Molecular interactions of ruthenium complexes in isolated mammalian nuclei and cytotoxicity on V79 cells in culture

Mutation Research 423 Ž1999. 171–181

Molecular interactions of ruthenium complexes in isolated mammalian nuclei and cytotoxicity on V79 cells in culture Andrea Barca a , Bianca Pani a , Marisa Tamaro b, Elio Russo a

a,)

Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, UniÕersita` di Trieste, Via L. Giogieri 1, 34127 Trieste, Italy b Dipartimento di Scienze Biomediche, UniÕersita` di Trieste, Trieste, Italy Received 15 July 1998; revised 17 November 1998; accepted 18 November 1998

Abstract In this paper, the molecular interactions in isolated mammalian nuclei of three ruthenium complexes, which are putative antineoplastic chemotherapeutic agents effective in reducing metastatic tumours in vivo, have been investigated and compared with the well-known antitumour drug CDDP Ž cis-diamminedichloroplatinum.. The compounds studied are: Na trans-RuCl 4ŽDMSO.Imidazole ŽNAMI., Na trans-RuCl 4ŽDMSO.Oxazole ŽNAOX. and Na trans-RuCl 4ŽTMSO.Isoquinoline ŽTEQU.. This study shows that the drugs bind to DNA but induce few, if any, DNA interstrand crosslinks, which are considered as the main biological lesions involved in the cytotoxic activity of several already known antitumour drugs, whilst in the same experimental conditions, CDDP is confirmed to induce them. On the other hand, proteins appear to be an important target in the cell for these drugs, since proteins-DNA crosslinks are shown to be induced by the complexes. Moreover, we investigated Ru complexes for their direct cytotoxicity on V79 cells in culture, showing that two of them ŽNAMI and NAOX. do not significantly reduce the cloning efficiency of the cells even at concentrations as high as 2–3 mgrml: only TEQU both reduces cloning efficiency and induces a significant number of mutants in V79 cells in culture. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Cytotoxicity; DNA–DNA crosslink; DNA–protein crosslink; RutheniumŽIII. complex

1. Introduction Heavy metals coordination complexes such as platinum, gold, ruthenium, etc. have been investiAbbreviations: CDDP, cis-Diamminedichloroplatinum; NAMI, N a trans-R uC l 4 Ž D M S O . im idazole; N A O X , N a transRuCl 4 ŽDMSO.oxazole; TEQU, Na trans-RuCl 4 ŽTMSO.isoquinoline; CFC, Colony forming cells; TMSO, Tetramethilenesulfoxide; FCS, Fetal calf serum; DMEM, Dulbecco’s minimal essential medium; 6-TG, 6-Thioguanine; HGPRT, Hypoxanthine–guanine phosphoribosyl transferase ) Corresponding author. Tel.: q39-040-676-3679; Fax: q39040-676-3691; E-mail: [email protected]

gated for their possible antitumour activity. Among the most studied metal complexes, platinum derivatives have been shown to be the most promising chemotherapeutic agents against mammalian tumours: at present, CDDP has proven to be very effective in clinical therapy of several human solid tumours such as testicular carcinomas, ovarian tumours, head and neck cancers, bladder tumours and osteosarcomas; however, it shows only a weak effect against many other malignancies of relevant social incidence such as breast cancers, lung and colorectal adenocarcinomas w1,2x. For these reasons, new metal coordination complexes have been studied in order

0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 8 . 0 0 2 4 0 - 1

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to find compounds active against tumours that do not respond, or show resistance, to CDDP and with less toxic side effects. In particular, some ruthenium derivatives have been in recent years synthetized and screened for their antineoplastic activity and for their cytotoxic effects w3x. Two complexes, namely ciswRuCl 2 ŽNH 3 .4 xCl and facwRuCl 3 ŽNH 3 . 3 x have been shown to be active against P388 mouse leukemia. Since then, many other RuŽIII. and RuŽII. derivatives have been synthetized and studied, with different ligands like heterocyclic nitrogen ligands Žimidazole or indazole. or sulfoxides. The release of chloride ligands should allow the interaction with biological targets by formation of covalent bonds; so the nature of ligands affects the biological activity of ruthenium compounds. A mechanism of ‘activation by reduction’ was claimed to explain their activity: according to this hypothesis RuŽIII. complexes can be considered as prodrugs which should be activated by reduction to the corresponding RuŽII. species which in turn should act as the true biological reagents w4,5x. It should be noted that in solid tumour tissues the environment is considered to be hypoxic, so that the reduction of RuŽIII. to the active species RuŽII. is facilitated and at the same time, its reoxidation becomes unlikely. This fact could induce an accumulation of active species of RuŽII. compounds just inside the solid tumour tissues, hence, making the cytotoxicity of these molecules against tumours selective with respect to the normal tissues. Moreover, because of the similarities between iron and ruthenium, the latter seems to enter the cells through the Fe-transferrin system: since this transport protein is more expressed in rapidly growing cells which show an increased iron requirement, ruthenium accumulates into neoplastic cells w5x. DNA is generally considered the main target for antineoplastic drugs acting as alkylating drugs like metallic complexes w1,6–8x, but probably other molecular interactions are relevant for their biological effects. In this paper, three Ru complexes, namely: Na trans-RuCl 4 ŽDMSO.Imidazole ŽNAMI., Na transRuCl 4 ŽDMSO.Oxazole ŽNAOX., and Na transRuCl 4ŽTMSO.Isoquinoline ŽTEQU., whose formulas are shown in Fig. 1, have been investigated for their interactions with nuclear chromatin, looking for the formation of DNA–DNA and DNA–proteins

Fig. 1. Chemical formulas of ruthenium complexes investigated. Abbreviations: Im s imidazole; Iq s Isoquinoline; Ox sOxazole; DMSOs Dimethylsulfoxide; TMSOs Tetramethylenesulfoxide.

crosslinks. This series of rutheniumŽIII. complexes, structurally related and characterized by the presence of sulfoxide and nitrogen-donor ligands, were tested on TLX5 lymphoma and some of them on MCa mammary carcinoma in order to evaluate the relationship of cytotoxicity and anti-metastatic activity with their respective chemical properties. The drugs were shown to be cytotoxic only at high concentrations Ž) 10y4 M. and their cytotoxicity is related to lipophilicity. The comparison of the in vitro cytotoxicity and in vivo antitumour and antimetastatic activity showed that the reduction of metastasis formation is not related to a direct cytotoxicity on tumour cells. In particular, the most cytotoxic compound, TEQU, is the least effective in reducing metastases, whilst NAMI which is very effective in reducing metastases formation is slightly cytotoxic on tumour cells in vitro w9x. In this paper, the cytotoxic activity of the drugs has been also studied on mammalian cells in culture and the mutagenic activity of the most cytotoxic one has been evaluated in the V79rHGPRT system. 2. Materials and methods 2.1. Chemicals CDDP, DMEM and DNase I were purchased from Sigma, pBR322, HindIII, RNase and Pro-

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teinase K from Boehringer Mannheim, micrococcal nuclease from Amersham International, trypsin from Difco Laboratories, FCS from BioSpa. Ruthenium complexes were synthetized as described and kindly supplied by Prof. Mestroni ŽDipartimento di Scienze Chimiche, University of Trieste, Italy. w10x. The drugs were dissolved in water just before use at the appropriate concentrations. All other chemicals were purchased from chemical sources. 2.2. Reactions with complexes pBR322 Plasmid was linearized with HindIII, according to standard protocols w11x, and purified by spinning 20 min at 500 g on Microcon 100 ŽAmicon Grace., changing buffer to 40 mM Tris pH 7.5, 1 mM EDTA. 100 ng DNA Žsupercoiled or linear. at the concentration of 25 ngrml were reacted for 1 h at 378C with metal complexes at the desired molar ratio. The reactions were quenched with 1.5 M AcONa and 100 mM EDTA, DNA was collected by precipitation with ethanol, dissolved in 40 mM Tris–AcONa pH 8.2, 1 mM EDTA, 10% glycerol and analysed by 1% agarose gel electrophoresis in the presence of 0.5 mgrml Ethidium Bromide for 1 h at 50 V. Samples for denaturation experiments were heated 2 min at 908C in the presence of 30% DMSO and immediately cooled on ice just before loading w12x. The following 30-bp double-stranded oligonucleotide was used for footprinting experiments: 5X-CCACCTCCCCCCGGCCCTCCCCTTCCTGCG 3X-GGTGGAGGGGGGCCGGGAGGGGAAGGACGC The two strands were synthetized separately by solid-phase procedures using standard phosphoroamidite chemistry ŽApplied Biosystem model 380 B DNA synthetizer.. The fully deprotected oligomers were purified by anion-exchange chromatography using a Mono-Q HR column ŽPharmacia. and eventually labeled with w g-32 PxATP and T4 polynucleotide kinase ŽPharmacia.. The two strands were mixed at the right stoichiometry to obtain the double helix oligonucleotides with only one strand labeled. A total of 0.4 pmoles were reacted 12 h at 378C with an excess of Ru complex Ž10 pmoles. in 10 ml Tris 50 mM–NaCl 10 mM. Then the solutions were made

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10 mM MgCl 2 and treated with 1 ng of DNase I at 378C. Aliquots were taken at different times, quenched with 100 mM EDTA and analyzed by electrophoresis in 20% denaturing polyacrylamide gels in the presence of 8 M urea at 458C. Following electrophoresis, the gels were transferred onto Whatman 3MM chromatographic paper, dried and exposed to autoradiography for about 8 h. Nuclei from rat thymus prepared as described w13x and stored in the presence of 50% glycerol at y208C were treated as follows: 80 = 10 6 nuclei Ž; 500 mg DNA. were incubated for 8 h at 378C with the complexes at different concentrations Žexpressed as molrbp., in 500 ml RSB Ž10 mM Tris–HCl pH 7.4, 10 mM NaCl, 5 mM MgCl 2 .. The reactions were stopped by cooling samples on ice, centrifuging at 1000 = g and resuspending in 500 ml cold RSB. For the digestion with micrococcal nuclease, samples Ž; 50 mg DNA each. were suspended in 50 ml 10 mM Tris–HCl pH 7.4, 1 mM CaCl 2 and digested at 378C with 25 units of micrococcal nuclease. Five minutes incubation were sufficient in order to obtain mainly mono- and a little amount of oligo-nucleosomes in untreated nuclei. The reactions were quenched by adding 5 ml 5% SDS and 50 mM EDTA and cooling on ice. Samples were then deproteinized by incubating twice for 3 h at 508C with 5 mg of Proteinase K, adjusted to 1 M NaCl and extracted with chloroformrisoamyl alcohol Ž24:1.. DNA, collected by precipitation with ethanol, was dissolved in 200 ml TBE Ž90 mM Tris, 80 mM H 3 BO 3 , 2.5 mM EDTA pH 8. with 10% glycerol and analysed by 1.5% agarose gel electrophoresis in TBE in the presence of 0.5 mgrml Ethidium Bromide for 2 h at 70 V. For the digestion with DNAse I, nuclei Ž; 200 mg DNA. were suspended in 200 ml 10 mM Tris– HCl pH 7.4, 5 mM EDTA, 0.5% SDS and digested for 1 h at 378C with 20 mg RNase, then twice for 3 h at 508C with 20 mg Proteinase K. Samples, adjusted to 1 M NaCl, were extracted with chloroformrisoamyl alcohol Ž24:1. and DNA was collected by precipitation with ethanol. A total of 25 mg purified DNA were digested at 208C with 0.5 units of DNase I in 1 ml 10 mM Tris–HCl pH 7.4, 2 mM MnCl 2 . The reaction was followed by a Jasco V550 spectrophotometer at 260 nm in a 1 cm quartz cell. Data were collected starting 30 s after the addition of the

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enzyme and plotted as absorbance increase against time, showing a linear trend and allowing the calculation of initial digestion rate. The percent inhibition Ž I . of digestion rate is expressed by the equation: I s Ž 1 y ÕtrÕc . 100 where Õt and Õc are initial DNA digestion rates from respectively treated and control nuclei. Nuclei Ž; 200 mg DNA each. were extracted overnight with 400 ml 0.4 M H 2 SO4 and free proteins were determined in the supernatant through the Bradford method w14x. 2.3. DNA melting Untreated and treated nuclei Ž; 50 mg DNA each. were suspended in 50 ml 10 mM Tris–HCl pH 7.4, 5 mM EDTA, 0.5% SDS and digested for 1 h at 378C with 20 mg RNase, then twice for 3 h at 508C with 20 mg Proteinase K. Samples, adjusted to 1 M NaCl, were extracted with chloroformrisoamyl alcohol Ž24:1. and DNA collected by pre-cipitation with ethanol was redissolved in 1.5 ml of SDS 1%, transferred in quartz cells and the absorbance at 260 nm was recorded by a Jasco V 550 spectrophotometer, changing the temperature by 18Crmin. 2.4. Cytotoxicity and mutagenicity assay on V79 Chinese hamster cells For the evaluation of cytotoxicity of Ru compounds, 2–2.5 = 10 6 V79 Chinese Hamster cells were plated on 2000 mm2 Petri dishes in DMEM supplemented with FCS, 100 UIrml penicillin and 100 grml streptomycin. After 24 h incubation at 378C in a humidified CO 2 incubator, the medium was replaced with fresh DMEM without FCS containing different concentrations of each Ru complex obtained by dilution of freshly prepared 10 mgrml solutions of each compound. All experiments included a control culture, in which the medium was replaced with fresh DMEM without FCS. After 1 h incubation, treated and untreated cultures were washed three times with medium without FCS, and then detached from the plates with trypsin. When necessary Žsee Section 4., the cells were detached mechanically with a rubber policeman. The cells of each treated or untreated culture were then re-sus-

pended in DMEM with FCS, the suspensions were counted in a Burker haemocytometer and replated at ¨ low density Ž200 cellsr2000 mm2 Petri dish, four replicates.. The cloning efficiency was evaluated after 7 days incubation by direct counting of more than 100 cells colonies after staining with 0.1% methylene blue. The cloning efficiency is expressed as percent CFC with respect to the control culture. The mutagenesis assay was performed according to O’Neill with minor modifications w15x. Together with the cultures performed to evaluate the cloning efficiency, subcultures of 7.5 = 10 5 cells in 8000 mm2 dishes were grown from each treated or untreated sample. Six to eight days later Žwhich is the previously determined appropriate expression time for HGPRT mutants., cells from each subculture were detached with trypsin, re-suspended in complete medium and re-plated as follows: Ža. 2 = 10 5 cells per 8000 mm2 dish Žfive replicates. to which, 1 h after plating, 6-TG Ž4 mgrml final concentration. was added in order to select HGPRT mutants. Žb. 2 = 10 2 cells per 2000 mm2 dish Žfour replicates. for the evaluation of CFC. After further 7 days incubation, colonies were scored by staining with methylene blue in both series of plates in order to evaluate the number of 6-TG resistant mutants per 10 6 CFC.

3. Results 3.1. Reaction with naked DNA We used linearized pBR322 DNA to investigate whether the complexes are able to induce crosslinks on DNA w12x. After treatment with the drugs at the desired molrbp ratio, samples were purified by precipitation with ethanol to remove the unreacted complexes and then thermally denaturated and renaturated just before loading on agarose gel. Denaturated linear single-stranded ŽSS. DNA is well resolved from double stranded DNA ŽDS. since the former migrates faster and is stained to a less extent by Ethidium Bromide. Fig. 2 shows that, as previously determined, in CDDP treated samples DS DNA is still present after denaturation, whilst in Ru treated samples DS DNA is found only in TEQU.

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Fig. 2. Agarose gel electrophoresis of linearized pBR322 plasmid treated with drugs and thermally denaturated. Drug concentrations are expressed as molrbp. DS s double strand DNA; SS s Single strand DNA.

When a double-strand break is introduced into DNA the supercoiled form ŽS. of the plasmid is transformed in the linear one ŽL., while in the case of the formation of single-strand nicks an increase in the amount of the relaxed form ŽR. is noted. These three forms are easily separated by agarose electrophoresis in the presence of Ethidium Bromide. Fig. 3 shows the electrophoresis of circular pBR322 plasmid treated with the drugs at different concentrations. It is evident that no compound examined is able to introduce double-strand breaks since L band does not appear in any case. The scanning of electrophoresis photos gives the ratio R formrtotal DNA. Without drugs, ; 20% of plasmid is in the R band and at a concentration of 0.5 molrbp that amount increases by 28% with CDDP, by 1% with NAMI, by 2% with NAOX and by 4% with TEQU. At our knowledge that is the first time that one demonstrates that CDDP introduces single strand nicks in DNA. Since the effect of the treatment with the Ru complexes is very slight, we investigated Ru drugs at higher concentrations obtaining increments in the R form of 4% for NAMI and 7% for NAOX at 2 molrbp, and of 12% for TEQU at 1 molrbp, so confirming the effects induced by these drugs.

Dnase I footprinting experiments were performed on a 30-bp DNA composed by a pyrimidine ŽPY. and a purine ŽPU. strand, that contains respectively

Fig. 3. Agarose gel electrophoresis of supercoiled pBR322 plasmid treated with drugs. Drug concentrations are expressed as molrbp. R s relaxed, Sssupercoiled, L s linear plasmid.

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74% of CT or of GA bases. The DNA fragment was alternatively labeled on one strand and the test was done to detect whether complexes bind to nucleotides and to stress preferences for bases. In Fig. 4, we show results obtained with TEQU. All treated oligonucleotides are less digested than untreated ones. The effect for the PU-strand is not very strong, but it is a little more evident for the PY-strand. These results suggest that the drug binds to DNA without altering too much its conformation and that there is more affinity towards pyrimidinic moieties. 3.2. Reaction with nuclei The in vivo situation was approached using purified nuclei to test complexes, extending incubation time to 8 h, since preliminary tests revealed no appreciable variations yet after 4 h. Nuclei washed

and collected through centrifugation to remove the unreacted drugs appeared yellow stained, confirming that complexes had bound to chromatin. We have limited maximum concentration range to 0.5 molrbp since molarity of drugs was relatively elevated and at higher values, self-polymerization of Ru complexes occurred w16x. Actually with NAOX that happened even at 0.5 molrbp, so to compromise further handling of samples especially in preventing the complete extraction of different components; therefore data obtained with NAOX at 0.5 molrbp are not well comparable with those obtained with other complexes. Micrococcal nuclease digestion is affected by modifications in the higher order chromatin, since its first target is linker DNA, without any sequence specificity. In fact, Pt treated nuclei are more resistant to the enzyme than untreated ones w17x. Fig. 5

Fig. 4. DNA footprinting with Dnase I. Lanes 1, 2, 5, 6, 9, 10, 13 and 14 contain 0.4 pmoles of the oligonucleotide with the PY strand X X 5 -labelled. Lanes 3, 4, 7, 8, 11, 12, 15 and 16 contain 0.4 pmoles of the oligonucleotide with the PU strand 5 -labelled. The untreated DNA is in the odd lanes, whilst the treated DNA is in the pair lanes. Digestion times with DNA I: lanes 1–4, 30 s; lanes 5–8, 45 s; lanes 9–12, 60 s; lanes 13–16, 120 s.

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Fig. 5. Agarose gel electrophoresis of nuclei digested with micrococcal nuclease. Drug concentrations are expressed as molrbp. A s 0; B s 0.125; C s 0.25; D s 0.5. M s mono, D s di-, T s tri- and Te s tetranucleosomes.

shows the results obtained when digestion times were adjusted in order to obtain mainly mono- ŽM. and di-nucleosomes ŽD. in control nuclei Žlanes A.. The electrophoretic patterns of samples from treated nuclei Žlanes B, C and D. show particles of increasing size like tri- ŽT. and tetra-nucleosomes ŽTe., with the characteristic 200 base pairs pattern. This effect is clearly concentration dependent and is very similar for NAMI and TEQU, showing some difference for NAOX at the highest concentration, as above discussed. These results reflect DNA distortions caused by the binding of the drugs to chromatin, as already demonstrated for CDDP w18x. The reduced activity of micrococcal nuclease in treated samples could depend on modifications induced in chromatin bound proteins; therefore, after the treatment with the complexes, we have digested all proteins with Proteinase K and have treated the purified DNA with DNase I using as metallic cofactor Mnqq, to minimize differences due to the DNA sequence w19x. The initial digestion rates plotted against drug concentration were linear from 30 until to at least 250 s after the addition of the enzyme. Fig. 6 shows the reduction of initial digestion rate due to the drugs. The effect is very low for TEQU, quite pronounced for NAOX, whilst NAMI and CDDP induce a comparable intermediate rate reduction. CDDP data are consistent with previously reported results w20x.

3.3. DNA melting The ability of Ru complexes to introduce DNA interstrand crosslinks in nuclei was investigated by melting experiments. Purified DNA was first heated

Fig. 6. Percent inhibition of the DNase I digestion of nuclei treated at different drugs concentrations.

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to 808C Žabove melting temperature., and then cooled to 408C. Absorbances at 260 nm were recorded, and normalized values were plotted against temperature ŽFig. 7.. In these conditions, the renaturation of mammalian genomic DNA is normally very limited, only 6–7% reverting to the native conformation, but the presence of interstrand crosslinks favours the exact base pairing so promoting a rapid renaturation w21x. The validity of determining the fraction of fast renaturing DNA after denaturation to measure crosslink amount has been demonstrated by Pera et al. w22x who have compared this method with alkaline elution and DNA sedimentation obtaining similar results. The spectrophotometric analysis shows also the cooperative trend of the event. Whilst DNA from CDDP treated nuclei shows an amount of fast renaturing DNA which increases with the drug concentration Žcurves B, C and D., DNA from nuclei treated with Ru complexes at 0.5 molrbp renatures very poorly, at an extent very similar for the three drugs Žcurves E, F and G. and for the untreated sample Žcurve A.. These data are not completely in agreement with previously described results ŽFig. 2., where we demonstrated that TEQU was able to introduce crosslinks in DNA.

Fig. 8. Percent retention of proteins extracted from nuclei treated at different drugs concentrations.

3.4. DNA–proteins crosslinks We investigated the ability of Ru compounds to induce DNA–protein crosslinks by measuring the total amount of freely extractable nuclear proteins. Fig. 8 shows the retention of proteins evaluated as difference of protein concentrations in solution between drug treated and untreated nuclei through the Bradford method w14x. The results obtained with CDDP confirm its ability to bind DNA to nuclear proteins w23x. On the other hand, ruthenium complexes induce a concentration dependent retention of proteins very similar to that of CDDP. Unfortunately, this experiment does not allow to recognize specific proteins bound to DNA. Therefore, we can only suppose that at these relatively high drug concentrations the binding involves mainly histones. 3.5. Genotoxic actiÕity on mammalian cells in culture

Fig. 7. Renaturation profiles of DNA extracted from nuclei treated at different drugs concentrations. Concentrations are expressed as molrbp. As untreated DNA; B s 0.125, C s 0.25, Ds 0.5 mol CDDPrbp; Es 0.5 mol NAMIrbp; F s 0.5 mol NAOXrbp; Gs 0.5 mol TEQUrbp.

The cytotoxic activity of the three Ru compounds was also evaluated on V79 Chinese Hamster cells, by measuring the cloning efficiency of cells plated at

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low density Ž200 cells per 50 mm dish. after treatment of confluence culture with the drugs. Fig. 9a shows the surviving fraction of V79 cells plotted against drug concentrations: it is noteworthy that among the three drugs only TEQU, which on the other hand had shown a very weak interaction with chromatin and DNA, seems to display a relevant cytotoxic activity, with a clear concentration-dependent exponential trend. The same compound was also tested for the mutagenic activity, showing also the ability to induce 6-TG resistant mutants in V79 cells, with a linear concentration-dependent trend ŽFig. 9b.. It should be noted that CDDP has also been studied for its cytotoxic and mutagenic activity on V79 cells showing that this antitumour drug is very active at concentrations as low as 10y5 M w24x while TEQU, which is the most cytotoxic of the three compounds tested in this study, reduces the surviving fraction of less than 10% only at concentration as high as 10y3 M. On the other hand, NAMI

Fig. 9. Cytotoxic and mutagenic activity of drugs on V79 cells. Ža. Log of the surviving fraction; ' s NAMI, v s NAOX, B s TEQU. Žb. Number of mutantsr10 6 CFC in TEQU-treated cells. Points are the mean values from at least three independent experiments.

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and NAOX in a range between 1 and 5 = 10y3 M are almost devoid of cytotoxicity on V79 cells.

4. Discussion In this paper, we compare three ruthenium compounds, which have shown promising antitumour and antimetastatic activity on experimental tumours w9x, with the well-known antitumour drug CDDP. Since previous studies had shown that the main target for this drug is DNA w1x, we have focused our attention on the characterization of DNA adducts induced by the compounds. It is widely accepted that the biological activity of antitumour drugs like CDDP correlates with their ability to interact with DNA, but there is a large debate on which adducts are responsible for the different biological effects w8x. Many authors w25,26x suppose that the more relevant effect of CDDP is the formation of interstrand crosslinks, even if they represent no more than 5% of total DNA–DNA crosslinks introduced by the drug; moreover a recent paper on polypyridyl ruthenium complexes shows a correlation between cytotoxicity in mammalian cells in culture and interstrand crosslinks formation w27x. NAMI, NAOX and TEQU bind covalently to DNA as demonstrated by the inhibition of nucleases, but of them only TEQU induces interstrand crosslinks. In fact, the renaturation profiles of thermally denaturated DNA after treatment with the drugs show no variation with respect to untreated DNA, while the experiments with linear pBR322 show that in TEQU treated DNA there are interstrand crosslinks. The discrepancy may be ascribed to the different sensibility between the two methods involved, as demonstrated in the case of the drug angelicin w28,29x, so suggesting that the total number of interstrand crosslinks is low. In a previous work on the same Ru compounds w16x, the authors had shown the formation of DNA–DNA interstrand crosslinks by all three complexes, in amounts comparable to that of CDDP. Their experiments were performed by the Ethidium Bromide method w30x, so we suggest that the reduced ability of treated DNA to uptake the dye was determined by the presence of intra-strand crosslinks, which prevent helix extension and unwinding to accommodate the intercalating

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molecules. In this view their results are in agreement with our data on digestion with DNase I, since in both experiments NAMI and NAOX give results very similar to those of CDDP, while TEQU shows a less pronounced inhibition of the enzyme activity. Furthermore, CDDP has been previously demonstrated to induce covalent protein–DNA crosslinks: this ability is also confirmed by our data on protein retention, which demonstrate that also NAMI, NAOX and TEQU are able to crosslink proteins to DNA in treated nuclei. The reaction is supposed to involve mainly histones, even if the retention of specific proteins present in very small amount cannot be demonstrated by the method used. It is difficult to find a correlation between these results and the demonstrated activity of these drugs against tumour cells in vitro and tumour growth and metastases in vivo w9x. In fact, TEQU displayed a significant cytotoxic activity against TXL5 lymphoma cells in vitro but only a weak activity in vivo against tumour growth and lung metastases. On the contrary, NAMI and NAOX are completely devoid of in vitro cytotoxic activity; conversely, they are able to reduce primary tumours growth in vivo and to inhibit even more significantly the formation of lung metastases in mice w9,31x. It should be noted that the cytotoxic activity of the three Ru compounds against tumour cell lines in vitro w9x is consistent with our reported in vitro cytotoxic activity on V79 cells. Among the three drugs tested, only TEQU has been shown to be cytotoxic on these cells; on the same cell line it showed also the ability to induce point mutations. We want to stress the fact that TEQU cytotoxic activity correlates with its ability to induce covalent bridges between the two DNA strands, so reinforcing the hypothesis that binds cytotoxicity to interstrand crosslinks formation. Our results do not approach the effect of reduction of Ru ions. At present they are inadequate to fully explain the biological activity of NAMI, NAOX and TEQU. They indicate DNA as the most relevant but not the only biological target for the three ruthenium compounds and they correlate roughly to the effects on primary tumours in mice, but they do not provide any explanation for the antimetastatic activity. In this respect we want to mention that V79 cells, after treatment with these drugs for cytotoxic-

ity and mutagenicity assays, became hardly detachable from dishes by trypsin Ždata not shown.. Of course, this finding needs further investigation to be explained, but it could suggest that cell matrix molecules are modified by the drugs, changing cell adhesion properties, which are critical for the metastasis of tumour cells; in any case our observation supports the hypothesis that in addition to DNA other relevant molecules in the cell are involved in the antitumour activity of ruthenium complexes.

Acknowledgements This work was supported by the Italian Ministero dell’Universita` e della Ricerca Scientifica.

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