Using electrophoresis and replication mapping, we show that the presence of DNA adducts of bifunctional antitumor cisplatin or monodentate [PtCl(dien)]Cl (dien = diethylenetriamine) in the substrate DNA inhibits eukaryotic topoisomerase 1 (top1) action, the adducts of cisplatin being more effective. The presence of camptothecin in the samples of platinated DNA markedly enhances effects of Pt-DNA adducts on top1 activity. Interestingly, the effects of Pt-DNA adducts on the catalytic activity of top1 in the presence of camptothecin differ depending on the sequence context. A multiple metallation of the short nucleotide sequences on the scissile strand, immediately downstream of the cleavage site impedes the cleavage by top1. On the other hand, DNA cleavage by top1 at some cleavage sites which were not platinated in their close proximity is notably enhanced as a consequence of global platination of DNA. We suggest that this enhancement of DNA cleavage by top1 may consist in its inability to bind to other cleavage sites platinated in their close neighborhood; thus, more molecules of top1 may become available for cleavage at the sites where top1 normally cleaves and where platination does not interfere.

• MeSH
• adukty DNA chemie   farmakologie   MeSH
• cisplatina analogy a deriváty   chemie   farmakologie   MeSH
• DNA-topoisomerasy I metabolismus   MeSH
• DNA chemie   metabolismus   MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• inhibitory topoisomerasy I * MeSH
• protinádorové látky chemie   farmakologie   MeSH
• štěpení DNA MeSH
• superhelikální DNA metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• chlorodiethylenetriamine platinum MeSH Prohlížeč
• cisplatin-DNA adduct MeSH Prohlížeč
• cisplatina MeSH
• DNA-topoisomerasy I MeSH
• DNA MeSH
• inhibitory enzymů MeSH
• inhibitory topoisomerasy I * MeSH
• protinádorové látky MeSH
• superhelikální DNA MeSH

The global modification of mammalian and plasmid DNAs by the novel platinum compounds cis-[PtCl(2)(isopropylamine)(1-methylimidazole)] and trans-[PtCl(2)(isopropylamine)(1-methylimidazole)] and the reactivity of these compounds with reduced glutathione (GSH) were investigated in cell-free media using various biochemical and biophysical methods. Earlier cytotoxicity studies had revealed that the replacement of the NH(3) groups in cisplatin by the azole and isopropylamine ligands lowers the activity of cisplatin in both sensitive and resistant cell lines. The results of the present work show that this replacement does not considerably affect the DNA modifications by this drug, recognition of these modifications by HMGB1 protein, their repair, and reactivity of the platinum complex with GSH. These results were interpreted to mean that the reduced activity of this analog of cisplatin in tumor cell lines is due to factors that do not operate at the level of the target DNA. In contrast, earlier studies had shown that the replacement of the NH(3) groups in the clinically ineffective trans isomer (transplatin) by the azole and isopropylamine ligands results in a radical enhancement of its activity in tumor cell lines. Importantly, this replacement also markedly alters the DNA binding mode of transplatin, which is distinctly different from that of cisplatin, but does not affect reactivity with GSH. Hence, the results of the present work are consistent with the view and support the hypothesis systematically tested by us and others that platinum drugs that bind to DNA in a fundamentally different manner from that of conventional cisplatin may have altered pharmacological properties.

• MeSH
• bezbuněčný systém MeSH
• cirkulární dichroismus MeSH
• DNA chemie   účinky léků   MeSH
• glutathion chemie   účinky léků   MeSH
• kultivační média chemie   MeSH
• lidé MeSH
• organoplatinové sloučeniny chemie   farmakologie   MeSH
• protinádorové látky chemie   farmakologie   MeSH
• spektrofotometrie ultrafialová MeSH
• stereoizomerie MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dichloro(isopropylamine)(1-methylimidazole)diplatinum(II) MeSH Prohlížeč
• DNA MeSH
• glutathion MeSH
• kultivační média MeSH
• organoplatinové sloučeniny MeSH
• protinádorové látky MeSH

Clinically ineffective transplatin [trans-diamminedichloridoplatinum(II)] is used in the studies of the structure-pharmacological activity relationship of platinum compounds. In addition, a number of transplatin analogs exhibit promising toxic effects in several tumor cell lines including those resistant to conventional antitumor cisplatin. Moreover, transplatin-modified oligonucleotides have been shown to be effective modulators of gene expression. Owing to these facts and because DNA is also considered the major pharmacological target of platinum complexes, interactions between transplatin and DNA are of great interest. We examined, using biophysical and biochemical methods, the stability of 1,3-GNG intrastrand cross-links (CLs) formed by transplatin in short synthetic oligodeoxyribonucleotide duplexes and natural double-helical DNA. We have found that transplatin forms in double-helical DNA 1,3-GNG intrastrand CLs, but their stability depends on the sequence context. In some sequences the 1,3-GNG intrastrand CLs formed by transplatin in double-helical DNA readily rearrange into interstrand CLs. On the other hand, in a number of other sequences these intrastrand CLs are relatively stable. We show that the stability of 1,3-GNG intrastrand CLs of transplatin correlates with the extent of conformational distortion and thermodynamic destabilization induced in double-helical DNA by this adduct.

• MeSH
• biofyzikální jevy * MeSH
• cisplatina metabolismus   MeSH
• DNA chemie   genetika   metabolismus   MeSH
• kalorimetrie MeSH
• konformace nukleové kyseliny MeSH
• oligodeoxyribonukleotidy chemie   genetika   metabolismus   MeSH
• reagencia zkříženě vázaná metabolismus   MeSH
• sekvence nukleotidů MeSH
• termodynamika MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cisplatina MeSH
• DNA MeSH
• oligodeoxyribonukleotidy MeSH
• reagencia zkříženě vázaná MeSH
• transplatin MeSH Prohlížeč

DNA-protein cross-links are formed by various DNA-damaging agents including antitumor platinum drugs. The natures of these ternary DNA-Pt-protein complexes (DPCLs) can be inferred, yet much remains to be learned about their structures and mechanisms of formation. We investigated the origin of these DPCLs and their cellular processing on molecular level using gel electrophoresis shift assay. We show that in cell-free media cisplatin [cis-diamminedichloridoplatinum(II)] forms DPCLs more effectively than ineffective transplatin [trans-diamminedichloridoplatinum(II)]. Mechanisms of transformation of individual types of plain DNA adducts of the platinum complexes into the DPCLs in the presence of several DNA-binding proteins have been also investigated. The DPCLs are formed by the transformation of DNA monofunctional and intrastrand cross-links of cisplatin. In contrast, interstrand cross-links of cisplatin and monofunctional adducts of transplatin are stable in presence of the proteins. The DPCLs formed by cisplatin inhibit DNA polymerization or removal of these ternary lesions from DNA by nucleotide excision repair system more effectively than plain DNA intrastrand or monofunctional adducts. Thus, the bulky DNA-protein cross-links formed by cisplatin represent a more distinct and persisting structural motif recognized by the components of downstream cellular systems processing DNA damage considerably differently than the plain DNA adducts of this metallodrug.

• MeSH
• adukty DNA chemie   MeSH
• cisplatina chemie   toxicita   MeSH
• DNA vazebné proteiny účinky léků   MeSH
• DNA biosyntéza   účinky léků   MeSH
• oprava DNA MeSH
• protinádorové látky chemie   toxicita   MeSH
• reagencia zkříženě vázaná chemie   toxicita   MeSH
• retardační test MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• cisplatin-DNA adduct MeSH Prohlížeč
• cisplatina MeSH
• DNA vazebné proteiny MeSH
• DNA MeSH
• protinádorové látky MeSH
• reagencia zkříženě vázaná MeSH
• transplatin MeSH Prohlížeč

Effects of adducts of [PtCl(NH3)3]Cl or chlorodiethylenetriamineplatinum(II) on DNA stability were studied with emphasis on thermodynamic origins of that stability. Oligodeoxyribonucleotide duplexes (15-bp) containing the single, site-specific monofunctional adduct at G-residues of the central sequences TGT/ACA or 5'-AGT/5'-ACT were prepared and analyzed by differential scanning calorimetry, temperature-dependent ultraviolet absorption and circular dichroism. The unfolding of the platinated duplexes was accompanied by relatively small unfavorable free energy terms. This destabilization was enthalpic in origin. On the other hand, a relatively large reduction of melting temperature (T(m)) was observed as a consequence of the monofunctional adduct in the TGT sequence, whereas T(m) due to the adduct in the AGT sequence was reduced only slightly. We also examined the efficiency of the mammalian nucleotide excision repair system to remove from DNA the monofunctional adducts and found that these lesions were not recognized by this repair system. Thus, rather thermodynamic than thermal characterization of DNA adducts of monofunctional platinum compounds is a property implicated in the modulation of downstream effects such as protein recognition and repair.

• MeSH
• adukty DNA analýza   chemie   MeSH
• denaturace nukleových kyselin MeSH
• DNA analýza   chemie   MeSH
• kinetika MeSH
• oprava DNA * MeSH
• platina analýza   chemie   MeSH
• přenos energie MeSH
• teplota MeSH
• termodynamika MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• DNA MeSH
• platina MeSH

The structure-pharmacological activity relationships generally accepted for antitumor platinum compounds stressed the necessity for the cis-[PtX(2)(amine)(2)] structure while the trans-[PtX(2)(amine)(2)] structure was considered inactive. However, more recently, several trans-platinum complexes have been identified which are potently toxic, antitumor-active and demonstrate activity distinct from that of conventional cisplatin (cis-[PtCl(2)(NH(3))(2)]). We have shown in the previous report that the replacement of ammine ligands by iminoether in transplatin (trans-[PtCl(2)(NH(3))(2)]) results in a marked enhancement of its cytotoxicity so that it is more cytotoxic than its cis congener and exhibits significant antitumor activity, including activity in cisplatin-resistant tumor cells. In addition, we have also shown previously that this new trans compound (trans-[PtCl(2)(E-iminoether)(2)]) forms mainly monofunctional adducts at guanine residues on DNA, which is generally accepted to be the cellular target of platinum drugs. In order to shed light on the mechanism underlying the antitumor activity of trans-[PtCl(2)(E-iminoether)(2)] we examined oligodeoxyribonucleotide duplexes containing a single, site-specific, monofunctional adduct of this transplatin analog by the methods of molecular biophysics. The results indicate that major monofunctional adducts of trans-[PtCl(2)(E-iminoether)(2)] locally distort DNA, bend the DNA axis by 21 degrees toward the minor groove, are not recognized by HMGB1 proteins and are readily removed from DNA by nucleotide excision repair (NER). In addition, the monofunctional adducts of trans-[PtCl(2)(E-iminoether)(2)] readily cross-link proteins, which markedly enhances the efficiency of this adduct to terminate DNA polymerization by DNA polymerases in vitro and to inhibit removal of this adduct from DNA by NER. It is suggested that DNA-protein ternary cross-links produced by trans-[PtCl(2)(E-iminoether)(2)] could persist considerably longer than the non-cross-linked monofunctional adducts, which would potentiate toxicity of this antitumor platinum compound toward tumor cells sensitive to this drug. Thus, trans-[PtCl(2)(E-iminoether)(2)] represents a quite new class of platinum antitumor drugs in which activation of trans geometry is associated with an increased efficiency to form DNA-protein ternary cross-links thereby acting by a different mechanism from 'classical' cisplatin and its analogs.

• MeSH
• adukty DNA chemie   metabolismus   MeSH
• CHO buňky MeSH
• cisplatina analogy a deriváty   chemie   farmakologie   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA chemie   účinky léků   metabolismus   MeSH
• domény HMG-Box MeSH
• HeLa buňky MeSH
• konformace nukleové kyseliny účinky léků   MeSH
• křečci praví MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• makromolekulární látky MeSH
• oligonukleotidy chemie   metabolismus   MeSH
• protein HMGB1 chemie   metabolismus   MeSH
• reagencia zkříženě vázaná chemie   farmakologie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• cisplatina MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• makromolekulární látky MeSH
• oligonukleotidy MeSH
• protein HMGB1 MeSH
• reagencia zkříženě vázaná MeSH

Negatively supercoiled, relaxed and linearized forms of pSP73 DNA were modified in cell-free medium by cis-diamminedichloroplatinum(II) (cisplatin). The frequency of interstrand cross-links (ICLs) formed in these DNAs has been determined by: (i) immunochemical analysis; (ii) an assay employing NaCN as a probe of DNA ICLs of cisplatin; (iii) gel electrophoresis under denaturing conditions. At low levels of the modification of DNA (<1 Pt atom fixed per 500 bp) the number of ICLs formed by cisplatin was radically enhanced in supercoiled in comparison with linearized or relaxed DNA. At these low levels of modification, the frequency of ICLs in supercoiled DNA was enhanced with increasing level of negative supercoiling or with decreasing level of modification. In addition, the replication mapping of DNA ICLs of cisplatin was consistent with these lesions being preferentially formed in negatively supercoiled DNA between guanine residues in both the 5'-d(GC)-3' and the 5'-d(CG)-3' sites. Among the DNA adducts of cisplatin the ICL has the markedly greatest capability to unwind the double helix. We suggest that the formation of ICLs of cisplatin is thermodynamically more favored in negatively supercoiled DNA owing mainly to the relaxation of supercoils.

• MeSH
• adukty DNA metabolismus   MeSH
• cisplatina farmakologie   MeSH
• denaturace nukleových kyselin MeSH
• elektroforéza v agarovém gelu MeSH
• ELISA MeSH
• kompetitivní vazba MeSH
• konformace nukleové kyseliny MeSH
• kruhová DNA metabolismus   MeSH
• kyanid sodný farmakologie   MeSH
• molekulární sekvence - údaje MeSH
• plazmidy účinky léků   metabolismus   MeSH
• protinádorové látky farmakologie   MeSH
• reagencia zkříženě vázaná metabolismus   MeSH
• restrikční mapování MeSH
• sekvence nukleotidů MeSH
• superhelikální DNA účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• cisplatina MeSH
• kruhová DNA MeSH
• kyanid sodný MeSH
• protinádorové látky MeSH
• reagencia zkříženě vázaná MeSH
• superhelikální DNA MeSH

It has been shown recently that some analogues of clinically ineffective trans-diamminedichloroplatinum (II) (transplatin) exhibit antitumor activity. This finding has inverted the empirical structure-antitumor activity relationships delineated for platinum(II) complexes, according to which only the cis geometry of leaving ligands in the bifunctional platinum complexes is therapeutically active. As a result, interactions of trans platinum compounds with DNA, which is the main pharmacological target of platinum anticancer drugs, are of great interest. The present paper describes the DNA binding of antitumor trans-[PtCl(2)(E-imino ether)(2)] complex (trans-EE) in a cell-free medium, which has been investigated using three experimental approaches. They involve thiourea as a probe of monofunctional DNA adducts of platinum (II) complexes with two leaving ligands in the trans configuration, ethidium bromide as a probe for distinguishing between monofunctional and bifunctional DNA adducts of platinum complexes and HPLC analysis of the platinated DNA enzymatically digested to nucleosides. The results show that bifunctional trans-EE preferentially forms monofunctional adducts at guanine residues in double-helical DNA even when DNA is incubated with the platinum complex for a relatively long time (48 h at 37 degrees C in 10 mM NaCIO(4). It implies that antitumor trans-EE modifies DNA in a different way than clinically ineffective transplatin, which forms prevalent amount of bifunctional DNA adducts after 48 h. This result has been interpreted to mean that the major adduct of trans-EE, occurring in DNA even after long reaction times, is a monofunctional adduct in which the reactivity of the second leaving group is markedly reduced. It has been suggested that the different properties of the adducts formed on DNA by transplatin and trans-EE are relevant to their distinct clinical efficacy.

• MeSH
• adukty DNA chemická syntéza   metabolismus   MeSH
• cisplatina analogy a deriváty   metabolismus   MeSH
• denaturace nukleových kyselin MeSH
• deoxyguanosin chemie   MeSH
• DNA chemie   metabolismus   MeSH
• ethidium MeSH
• fluorescenční barviva MeSH
• molekulární sondy MeSH
• organoplatinové sloučeniny chemická syntéza   metabolismus   MeSH
• skot MeSH
• thiomočovina MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• cisplatina MeSH
• deoxyguanosin MeSH
• DNA MeSH
• ethidium MeSH
• fluorescenční barviva MeSH
• molekulární sondy MeSH
• organoplatinové sloučeniny MeSH
• thiomočovina MeSH