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

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<ul><li><p> .Mutation Research 423 1999 171181</p><p>Molecular interactions of ruthenium complexes in isolatedmammalian nuclei and cytotoxicity on V79 cells in culture</p><p>Andrea Barca a, Bianca Pani a, Marisa Tamaro b, Elio Russo a,)a Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Uniersita di Trieste, Via L. Giogieri 1, 34127 Trieste, Italy`</p><p>b Dipartimento di Scienze Biomediche, Uniersita di Trieste, Trieste, Italy`</p><p>Received 15 July 1998; revised 17 November 1998; accepted 18 November 1998</p><p>Abstract</p><p>In this paper, the molecular interactions in isolated mammalian nuclei of three ruthenium complexes, which are putativeantineoplastic chemotherapeutic agents effective in reducing metastatic tumours in vivo, have been investigated and</p><p> .compared with the well-known antitumour drug CDDP cis-diamminedichloroplatinum . The compounds studied are: . . . . .Na trans-RuCl DMSO Imidazole NAMI , Na trans-RuCl DMSO Oxazole NAOX and Na trans-RuCl TMSO -4 4 4</p><p> .Isoquinoline TEQU . 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 antitumourdrugs, whilst in the same experimental conditions, CDDP is confirmed to induce them. On the other hand, proteins appear tobe 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 .NAMI and NAOX do not significantly reduce the cloning efficiency of the cells even at concentrations as high as 23mgrml: 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.</p><p> .Keywords: Cytotoxicity; DNADNA crosslink; DNAprotein crosslink; Ruthenium III complex</p><p>1. Introduction</p><p>Heavy metals coordination complexes such asplatinum, gold, ruthenium, etc. have been investi-</p><p>Abbreviations: CDDP, cis-Diamminedichloroplatinum; NAMI, .N a trans-RuCl D M SO im idazole; N A O X , N a trans-4</p><p> . .RuCl DMSO oxazole; TEQU, Na trans-RuCl TMSO isoquino-4 4line; CFC, Colony forming cells; TMSO, Tetramethilenesulfoxide;FCS, Fetal calf serum; DMEM, Dulbeccos minimal essentialmedium; 6-TG, 6-Thioguanine; HGPRT, Hypoxanthineguaninephosphoribosyl transferase</p><p>) Corresponding author. Tel.: q39-040-676-3679; Fax: q39-040-676-3691; E-mail: russelio@bbcm.univ.trieste.it</p><p>gated for their possible antitumour activity. Amongthe most studied metal complexes, platinum deriva-tives have been shown to be the most promisingchemotherapeutic agents against mammalian tu-mours: at present, CDDP has proven to be veryeffective in clinical therapy of several human solidtumours such as testicular carcinomas, ovarian tu-mours, head and neck cancers, bladder tumours andosteosarcomas; however, it shows only a weak effectagainst many other malignancies of relevant socialincidence such as breast cancers, lung and colorectal</p><p>w xadenocarcinomas 1,2 . For these reasons, new metalcoordination complexes have been studied in order</p><p>0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. .PII: S0027-5107 98 00240-1</p></li><li><p>( )A. Barca et al.rMutation Research 423 1999 171181172</p><p>to find compounds active against tumours that do notrespond, or show resistance, to CDDP and with lesstoxic side effects. In particular, some rutheniumderivatives have been in recent years synthetized andscreened for their antineoplastic activity and for their</p><p>w xcytotoxic effects 3 . Two complexes, namelyw . x w . xcis RuCl NH Cl and fac RuCl NH have2 3 4 3 3 3</p><p>been shown to be active against P388 mouse . .leukemia. Since then, many other Ru III and Ru II</p><p>derivatives have been synthetized and studied, withdifferent ligands like heterocyclic nitrogen ligands .imidazole or indazole or sulfoxides. The release ofchloride ligands should allow the interaction withbiological targets by formation of covalent bonds; sothe nature of ligands affects the biological activity ofruthenium compounds. A mechanism of activationby reduction was claimed to explain their activity:</p><p> .according to this hypothesis Ru III complexes canbe considered as prodrugs which should be activated</p><p> .by reduction to the corresponding Ru II specieswhich in turn should act as the true biological</p><p>w xreagents 4,5 . It should be noted that in solid tumourtissues the environment is considered to be hypoxic,</p><p> .so that the reduction of Ru III to the active species .Ru II is facilitated and at the same time, its reoxida-</p><p>tion becomes unlikely. This fact could induce an .accumulation of active species of Ru II compounds</p><p>just inside the solid tumour tissues, hence, makingthe cytotoxicity of these molecules against tumoursselective with respect to the normal tissues. More-over, because of the similarities between iron andruthenium, the latter seems to enter the cells throughthe Fe-transferrin system: since this transport proteinis more expressed in rapidly growing cells whichshow an increased iron requirement, ruthenium accu-</p><p>w xmulates into neoplastic cells 5 .DNA is generally considered the main target for</p><p>antineoplastic drugs acting as alkylating drugs likew xmetallic complexes 1,68 , but probably other</p><p>molecular interactions are relevant for their biologi-cal effects.</p><p>In this paper, three Ru complexes, namely: Na- . .trans-RuCl DMSO Imidazole NAMI , Na trans-4</p><p> . .RuCl DMSO Oxazole NAOX , and Na trans-4 . .RuCl TMSO Isoquinoline TEQU , whose formulas4</p><p>are shown in Fig. 1, have been investigated for theirinteractions with nuclear chromatin, looking for theformation of DNADNA and DNAproteins</p><p>Fig. 1. Chemical formulas of ruthenium complexes investigated.Abbreviations: Ims imidazole; Iqs Isoquinoline; Ox sOxazole;DMSOsDimethylsulfoxide; TMSOsTetramethylenesulfoxide.</p><p> .crosslinks. This series of ruthenium III complexes,structurally related and characterized by the presenceof sulfoxide and nitrogen-donor ligands, were testedon TLX5 lymphoma and some of them on MCamammary carcinoma in order to evaluate the rela-tionship of cytotoxicity and anti-metastatic activitywith their respective chemical properties. The drugswere shown to be cytotoxic only at high concentra-</p><p> y4 .tions )10 M and their cytotoxicity is related tolipophilicity. The comparison of the in vitro cytotox-icity and in vivo antitumour and antimetastatic activ-ity showed that the reduction of metastasis formationis not related to a direct cytotoxicity on tumour cells.In particular, the most cytotoxic compound, TEQU,is the least effective in reducing metastases, whilstNAMI which is very effective in reducing metastasesformation is slightly cytotoxic on tumour cells in</p><p>w xvitro 9 .In this paper, the cytotoxic activity of the drugs</p><p>has been also studied on mammalian cells in cultureand the mutagenic activity of the most cytotoxic onehas been evaluated in the V79rHGPRT system.</p><p>2. Materials and methods</p><p>2.1. Chemicals</p><p>CDDP, DMEM and DNase I were purchasedfrom Sigma, pBR322, HindIII, RNase and Pro-</p></li><li><p>( )A. Barca et al.rMutation Research 423 1999 171181 173</p><p>teinase K from Boehringer Mannheim, micrococcalnuclease from Amersham International, trypsin fromDifco Laboratories, FCS from BioSpa. Rutheniumcomplexes were synthetized as described and kindly</p><p>supplied by Prof. Mestroni Dipartimento di Scienze. w xChimiche, University of Trieste, Italy 10 . The</p><p>drugs were dissolved in water just before use at theappropriate concentrations. All other chemicals werepurchased from chemical sources.</p><p>2.2. Reactions with complexes</p><p>pBR322 Plasmid was linearized with HindIII,w xaccording to standard protocols 11 , and purified by</p><p>spinning 20 min at 500 g on Microcon 100 Amicon.Grace , changing buffer to 40 mM Tris pH 7.5, 1</p><p> .mM EDTA. 100 ng DNA supercoiled or linear atthe concentration of 25 ngrml were reacted for 1 hat 378C with metal complexes at the desired molarratio. The reactions were quenched with 1.5 MAcONa and 100 mM EDTA, DNA was collected byprecipitation with ethanol, dissolved in 40 mMTrisAcONa pH 8.2, 1 mM EDTA, 10% glyceroland analysed by 1% agarose gel electrophoresis inthe presence of 0.5 mgrml Ethidium Bromide for 1h at 50 V. Samples for denaturation experimentswere heated 2 min at 908C in the presence of 30%DMSO and immediately cooled on ice just before</p><p>w xloading 12 . The following 30-bp double-strandedoligonucleotide was used for footprinting experi-ments:</p><p>5X-CCACCTCCCCCCGGCCCTCCCCTTCCTGCG3X-GGTGGAGGGGGGCCGGGAGGGGAAGGA-</p><p>CGCThe two strands were synthetized separately by</p><p>solid-phase procedures using standard phospho-roamidite chemistry Applied Biosystem model 380</p><p>.B DNA synthetizer . The fully deprotected oligomerswere purified by anion-exchange chromatography</p><p> .using a Mono-Q HR column Pharmacia and even-w 32 xtually labeled with g- P ATP and T4 polynu-</p><p> .cleotide kinase Pharmacia . The two strands weremixed at the right stoichiometry to obtain the doublehelix oligonucleotides with only one strand labeled.A total of 0.4 pmoles were reacted 12 h at 378C with</p><p> .an excess of Ru complex 10 pmoles in 10 ml Tris50 mMNaCl 10 mM. Then the solutions were made</p><p>10 mM MgCl and treated with 1 ng of DNase I at2378C. Aliquots were taken at different times,quenched with 100 mM EDTA and analyzed byelectrophoresis in 20% denaturing polyacrylamidegels in the presence of 8 M urea at 458C. Followingelectrophoresis, the gels were transferred onto What-man 3MM chromatographic paper, dried and ex-posed to autoradiography for about 8 h.</p><p>w xNuclei from rat thymus prepared as described 13and stored in the presence of 50% glycerol at y208C</p><p>6 were treated as follows: 80=10 nuclei ;500 mg.DNA were incubated for 8 h at 378C with the</p><p>complexes at different concentrations expressed as. molrbp , in 500 ml RSB 10 mM TrisHCl pH 7.4,</p><p>.10 mM NaCl, 5 mM MgCl . The reactions were2stopped by cooling samples on ice, centrifuging at1000=g and resuspending in 500 ml cold RSB.</p><p>For the digestion with micrococcal nuclease, sam- .ples ;50 mg DNA each were suspended in 50 ml</p><p>10 mM TrisHCl pH 7.4, 1 mM CaCl and digested2at 378C with 25 units of micrococcal nuclease. Fiveminutes incubation were sufficient in order to obtainmainly mono- and a little amount of oligo-nucleo-somes in untreated nuclei. The reactions werequenched by adding 5 ml 5% SDS and 50 mMEDTA and cooling on ice. Samples were then depro-teinized by incubating twice for 3 h at 508C with 5mg of Proteinase K, adjusted to 1 M NaCl and</p><p> .extracted with chloroformrisoamyl alcohol 24:1 .DNA, collected by precipitation with ethanol, was</p><p>dissolved in 200 ml TBE 90 mM Tris, 80 mM.H BO , 2.5 mM EDTA pH 8 with 10% glycerol3 3</p><p>and analysed by 1.5% agarose gel electrophoresis inTBE in the presence of 0.5 mgrml Ethidium Bro-mide for 2 h at 70 V.</p><p>For the digestion with DNAse I, nuclei ;200.mg DNA were suspended in 200 ml 10 mM Tris</p><p>HCl pH 7.4, 5 mM EDTA, 0.5% SDS and digestedfor 1 h at 378C with 20 mg RNase, then twice for 3 hat 508C with 20 mg Proteinase K. Samples, adjustedto 1 M NaCl, were extracted with chloroformriso-</p><p> .amyl alcohol 24:1 and DNA was collected byprecipitation with ethanol. A total of 25 mg purifiedDNA were digested at 208C with 0.5 units of DNaseI in 1 ml 10 mM TrisHCl pH 7.4, 2 mM MnCl .2The reaction was followed by a Jasco V550 spectro-photometer at 260 nm in a 1 cm quartz cell. Datawere collected starting 30 s after the addition of the</p></li><li><p>( )A. Barca et al.rMutation Research 423 1999 171181174</p><p>enzyme and plotted as absorbance increase againsttime, showing a linear trend and allowing the calcu-lation of initial digestion rate. The percent inhibition .I of digestion rate is expressed by the equation:Is 1yr 100 .t cwhere and are initial DNA digestion rates fromt crespectively treated and control nuclei.</p><p> .Nuclei ;200 mg DNA each were extractedovernight with 400 ml 0.4 M H SO and free pro-2 4teins were determined in the supernatant through the</p><p>w xBradford method 14 .</p><p>2.3. DNA melting</p><p> .Untreated and treated nuclei ;50 mg DNA eachwere suspended in 50 ml 10 mM TrisHCl pH 7.4, 5mM EDTA, 0.5% SDS and digested for 1 h at 378Cwith 20 mg RNase, then twice for 3 h at 508C with20 mg Proteinase K. Samples, adjusted to 1 M NaCl,were extracted with chloroformrisoamyl alcohol .24:1 and DNA collected by pre-cipitation withethanol was redissolved in 1.5 ml of SDS 1%,transferred in quartz cells and the absorbance at 260nm was recorded by a Jasco V 550 spectrophotom-eter, changing the temperature by 18Crmin.</p><p>2.4. Cytotoxicity and mutagenicity assay on V79Chinese hamster cells</p><p>For the evaluation of cytotoxicity of Ru com-pounds, 22.5=106 V79 Chinese Hamster cellswere plated on 2000 mm2 Petri dishes in DMEMsupplemented with FCS, 100 UIrml penicillin and100 grml streptomycin. After 24 h incubation at378C in a humidified CO incubator, the medium2was replaced with fresh DMEM without FCS con-taining different concentrations of each Ru complexobtained by dilution of freshly prepared 10 mgrmlsolutions of each compound. All experiments in-cluded a control culture, in which the medium wasreplaced with fresh DMEM without FCS. After 1 hincubation, treated and untreated cultures werewashed three times with medium without FCS, andthen detached from the plates with trypsin. When</p><p> .necessary see Section 4 , the cells were detachedmechanically with a rubber policeman. The cells ofeach treated or untreated culture were then re-sus-</p><p>pended in DMEM with FCS, the suspensions werecounted in a Burker haemocytometer and replated at</p><p> 2low density 200 cellsr2000 mm Petri dish, four.replicates .</p><p>The cloning efficiency was evaluated after 7 daysincubation by direct counting of more than 100 cellscolonies after staining with 0.1% methylene blue.The cloning efficiency is expressed as percent CFCwith respect to the control culture.</p><p>The mutagenesis assay was performed accordingw xto ONeill with minor modifications 15 . Together</p><p>with the cultures performed to evaluate the cloningefficiency, subcultures of 7.5=105 cells in 8000mm2 dishes were grown from each treated or un-</p><p>treated sample. Six to eight days later which is thepreviously determined appropriate expression time</p><p>.for HGPRT mutants , cells from each subculturewere detached with trypsin, re-suspended in com-</p><p> . 5plete medium and re-plated as follows: a 2=102 .cells per 8000 mm dish five replicates to which, 1</p><p> .h after plating, 6-TG 4 mgrml final concentration .was added in order to select HGPRT mutants. b</p><p>2 2 .2=10 cells per 2000 mm dish four replicates forthe evaluation of CFC.</p><p>After further 7 days incubation, colonies werescored by staining with methylene blue in both seriesof plates in order to evaluate the number of 6-TGresistant mutants per 106 CFC.</p><p>3. Results</p><p>3.1. Reaction with naked DNA</p><p>We used linearized pBR322 DNA to investigatewhether the complexes are able to induce crosslinks</p><p>w xon DNA 12 . After treatment with the drugs at thedesired molrbp ratio, samples were purified by pre-cipita...</p></li></ul>