RESUMEN
Self-assembled enantiomers of an asymmetric di-iron metallohelix differ in their antiproliferative activities against HCT116 colon cancer cells such that the compound with Λ-helicity at the metals becomes more potent than the Δ compound with increasing exposure time. From concentration- and temperature-dependent 57Fe isotopic labelling studies of cellular accumulation we postulate that while the more potent Λ enantiomer undergoes carrier-mediated efflux, for Δ the process is principally equilibrative. Cell fractionation studies demonstrate that both enantiomers localise in a similar fashion; compound is observed mostly within the cytoskeleton and/or genomic DNA, with significant amounts also found in the nucleus and membrane, but with negligible concentration in the cytosol. Cell cycle analyses using flow cytometry reveal that the Δ enantiomer induces mild arrest in the G1 phase, while Λ causes a very large dose-dependent increase in the G2/M population at a concentration significantly below the relevant IC50. Correspondingly, G2-M checkpoint failure as a result of Λ-metallohelix binding to DNA is shown to be feasible by linear dichroism studies, which indicate, in contrast to the Δ compound, a quite specific mode of binding, probably in the major groove. Further, spindle assembly checkpoint (SAC) failure, which could also be responsible for the observed G2/M arrest, is established as a feasible mechanism for the Λ helix via drug combination (synergy) studies and the discovery of tubulin and actin inhibition. Here, while the Λ compound stabilizes F-actin and induces a distinct change in tubulin architecture of HCT116 cells, Δ promotes depolymerization and more subtle changes in microtubule and actin networks.
Asunto(s)
Neoplasias del Colon , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/metabolismo , Actinas , Microtúbulos , Neoplasias del Colon/tratamiento farmacológico , ADN/químicaRESUMEN
The oxidation of cis-[Pt(NH3 )2 (OAc)2 ] with H2 O2 yields a mixture of two isomers: ctc-[Pt(NH3 )2 (OH)2 (OAc)2 ] and ctc-[Pt(NH3 )2 (OH)(OAc)(OH)(OAc)]. Following modification with 4-phenylbutyric (PhB) anhydride, two isomers were separated and characterized; the symmetric ctc-[Pt(NH3 )2 (PhB)2 (OAc)2 ] (1) and the nonsymmetric ctc-[Pt(NH3 )2 (PhB)(OAc)(PhB)(OAc)] (2). They differ in their log P values and despite having similar cellular uptake and similar DNA platination levels, the symmetric ctc-[Pt(NH3 )2 (OH)2 (OAc)2 ] is more than 4-fold more potent than the nonsymmetric isomer in a panel of 4 cancer cell lines.
RESUMEN
Polynuclear platinum complexes represent a unique structural class of DNA-binding agents of biological significance. They contain at least two platinum coordinating units bridged by a linker, which means that the formation of double-base lesions (cross-links) in DNA is possible. Here, we show that the lead compound, bifunctional [{trans-PtCl(NH3)2}2µ-trans-Pt(NH3)2{H2N(CH2)6NH2}2]4+ (Triplatin or BBR3464), forms in DNA specific double-base lesions which affect the biophysical and biochemical properties of DNA in a way fundamentally different compared to the analogous double-base lesions formed by two adducts of monofunctional chlorodiethylenetriamineplatinum(ii) chloride (dienPt). We find concomitantly that translesion DNA synthesis by the model A-family polymerase, the exonuclease deficient Klenow fragment, across the double-base lesions derived from the intrastrand CLs of Triplatin was markedly less extensive than that across the two analogous monofunctional adducts of dienPt. Collectively, these data provide convincing support for the hypothesis that the central noncovalent tetraamine platinum linker of Triplatin, capable of hydrogen-bonding and electrostatic interactions with DNA and bridging the two platinum adducts, represents an important factor responsible for the markedly lowered tolerance of DNA double-base adducts of Triplatin by DNA polymerases.
Asunto(s)
Antineoplásicos/química , Reactivos de Enlaces Cruzados/química , Aductos de ADN/química , ADN/química , Compuestos Organoplatinos/química , Antineoplásicos/metabolismo , Unión Competitiva , Reactivos de Enlaces Cruzados/metabolismo , ADN/metabolismo , Aductos de ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Conformación de Ácido Nucleico , Compuestos Organoplatinos/metabolismo , TermodinámicaRESUMEN
In this paper, we describe the biochemical properties and biological activity of a series of cholinesterase reactivators (symmetrical bisquaternary xylene-linked compounds, K106-K114) with ctDNA. The interaction of the studied derivatives with ctDNA was investigated using UV-Vis, fluorescence, CD and LD spectrometry, and electrophoretic and viscometric methods. The binding constants K were estimated to be in the range 1.05 × 10(5)-5.14 × 10(6) M(-1) and the percentage of hypochromism was found to be 10.64-19.28% (from UV-Vis titration). The used methods indicate that the studied samples are groove binders. Electrophoretic methods proved that the studied compounds clearly influence calf thymus Topo I (at 5 µM concentration, except for compounds K107, K111 and K114 which were effective at higher concentrations) and human Topo II (K110 partially inhibited Topo II effects even at 5 µM concentration) activity.
Asunto(s)
Reactivadores de la Colinesterasa/química , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo I/química , ADN/química , Reactivadores de la Colinesterasa/farmacología , Dicroismo Circular , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Estructura Molecular , Desnaturalización de Ácido Nucleico , Unión Proteica , Análisis Espectral , ViscosidadRESUMEN
Consistency of literature results with the transformation of trans-diamminedichloridoplatinum(ii) (transplatin) into cis-diammine-dichloridoplatinum(ii) (cisplatin) under UVA irradiation claimed in the article recently published in this journal is questioned on the basis of previous and new experimental data.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Cisplatino/administración & dosificación , Cisplatino/farmacología , Profármacos/administración & dosificación , Profármacos/farmacología , HumanosRESUMEN
NAMI-A is a ruthenium-based drug endowed with the unique property of selectively targeting solid tumour metastases. Although two clinical studies had already been completed, limited information exists on the behavior of NAMI-A after injection into the bloodstream. PK data in humans informs us of a rather low free drug concentration, of a relatively high half-life time of elimination and of a linear relationship between the administered dose and the corresponding AUC for up to toxic doses. In the present study, we examined the chemical kinetics of albumin binding with or without the presence of reducing agents, and we evaluated how these chemical aspects might influence the in vivo PK and the in vitro ability of NAMI-A to inhibit cell migration, which is a bona fide, rapid and easy way to suggest anti-metastatic properties. The experimental data support the binding of NAMI-A to serum albumin. The reaction is facilitated when the drug is in its reduced form and, in agreement with already reported data, the adduct formed with albumin maintains the biological activity of the ruthenium drug. The formation of the adduct is favored by low ratios of NAMI-A : HSA and by the reduction of the drug with ascorbic acid. The difference in in vivo PK and the faster binding to albumin of the reduced NAMI-A seem to suggest that the drug is not rapidly reduced immediately upon injection, even at low doses. Most probably, cell and protein binding prevail over the reduction of the drug. This observation supports the thesis that the reduction of the drug before injection must be considered relevant for the pharmacological activity of NAMI-A against tumour metastases.
Asunto(s)
Antineoplásicos , Dimetilsulfóxido/análogos & derivados , Compuestos Organometálicos , Albúmina Sérica/química , Albúmina Sérica/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Ácido Ascórbico/química , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Dimetilsulfóxido/química , Dimetilsulfóxido/farmacocinética , Dimetilsulfóxido/farmacología , Humanos , Riñón/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Masculino , Ratones Endogámicos ICR , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacocinética , Compuestos Organometálicos/farmacología , Oxidación-Reducción , Rodaminas/metabolismo , Rutenio/sangre , Rutenio/metabolismo , Compuestos de RutenioRESUMEN
A series of lichen secondary metabolites (parietin, atranorin, usnic and gyrophoric acid) and their interactions with calf thymus DNA were investigated using molecular biophysics and biochemical methods. The binding constants K were estimated to range from 4.3×10(5) to 2.4×10(7)M(-1) and the percentage of hypochromism was found to be 16-34% (from spectral titration). The results of spectral measurement indicate that the compounds act as effective DNA-interacting agents. Electrophoretic separation studies prove that from all the metabolites tested in this study, only gyrophoric acid exhibited an inhibitory effect on Topo I (25µM).
Asunto(s)
ADN/efectos de los fármacos , Líquenes/química , Líquenes/metabolismo , Animales , Benzoatos/farmacología , Benzofuranos/farmacología , Bovinos , Dicroismo Circular , ADN/metabolismo , Emodina/análogos & derivados , Emodina/farmacología , Humanos , Hidroxibenzoatos/farmacología , Sustancias Intercalantes/farmacología , Cinética , Conformación de Ácido Nucleico , Espectrofotometría Ultravioleta , Inhibidores de Topoisomerasa I/farmacología , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
The multiprotein factor composed of XPA and replication protein A (RPA) is an essential subunit of the mammalian nucleotide excision repair system. Although XPA-RPA has been implicated in damage recognition, its activity in the DNA repair pathway remains controversial. By replacing DNA adducts with mispaired bases or non-hybridizing analogues, we found that the weak preference of XPA and RPA for damaged substrates is entirely mediated by indirect readout of DNA helix conformations. Further screening with artificially distorted substrates revealed that XPA binds most efficiently to rigidly bent duplexes but not to single-stranded DNA. Conversely, RPA recognizes single-stranded sites but not backbone bending. Thus, the association of XPA with RPA generates a double-check sensor that detects, simultaneously, backbone and base pair distortion of DNA. The affinity of XPA for sharply bent duplexes, characteristic of architectural proteins, is not compatible with a direct function during recognition of nucleotide lesions. Instead, XPA in conjunction with RPA may constitute a regulatory factor that monitors DNA bending and unwinding to verify the damage-specific localization of repair complexes or control their correct three-dimensional assembly.
Asunto(s)
Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , ADN/química , Proteínas de Unión al ARN/metabolismo , Disparidad de Par Base , Secuencia de Bases , Sitios de Unión , ADN/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli , Humanos , Enlace de Hidrógeno , Cinética , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/química , Ácidos Nucleicos Heterodúplex/metabolismo , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteína de Replicación A , Proteína de la Xerodermia Pigmentosa del Grupo ARESUMEN
Bifunctional polynuclear platinum compounds represent a novel class of metal-based antitumor drugs which are currently undergoing preclinical development. A typical agent is [(trans-PtCl(NH(3))(2))(2)H(2)N(CH(2))(4)NH(2)]Cl(2) (1,1/t,t), which coordinates to bases in DNA and forms various types of covalent crosslinks. It also forms a 1,2-d(GpG) intrastrand adduct, the equivalent of the major DNA lesion of 'classical' cisplatin. In the present study differential scanning calorimetry and spectroscopic techniques were employed to characterize the influence of this crosslink on the thermal stability and energetics of 20 bp DNA duplexes site-specifically modified by 1,1/t,t. Thermal denaturation data revealed that the crosslink of 1,1/t,t reduced thermal and thermodynamical stability of the duplex noticeably more than that of 'classical' cisplatin. The energetic consequences of the intrastrand crosslink at the d(GG) site are discussed in relation to the structural distortions induced by this adduct in DNA and to its recognition and binding by HMG domain proteins. It has been suggested that the results of the present work are consistent with different DNA binding modes of cisplatin and polynuclear bifunctional DNA-binding drugs, which might be relevant to their distinct biological effectiveness.
Asunto(s)
Antineoplásicos/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Aductos de ADN/química , Aductos de ADN/metabolismo , Mutagénesis Sitio-Dirigida , Platino (Metal)/metabolismo , Antineoplásicos/química , Secuencia de Bases , Sitios de Unión , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Cisplatino/metabolismo , Reactivos de Enlaces Cruzados/química , Aductos de ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Entropía , Guanina/metabolismo , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/metabolismo , Desnaturalización de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Platino (Metal)/química , Estructura Terciaria de Proteína , Espectrofotometría Ultravioleta , Temperatura , TermodinámicaRESUMEN
Modifications of natural DNA by three anticancer heterocyclic ruthenium(III) compounds were studied by methods of molecular biophysics. These methods included DNA binding studies using atomic absorption spectrophotometry, inhibition of restriction endonucleases, mapping of DNA adducts by transcription assay, interstrand cross-linking employing gel electrophoresis under denaturing conditions, DNA unwinding studied by gel electrophoresis, circular dichroism analysis of the B-->Z transition in DNA, and DNA melting curves measured by absorption spectrophotometry. The results indicate that the complexes HIm[trans-Cl4Im2RuIII], HInd[trans-Cl4Ind2RuIII], and Na[trans-Cl4Im(Me2SO)RuIII] (Im and Ind stand for imidazole and indazole, respectively) coordinate irreversibly to DNA. Their DNA binding mode is, however, different from that of cisplatin. Interestingly, Na[trans-Cl4Im(Me2SO)RuIII] binds to DNA considerably faster than the other two ruthenium compounds and cisplatin. In addition, when Na[trans-Cl4Im(Me2SO)RuIII] binds to DNA it exhibits an enhanced base sequence specificity in comparison with the other two ruthenium complexes. Na[trans-Cl4Im(Me2SO)RuIII] also forms bifunctional intrastrand adducts on double-helical DNA which are capable of terminating RNA synthesis in vitro, while the capability of the other two ruthenium compounds to form such adducts is markedly lower. This observation has been interpreted to mean that the bifunctional adducts of HInd[trans-Cl4Ind2RuIII] and Na[trans-Cl4Im2RuIII] formed on rigid double-helical DNA are sterically more crowded by their octahedral geometry than those of Na[trans-Cl4Im(Me2SO)RuIII]. In addition, the adducts of all three ruthenium compounds affect the conformation of DNA, Na[trans-Cl4Im(Me2SO)RuIII] being most effective. It has been suggested that the altered DNA binding mode of ruthenium compounds in comparison with cisplatin might be an important factor responsible for the altered cytostatic activity of this class of ruthenium compounds in tumor cells.
Asunto(s)
Antineoplásicos/metabolismo , ADN/química , ADN/metabolismo , Compuestos Organometálicos/metabolismo , Compuestos de Rutenio/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Biofisica/métodos , Sistema Libre de Células , Cisplatino/metabolismo , Reactivos de Enlaces Cruzados/química , ADN/efectos de los fármacos , Aductos de ADN/química , Aductos de ADN/genética , Desoxirribonucleasa BamHI/química , Desoxirribonucleasa BamHI/metabolismo , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/metabolismo , Compuestos Heterocíclicos/farmacología , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Compuestos de Rutenio/química , Compuestos de Rutenio/farmacología , Transcripción GenéticaRESUMEN
The new antitumor trinuclear platinum compound [(trans-PtCl(NH(3))(2))(2)mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)](4+) (designated as BBR3464) is currently in phase II clinical trials. DNA is generally considered the major pharmacological target of platinum drugs. As such it is of considerable interest to understand the patterns of DNA damage. The bifunctional DNA binding of BBR3464 is characterized by the rapid formation of long range intra- and interstrand cross-links. We examined how the structures of the various types of the intrastrand cross-links of BBR3464 affect conformational properties of DNA, and how these adducts are recognized by high mobility group 1 protein and removed from DNA during in vitro nucleotide excision repair reactions. The results have revealed that intrastrand cross-links of BBR3464 create a local conformational distortion, but none of these cross-links results in a stable curvature. In addition, we have observed no recognition of these cross-links by high mobility group 1 proteins, but we have observed effective removal of these adducts from DNA by nucleotide excision repair. These results suggest that the processing of the intrastrand cross-links of BBR3464 in tumor cells sensitive to this drug may not be relevant to its antitumor effects. Hence, polynuclear platinum compounds apparently represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues.
Asunto(s)
Aductos de ADN/metabolismo , Reparación del ADN , ADN/efectos de los fármacos , Proteínas del Grupo de Alta Movilidad/metabolismo , Conformación de Ácido Nucleico , Compuestos Organoplatinos/farmacología , Secuencia de Bases , Sitios de Unión , ADN/química , Sondas Moleculares , Compuestos Organoplatinos/químicaRESUMEN
The p53 gene encodes a nuclear phosphoprotein that is biologically activated in response to genotoxic stresses including treatment with anticancer platinum drugs. The DNA binding activity of p53 protein is crucial for its tumor suppressor function. DNA interactions of active wild-type human p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by antitumor cisplatin and its clinically ineffective trans isomer (transplatin) were investigated by using a gel mobility shift assay. It was found that DNA adducts of cisplatin reduced binding affinity of the consensus DNA sequence to p53, whereas transplatin adducts did not. This result was interpreted to mean that the precise steric fit required for the formation and stability of the tetrameric complex of p53 with the consensus sequence cannot be attained, as a consequence of severe conformational perturbations induced in DNA by cisplatin adducts. The results also demonstrate an increase of the binding affinity of p53 to DNA lacking the consensus sequence and modified by cisplatin but not by transplatin. In addition, only major 1,2-GG intrastrand cross-links of cisplatin are responsible for this enhanced binding affinity of p53. The data base on structures of various DNA adducts of cisplatin and transplatin reveals distinctive structural features of 1,2-intrastrand cross-links of cisplatin, suggesting a unique role for this adduct in the binding of p53 to DNA lacking the consensus sequence. The results support the hypothesis that the mechanism of antitumor activity of cisplatin may also be associated with its efficiency to affect the binding affinity of platinated DNA to active p53 protein.
Asunto(s)
Cisplatino/metabolismo , Aductos de ADN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Secuencia de Consenso , Humanos , Isomerismo , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Proteínas Recombinantes/metabolismo , Spodoptera , Especificidad por Sustrato , TransfecciónRESUMEN
The effect of the single, site-specific interstrand cross-link formed by cisplatin or transplatin on the thermal stability and energetics of a 20-base pair DNA duplex is reported. The cross-linked or unplatinated 20-base pair duplexes were investigated with the aid of differential scanning calorimetry, temperature-dependent UV absorption, and circular dichroism. The cross-link of both platinum isomers increases the thermal stability of the modified duplexes by changing the molecularity of denaturation. The structural perturbation resulting from the interstrand cross-link of cisplatin increases entropy of the duplex and in this way entropically stabilizes the duplex. This entropic cross-link-induced stabilization of the duplex is partially but not completely compensated by the enthalpic destabilization of the duplex. The net result of these enthalpic and entropic effects is that the structural perturbation resulting from the formation of the interstrand cross-link by cisplatin induces a decrease in duplex thermodynamic stability, with this destabilization being enthalpic in origin. By contrast, the interstrand cross-link of transplatin is enthalpically almost neutral with the cross-link-induced destabilization entirely entropic in origin. These differences are consistent with distinct conformational distortions induced by the interstrand cross-links of the two isomers. Importantly, for the duplex cross-linked by cisplatin relative to that cross-linked by transplatin, the compensating enthalpic and entropic effects almost completely offset the difference in cross-link-induced energetic destabilization. It has been proposed that the results of the present work further support the view that the impact of the interstrand cross-links of cisplatin and transplatin on DNA is different for each and might also be associated with the distinctly different antitumor effects of these platinum compounds.
Asunto(s)
Cisplatino/química , Cisplatino/farmacología , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/farmacología , ADN/química , Antineoplásicos/química , Antineoplásicos/farmacología , Secuencia de Bases , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Entropía , Modelos Químicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Espectrofotometría , Estereoisomerismo , Temperatura , Termodinámica , Rayos UltravioletaRESUMEN
The sensitivity to the lethal effect of gamma-rays, cis- and trans-diamminedichloroplatinum (DDP), cis- and trans-iminoethers of DDP (IE) was compared in two groups of E. coli--K12 and B. In all experiments, cells of wild types appeared to be most resistant to these agents. gamma-Resistant and gamma-sensitivity/hypersensitive strains occupy an intermediate position according to their sensitivity to cis-DDP derivatives. In almost all the cases, both single and especially double mutants defective for the systems of nucleotide excision repair, recombination repair, and inducible SOS-repair are most sensitive to DDP derivatives. The data obtained show that in E. coli the repair of lethal lesions after cis-DDP action is more complicated than after gamma-irradiation. Of DDP derivatives cis-DDP is most effective, while trans-DDP is less effective, and cis- and trans-IE are considerably less effective, respectively. It is shown that the effects of ionizing radiation in low doses (more than 10 different regimes), or of treatment with cis-DDP in low concentrations do not change the survival of E. coli after their respective effects in high doses. In other words, under the effect of ionizing radiation and cis-DDP no adaptive response for the lethal action was found in E. coli.
Asunto(s)
Adaptación Biológica/efectos de los fármacos , Adaptación Biológica/efectos de la radiación , Cisplatino/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/efectos de la radiación , Fármacos Sensibilizantes a Radiaciones/farmacología , Cisplatino/química , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Escherichia coli/fisiología , Rayos gamma , IsomerismoRESUMEN
Modifications of natural DNA in cell-free media by the antitumor ruthenium compounds cis- and trans-[Cl(2)(Me(2)SO(4))(4)Ru] were studied by various biochemical and biophysical methods. These methods included: binding studies by means of flameless atomic absorption spectrophotometry, mapping of DNA adducts by means of transcription assay, use of ethidium bromide as a fluorescent probe of DNA adducts of metal complexes, an interstrand cross-linking assay employing gel electrophoresis under denaturing conditions, measurements of DNA unwinding by gel electrophoresis, differential pulse polarographic analysis of DNA conformation, and analysis of liquid crystalline dispersions of DNA by circular dichroism. The results indicated that both ruthenium compounds irreversibly coordinated to DNA; the rate of binding of the cis isomer was considerably lower than that of the trans isomer. The DNA-binding mode of trans-[Cl(2)(Me(2)SO(4))(4)Ru] included formation of bifunctional adducts such as intrastrand cross-links between neighboring purine residues and a small amount ( approximately 1%) of interstrand cross-links. cis-[Cl(2)(Me(2)SO(4))(4)Ru] formed mainly monofunctional lesions on natural DNA. Both ruthenium isomers induced conformational alterations of non-denaturational character in DNA, the trans compound being more effective. In addition, DNA adducts of trans-[Cl(2)(Me(2)SO(4))(4)Ru] were capable of inhibiting RNA synthesis by DNA-dependent RNA polymerases, while the adducts of the cis isomer were not. Thus, several features of the DNA-binding mode of trans-[Cl(2)(Me(2)SO(4))(4)Ru] were similar to those of antitumor cis-diamminedichloroplatinum (II), which may be relevant to the biological effects of this antitumor ruthenium drug. On the other hand, the different DNA-binding mode of cis-[Cl(2)(Me(2)SO(4))(4)Ru] was consistent with its less pronounced biological effects.
Asunto(s)
Antineoplásicos/farmacología , ADN/efectos de los fármacos , Compuestos de Rutenio/farmacología , Secuencia de Bases , Sistema Libre de Células , Reactivos de Enlaces Cruzados , Medios de Cultivo/farmacología , ADN/química , ADN/metabolismo , Aductos de ADN/análisis , Aductos de ADN/química , Etidio , Colorantes Fluorescentes , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos/genética , Polarografía , Transcripción GenéticaRESUMEN
The requirement for novel platinum antitumor drugs led to the concept of synthesis of novel platinum drugs based on targeting cisplatin to various carrier molecules. We have shown [Loskotova, H., and Brabec, V. (1999) Eur. J. Biochem. 266, 392-402] that attachment of DNA minor-groove-binder distamycin to cisplatin changes several features of DNA-binding mode of the parent platinum drug. Major differences comprise different conformational changes in DNA and a considerably higher interstrand cross-linking efficiency. The studies of the present work have been directed to the analysis of oligodeoxyribonucleotide duplexes containing single, site-specific adducts of platinum-distamycin conjugates. These uniquely modified duplexes were analyzed by Maxam-Gilbert footprinting, phase-sensitive gel electrophoresis bending assay and chemical probes of DNA conformation. The results have indicated that the attachment of distamycin to cisplatin mainly affects the sites involved in the interstrand cross-links so that these adducts are preferentially formed between complementary guanine and cytosine residues. This interstrand cross-link bends the helix axis by approximately 35 degrees toward minor groove, unwinds DNA by approximately 95 degrees and distorts DNA symmetrically around the adduct. In addition, CD spectra of restriction fragments modified by the cisplatin-distamycin conjugates have demonstrated that distamycin moiety in the interstrand cross-links of these compounds interacts with DNA. This interaction facilitates the formation of these adducts. Hence, the structural impact of the specific interstrand cross-link detected in this study deserves attention when biological behavior of cisplatin derivatives targeted by oligopeptide DNA minor-groove-binders is evaluated.
Asunto(s)
Cisplatino/química , Reactivos de Enlaces Cruzados/química , Aductos de ADN/química , ADN/química , Fosfatos de Dinucleósidos/química , Distamicinas/química , Conformación de Ácido Nucleico , Bromo/química , Dicroismo Circular , ADN de Cadena Simple/química , Dietil Pirocarbonato/química , Ácidos Nucleicos Heterodúplex/síntesis química , Ácidos Nucleicos Heterodúplex/química , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/química , Permanganato de Potasio/química , Nucleótidos de Pirimidina/síntesis química , Nucleótidos de Pirimidina/químicaRESUMEN
Recent findings that novel trans-dichloroplatinum(II) complexes exhibit antitumor activity violate the classical structure-activity relationships of platinum(II) complexes. These novel "nonclassical" trans platinum complexes also comprise those containing planar aromatic amines. Initial studies have shown that these compounds form a considerable amount of DNA interstrand cross-links (up to approximately 30%) with a rate markedly higher than clinically ineffective transplatin. The present work has shown, using Maxam-Gilbert footprinting, that trans-[PtCl2(NH3)(quinoline)] and trans-[PtCl2(NH3)(thiazole)], representatives of the group of new antitumor trans-dichloroplatinum complexes containing planar amines, preferentially form DNA interstrand cross-links between guanine residues at the 5'-GC-3' sites. Thus, DNA interstrand cross-linking by trans-[PtCl2(NH3)(quinoline)] and trans-[PtCl2(NH3)(thiazole)] is formally equivalent to that by antitumor cisplatin, but different from clinically ineffective transplatin which preferentially forms these adducts between complementary guanine and cytosine residues. This result shows for the first time that simple chemical modification of the structure of an inactive compound alters its DNA binding site into a DNA adduct of an active drug.
Asunto(s)
Antineoplásicos/química , Reactivos de Enlaces Cruzados/química , ADN/química , Oligonucleótidos/síntesis química , Secuencia de Bases , Cisplatino/metabolismo , Citosina/química , Aductos de ADN/metabolismo , Huella de ADN , Guanina/química , Ligandos , Conformación de Ácido Nucleico , Oligonucleótidos/química , Quinolinas/química , Estereoisomerismo , Relación Estructura-Actividad , Tiazoles/químicaRESUMEN
Interactions of high mobility group (HMG) domain proteins with DNA modified by cisplatin plays a role in mechanisms underlying its antitumor activity. A structural motif recognized by HMG domain proteins on cisplatin-modified DNA is a stable, directional bend of the helix axis. In the present work, bending induced in DNA by major adducts of a novel class of antitumor compounds, represented by the formula [¿trans-PtCl(NH(3))(2)¿H(2)N(CH(2))(2-6)NH(2)]Cl(2), was investigated. The oligodeoxyribonucleotide duplexes containing various site-specific interstrand cross-links of these bifunctional dinuclear platinum drugs were purified and characterized by Maxam-Gilbert footprinting, chemical probing, and phasing assay. It was demonstrated that the cross-links of the dinuclear compounds bent the helix much less than those of cisplatin. Gel retardation assay revealed very weak recognition of DNA adducts of dinuclear complexes by HMG1 protein. Hence, the mediation of antitumor properties of dinuclear platinum complexes by HMG domain proteins is unlikely so that polynuclear platinum compounds may represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues. A further understanding of how polynuclear platinum compounds modify DNA and how these modifications are processed in cells should provide a rational basis for the design of new platinum drugs rather than searching for cisplatin analogues.
Asunto(s)
Cisplatino/química , Aductos de ADN/química , Proteínas del Grupo de Alta Movilidad/química , Conformación de Ácido Nucleico , Antineoplásicos/química , Cisplatino/análogos & derivados , Reactivos de Enlaces Cruzados/química , Huella de ADN , Proteínas de Unión al ADN/química , Electroforesis en Gel de Poliacrilamida , Cinética , Oligodesoxirribonucleótidos/química , Compuestos de Platino/químicaRESUMEN
Modifications of natural DNA and synthetic oligodeoxyribonucleotide duplexes in a cell-free medium by analogs of antitumor cisplatin containing enantiomeric amine ligands, such as cis-[PtCl(2)(RR-DAB)] and cis-[PtCl(2)(SS-DAB)] (DAB = 2,3-diaminobutane), were studied by various methods of molecular biophysics and biophysical chemistry. These methods include DNA binding studies by pulse polarography and atomic absorption spectrophotometry, mapping of DNA adducts using transcription assay, interstrand cross-linking assay using gel electrophoresis under denaturing conditions, differential scanning calorimetry, chemical probing, and bending and unwinding studies of the duplexes containing single, site-specific cross-link. The major differences resulting from the modification of DNA by the two enantiomers are the thermodynamical destabilization and conformational distortions induced in DNA by the 1,2-d(GpG) intrastrand cross-link. It has been suggested that these differences are associated with a different biological activity of the two enantiomers observed previously. In addition, the results of the present work are also consistent with the view that formation of hydrogen bonds between the carbonyl oxygen of the guanine residues and the "quasi equatorial" hydrogen of the cis amine in the 1, 2-d(GpG) intrastrand cross-link plays an important role in determining the character of the distortion induced in DNA by this lesion.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Cisplatino/análogos & derivados , ADN/química , ADN/efectos de los fármacos , Aminas/química , Animales , Secuencia de Bases , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Rastreo Diferencial de Calorimetría , Bovinos , Cisplatino/química , Cisplatino/farmacología , Reactivos de Enlaces Cruzados , ADN/genética , Aductos de ADN/química , Técnicas In Vitro , Ligandos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estereoisomerismo , Termodinámica , Transcripción GenéticaRESUMEN
A general statistical test of the stability of measurement conditions was demonstrated on the beta-spectra of 241Pu cumulated during four years. The alpha- and gamma-ray spectroscopy indicated stability of the 241Pu source. Monte Carlo modelling of individual collision events clarified the role of electron scattering and energy losses within a radioactive source down to energies of several hundreds of eV. The impact ionization by beta-particles of carbon and oxygen atoms in a surface contamination layer on the 241Pu and 63Ni sources was observed.