'-GMP are mainly bis(5'-GMP)platinum(II) amine adducts.

The reaction products obtained from mixtures of 5'-GMP and platinum(IV) compounds with formula Pt(IV)Cl4(LL) and Pt(IV)Cl2(OH)2(LL) (LL representing two monodentate or one bidentate amine ligand) have been characterized by proton NMR spectroscopy. The amines used are NH3, H2N-CH2-CH2-NH2 (ethylenediamine, en), H2N-CH2-C(CH3)2-CH2-NH2 (2,2-dimethyl-1,3-diaminopropane, dmdap), and HC(CH3)2-NH2 (isopropylamine, ipa). Conditions varied during the reaction are pH (values of 4, 7, and 10), effect of visible light, and addition of vitamin C as a reducing agent. In all cases, the major product appeared to be the bis(5'-GMP)(LL)Pt(II) compound. The pH effect is limited; i.e., at pH 4 the reactions proceed somewhat faster than at neutral pH, while at pH 10 slower reactions occur. The illumination with visible light also induces only slight differences in the yields of the products. On the other hand, when vitamin C is present, the reactions proceed quite rapidly, resulting in the same main product but in higher yields (up to 80%). The facts that apparently no Pt(IV) adducts with 5'-GMP can be observed under these conditions and that the major products are bis(5'-GMP)(LL)Pt(II) compounds clearly support the hypothesis that the antitumor activity of certain platinum(IV) compounds is based upon in vivo reduction to the corresponding platinum(II) compounds.

Regeneration experiments of the platinated enzyme fumarase, using sodium diethyldithiocarbamate, thiourea, and sodium thiosulfate.

The enzyme fumarase is inhibited by [cis-Pt(NH3)2(H2O)2] (NO3)2. The Pt compound most likely binds at a S-methionine site. Sodium diethyldithiocarbamate (Naddtc) appears to be a powerful regenerator of enzymatic activity. Thiourea is less active, while sodium thiosulfate (STS) is almost inactive in restoring the activity of the enzyme. The regeneration phenomena are based on the dissociation of the Pt-S bonds of the methionine type, and formation of species like [Pt(ddtc)2]. In the model adduct [Pt(dien)GS-Me]2+ Naddtc, thiourea and STS easily break the Pt-S bond of the methionine type. It is concluded that the model system for Naddtc and thiourea does resemble fumarase quite well. S-donor ligands, which may be used as rescue agents in Pt antitumor therapy, are known to suppress nephrotoxicity caused by [cis-PtCl2(NH3)2]. A parallel is drawn between the enzyme reactivation, modeled by fumarase, and the [cis-PtCl2(NH3)2] nephrotoxicity suppression by rescue agents. It is proposed that a Pt-methionine type binding is broken by the rescue agents Naddtc and thiourea, but that the rescue agent STS only inhibits the nephrotoxicity by inactivating unbound Pt species in the cell.

A simple method for the inactivation of monofunctionally DNA-bound cis-diamminedichloroplatinum (II).

The effects of inorganic salts on the binding of cis-diamminedichloroplatinum(II) (= cis-Pt) to salmon sperm DNA have been investigated for various concentrations of NaCl, NaHCO3, and NH4HCO3. In concentrations of 0.01 M or higher, all three salts strongly inhibit the binding to DNA. However, only NH4HCO3, which may generate NH3, is able to block irreversibly the reactive coordination sites of cis-Pt. This was shown when monofunctionally bound cis-Pt-dGMP was treated with the salts, followed by incubation with an excess of dGMP, after dilution to reduce the salt concentration. The results indicated that monofunctionally bound cis-Pt-dGMP was preserved during the incubation with the excess of dGMP, when NH4HCO3 was used during the pretreatment. Similar treatments with NaCl or NaHCO3, on the other hand, could not prevent the subsequent formation of the bifunctionally bound adduct cis-Pt(NH3)2(dGMP)2. Evidence is given for the formation of Pt(NH3)3dGMP upon treatment of monofunctionally bound cis-Pt-dGMP with NH4HCO3.

Guanine-06 methylation reduces the reactivity of d(GpG) towards platinum complexes.

6-methylated guanine dinucleotides were used to study the influence of hydrogen bonding on the specific binding of the antitumor drug cDDP, cis-PtCl2(NH3)2, to DNA. In this interaction, the guanine-06 site appears to be important in explaining the preference for a pGpG-N7(1),N7(2) chelate, which results from H-bridge formation with the ammine ligand of cDDP. Guanine-06 methylated dinucleotides and the nonmodified dinucleotides were reacted with [Pt(dien)Cl]+, cis-PtCl2(NH3)2, and cis-[Pt(NH3)2(H2O)2]2+ and the reaction products were characterized by 1H NMR using pH titrations. Methylation at guanine-06 clearly reduces the preference for the guanine. In competition experiments monitored by NMR and experiments using UV spectrophotometry a decreasing reactivity towards [Pt(dien)(H2O)]2+ and cis-[Pt(NH3)2(H2O)2]2+ was found, in the order of d(GpG) greater than d(GomepG) greater than d(GpGome) greater than d(GomepGome). The difference in reactivity between 5' guanine methylation and 3' guanine methylation is ascribed to differences in the H-bond formation with the backbone phosphate. The resulting reduced stacking of the bases in both modified dinucleotides, compared to the bases in d(GpG), results in a preference for the 3' guanine over 5'.

The new antitumor compound, cis-[Pt(NH3)2(4-methylpyridine)Cl]Cl, does not form N7,N7-d(GpG) chelates with DNA. An unexpected preference for platinum binding at the 5'G in d(GpG).

The reaction of the antitumor active agent cis-[Pt(NH3)2(4-mepy)Cl]Cl (4-mepy stands for 4-methylpyridine) with d(GpG) has been investigated by 1H magnetic resonance spectroscopy. Initially, two mononuclear complexes cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(1)] 1 and cis-Pt(NH3)2(4-mepy)[d(GpG)-N7(2)] 2 are formed in an unexpected ratio 65:35, as determined by 1H NMR and enzymatic digestion techniques. Both products react further with a second equivalent of cis-[Pt(NH3)2(4-mepy)Cl]Cl forming the dinuclear platinum complex [cis-Pt(NH3)2(4-mepy)]2[mu-d(GpG)- N7(1),N7(2)] 3. With [Pt(dien)Cl]Cl and [Pt(NH3)3Cl]Cl similar complexes are formed. No evidence was found for the formation of chelates cis-Pt(NH3)(4-mepy) [d(GpG)-N7(1),N7(2)], which would be formed upon ammonia release from the mononuclear complexes 1 and 2. Even addition of strong nucleophiles, like sodium diethyldithiocarbamate, thiourea, cysteine, or methionine, before or after reaction, do not induce the formation of a chelate. Under all conditions the N-donor ligands remain coordinated to Pt in 1,2 and 3. In addition, the results of bacterial survival and mutagenesis experiments with E. coli strains show that the in vivo formation of bifunctional adducts in DNA, comparable to those induced by cis-Pt(NH3)2Cl2, by treatment of cells with cis-[Pt(NH3)2(4-mepy)Cl]Cl is unlikely. Also, a mechanism of binding and intercalation is not supported by experimental data. All experiments suggest that the mechanism of action of this new class of antitumor agents must be different from that of cis-Pt(NH3)2Cl2.

Sterically hindered cisplatin derivatives with multiple carboxylate auxiliary arms: synthesis and reactions with guanosine-5'-monophosphate and plasmid DNA.

Two novel sterically hindered cisplatin derivatives with the ligand L=NH(2)C(CH(2)CH(2)COOH)(3) were prepared: cis-PtCl(2)L(2) and cis-PtCl(2)L(NH(3)). The starting compound for the syntheses was NH(2)C(CH(2)CH(2)COOtBu)(3), also known as a building block for dendrimers. cis-PtCl(2)L(2) was prepared from K(2)PtCl(4) in an unusual two-phase reaction in water-chloroform, followed by deprotection of the tert-butyl protective groups with formic acid to yield a water-soluble complex. The mixed-ligand compound cis-PtCl(2)L(NH(3)) was prepared from [PPh(4)][PtCl(3)(NH(3))] in methanol, with subsequent deprotection in formic acid. DNA-binding properties of the two compounds were investigated using the model base guanosine-5'-monophosphate (5'-GMP) and pBR322 plasmid DNA. While cisplatin [cis-PtCl(2)(NH(3))(2)] induced an unwinding of 12 degrees in pBR322 plasmid DNA, cis-PtCl(2)L(NH(3)) induced only 3 degrees unwinding, which is indicative of a monofunctional binding mode. Remarkably, cis-PtCl(2)L(2) did not induce any distortion in plasmid DNA, which strongly suggests that the compound does not bind to DNA. Test reactions with 5'-GMP, monitored by 1H and 195Pt NMR, confirmed that cis-PtCl(2)L(2) is unable to bind to DNA, whereas cis-PtCl(2)L(NH(3)) binds only one nucleotide. Apparently, binding of platinum to nucleotides at the coordination site cis with respect to the ligand L is prevented by steric crowding. Thus, cis-PtCl(2)L(NH(3)) must bind DNA monofunctionally at the trans position. Besides, both compounds have a chloride replaced by one of the carboxylate arms, forming a a seven-membered chelate ring. In theory, cis-PtCl(2)L(2) could also form a second chelate ring, but this was not observed.

Solid-Phase Synthesis of a Monofunctional trans-a(2)Pt(II) Complex Tethered to a Single-Stranded Oligonucleotide.

Cross-linking ability is possible with the oligonucleotide-tethered, monofunctional trans-Pt(II) complex shown. It was synthesized by a novel solid-phase approach comprising conjugation of immobilized tetrathymidylic acid with a trans-a(2)Pt(II) building unit, ammonolysis, and transformation of the resulting complex (R=1-N-cyclohexylmethylthyminate) into the chloro derivative (R=Cl). a=NH(2)CH(3), T=thymine.