The effects of major DNA intrastrand cross-links of antitumor dinuclear Pt(II) complexes [{trans-PtCl(NH(3))(2)}(2)-μ-{trans-(H(2)N(CH(2))(6)NH(2)(CH(2))(2)NH(2)(CH(2))(6)NH(2))}](4+) (1) and [{PtCl(DACH)}(2)-μ-{H(2)N(CH(2))(6)NH(2)(CH(2))(2)NH(2)(CH(2))(6)NH(2))}](4+) (2) (DACH is 1,2-diaminocyclohexane) on DNA stability were studied with emphasis on thermodynamic origins of that stability. Oligodeoxyribonucleotide duplexes containing the single 1,2, 1,3, or 1,5 intrastrand cross-links at guanine residues in the central TGGT, TGTGT, or TGTTTGT sequences, respectively, were prepared and analyzed by differential scanning calorimetry. The unfolding of the platinated duplexes was accompanied by unfavorable free energy terms. The efficiency of the cross-links to thermodynamically destabilize the duplex depended on the number of base pairs separating the platinated bases. The trend was 1,5→1,2→1,3 cross-link of 1 and 1,5→1,3→1,2 cross-link of 2. Interestingly, the results showed that the capability of the cross-links to reduce the thermodynamic stability of DNA (ΔG(298)(0)) correlated with the extent of conformational distortions induced in DNA by various types of intrastrand cross-links of 1 or 2 determined by chemical probes of DNA conformation. We also examined the efficiency of the mammalian nucleotide excision repair systems to remove from DNA the intrastrand cross-links of 1 or 2. The efficiency of the excinucleases to remove the cross-links from DNA depended on the length of the cross-link; the trend was identical to that observed for the efficiency of the intrastrand cross-links to thermodynamically destabilize the duplex. Thus, the results are consistent with the thesis that an important factor that determines the susceptibility of the intrastrand cross-links of dinuclear platinum complexes 1 and 2 to be removed from DNA by nucleotide excision repair is the efficiency of these lesions to thermodynamically destabilize DNA.

• MeSH
• Calorimetry, Differential Scanning MeSH
• DNA chemistry   MeSH
• Intercalating Agents chemistry   pharmacology   MeSH
• Nucleic Acid Conformation drug effects   MeSH
• DNA Repair drug effects   MeSH
• Organoplatinum Compounds chemistry   pharmacology   MeSH
• Antineoplastic Agents chemistry   pharmacology   MeSH
• Base Sequence MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Intercalating Agents MeSH
• Organoplatinum Compounds MeSH
• Antineoplastic Agents MeSH

A combination of biophysical, biochemical, and computational techniques was used to delineate mechanistic differences between the platinum-acridine hybrid agent [PtCl(en)(L)](NO(3))(2) (complex 1, en = ethane-1,2-diamine, L = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) and a considerably more potent second-generation analogue containing L' = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine (complex 2). Calculations at the density functional theory level provide a rationale for the binding preference of both complexes for guanine-N7 and the relatively high level of adenine adducts observed for compound 1. A significant rate enhancement is observed for binding of the amidine-based complex 2 with DNA compared with the thiourea-based prototype 1. Studies conducted with chemical probes and on the bending and unwinding of model duplex DNA suggest that adducts of complex 2 perturb B-form DNA more severely than complex 1, however, without denaturing the double strand and significantly less than cisplatin. Circular and linear dichroism spectroscopies and viscosity measurements suggest that subtle differences exist between the intercalation modes and adduct geometries of the two complexes. The adducts formed by complex 2 most efficiently inhibit transcription of the damaged DNA by RNA polymerase II. Not only do complexes 1 and 2 cause less distortion to DNA than cisplatin, they also do not compromise the thermodynamic stability of the modified duplex. This leads to a decreased or negligible affinity of HMG domain proteins for the adducts formed by either Pt-acridine complex. In a DNA repair synthesis assay the lesions formed by complex 2 were repaired less efficiently than those formed by complex 1. These significant differences in DNA adduct formation, structure, and recognition between the two acridine complexes and cisplatin help to elucidate why compound 2 is highly active in cisplatin-resistant, repair proficient cancer cell lines.

• MeSH
• DNA Adducts chemistry   MeSH
• Acridines chemistry   metabolism   pharmacology   MeSH
• Amidines chemistry   metabolism   pharmacology   MeSH
• DNA, B-Form chemistry   metabolism   MeSH
• Cisplatin analogs & derivatives   chemistry   metabolism   pharmacology   MeSH
• DNA chemistry   metabolism   MeSH
• Transcription, Genetic drug effects   MeSH
• HeLa Cells MeSH
• Intercalating Agents chemistry   metabolism   pharmacology   MeSH
• Kinetics MeSH
• Nucleic Acid Conformation drug effects   MeSH
• Humans MeSH
• DNA Repair drug effects   MeSH
• Organoplatinum Compounds chemistry   metabolism   pharmacology   MeSH
• Protein Isoforms metabolism   MeSH
• HMGB1 Protein metabolism   MeSH
• Antineoplastic Agents chemistry   metabolism   pharmacology   MeSH
• Drug Design MeSH
• Thiourea chemistry   metabolism   pharmacology   MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Comparative Study MeSH
• Names of Substances
• DNA Adducts MeSH
• Acridines MeSH
• Amidines MeSH
• DNA, B-Form MeSH
• Cisplatin MeSH
• DNA MeSH
• Intercalating Agents MeSH
• Organoplatinum Compounds MeSH
• Protein Isoforms MeSH
• HMGB1 Protein MeSH
• Antineoplastic Agents MeSH
• Thiourea MeSH

Downstream processes that discriminate between DNA adducts of a third generation platinum antitumor drug oxaliplatin and conventional cisplatin are believed to be responsible for the differences in their biological effects. These different biological effects are explained by the ability of oxaliplatin to form DNA adducts more efficient in their biological effects. In this work conformation, recognition by HMG domain protein and DNA polymerization across the major 1,2-GG intrastrand cross-link formed by cisplatin and oxaliplatin in three sequence contexts were compared with the aid of biophysical and biochemical methods. The following major differences in the properties of the cross-links of oxaliplatin and cisplatin were found: i), the formation of the cross-link by oxaliplatin is more deleterious energetically in all three sequence contexts; ii), the cross-link of oxaliplatin bends DNA slightly but systematically less in all sequence contexts tested; iii), the affinity of HMG domain protein to the cross-link of oxaliplatin is considerably lower independent of the sequence context; and iv), the Klenow fragment of DNA polymerase I pauses considerably more at the cross-link of oxaliplatin in all sequence contexts tested. We have also demonstrated that the chirality at the carrier ligand of oxaliplatin can affect its biological effects.

• MeSH
• DNA Adducts chemistry   ultrastructure   MeSH
• Guanine chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Organoplatinum Compounds chemistry   MeSH
• Oxaliplatin MeSH
• Base Pairing MeSH
• Antineoplastic Agents chemistry   MeSH
• Cross-Linking Reagents MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• Guanine MeSH
• Organoplatinum Compounds MeSH
• Oxaliplatin MeSH
• Antineoplastic Agents MeSH
• Cross-Linking Reagents MeSH

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
• DNA Adducts chemistry   MeSH
• Cisplatin chemistry   toxicity   MeSH
• DNA-Binding Proteins drug effects   MeSH
• DNA biosynthesis   drug effects   MeSH
• DNA Repair MeSH
• Antineoplastic Agents chemistry   toxicity   MeSH
• Cross-Linking Reagents chemistry   toxicity   MeSH
• Electrophoretic Mobility Shift Assay MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• cisplatin-DNA adduct MeSH Browser
• Cisplatin MeSH
• DNA-Binding Proteins MeSH
• DNA MeSH
• Antineoplastic Agents MeSH
• Cross-Linking Reagents MeSH
• transplatin MeSH Browser

Replacement of one ammine in clinically ineffective trans-[PtCl2(NH3)2] (transplatin) by a planar N-heterocycle, thiazole, results in significantly enhanced cytotoxicity. Unlike 'classical' cisplatin {cis-[PtCl2(NH3)2]} or transplatin, modification of DNA by this prototypical cytotoxic transplatinum complex trans-[PtCl2(NH3)(thiazole)] (trans-PtTz) leads to monofunctional and bifunctional intra or interstrand adducts in roughly equal proportions. DNA fragments containing site-specific bifunctional DNA adducts of trans-PtTz were prepared. The structural distortions induced in DNA by these adducts and their consequences for high-mobility group protein recognition, DNA polymerization and nucleotide excision repair were assessed in cell-free media by biochemical methods. Whereas monofunctional adducts of trans-PtTz behave similar to the major intrastrand adduct of cisplatin [J. Kasparkova, O. Novakova, N. Farrell and V. Brabec (2003) Biochemistry, 42, 792-800], bifunctional cross-links behave distinctly differently. The results suggest that the multiple DNA lesions available to trans-planaramine complexes may all contribute substantially to their cytotoxicity so that the overall drug cytotoxicity could be the sum of the contributions of each of these adducts. However, acquisition of drug resistance could be a relatively rare event, since it would have to entail resistance to or tolerance of multiple, structurally dissimilar DNA lesions.

• MeSH
• DNA Adducts chemistry   metabolism   MeSH
• Cisplatin chemistry   toxicity   MeSH
• DNA biosynthesis   MeSH
• Nucleic Acid Conformation MeSH
• DNA Repair MeSH
• Organoplatinum Compounds chemistry   toxicity   MeSH
• High Mobility Group Proteins metabolism   MeSH
• Antineoplastic Agents chemistry   toxicity   MeSH
• Cross-Linking Reagents chemistry   toxicity   MeSH
• Thiazoles chemistry   toxicity   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• DNA Adducts MeSH
• Cisplatin MeSH
• DNA MeSH
• Organoplatinum Compounds MeSH
• High Mobility Group Proteins MeSH
• Antineoplastic Agents MeSH
• Cross-Linking Reagents MeSH
• Thiazoles MeSH
• trans-(PtCl2(NH3)(thiazole)) MeSH Browser
• transplatin MeSH Browser

1,2-GG intrastrand cross-links formed in DNA by the enantiomeric complexes [PtCl(2)(R,R-2,3-diaminobutane (DAB))] and [PtCl(2)(S,S-DAB)] were studied by biophysical methods. Molecular modeling revealed that structure of the cross-links formed at the TGGT sequence was affected by repulsion between the 5'-directed methyl group of the DAB ligand and the methyl group of the 5'-thymine of the TGGT fragment. Molecular dynamics simulations of the solvated platinated duplexes and our recent structural data indicated that the adduct of [PtCl(2)(R,R-DAB)] alleviated this repulsion by unwinding the TpG step, whereas the adduct of [PtCl(2)(S,S-DAB)] avoided the unfavorable methyl-methyl interaction by decreasing the kink angle. Electrophoretic retardation measurements on DNA duplexes containing 1,2-GG intrastrand cross-links of Pt(R,R-DAB)(2+) or Pt(S,S-DAB)(2+) at a CGGA site showed that in this sequence both enantiomers distorted the double helix to the identical extent similar to that found previously for the same sequence containing the cross-links of the parent antitumor cis-Pt(NH(3))(2)(2+) (cisplatin). In addition, the adducts showed similar affinities toward the high-mobility-group box 1 proteins. Hence, whereas the structural perturbation induced in DNA by 1,2-GG intrastrand cross-links of cisplatin does not depend largely on the bases flanking the cross-links, the perturbation related to GG cross-linking by bulkier platinum diamine derivatives does.

• MeSH
• DNA Adducts chemistry   genetics   MeSH
• Biophysics MeSH
• Biophysical Phenomena MeSH
• Cisplatin analogs & derivatives   chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Ligands MeSH
• Models, Molecular MeSH
• HMGB1 Protein chemistry   MeSH
• Cross-Linking Reagents MeSH
• Electrophoretic Mobility Shift Assay MeSH
• Base Sequence MeSH
• Stereoisomerism MeSH
• In Vitro Techniques MeSH
• Thermodynamics MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• cisplatin-DNA adduct MeSH Browser
• Cisplatin MeSH
• Ligands MeSH
• HMGB1 Protein MeSH
• Cross-Linking Reagents MeSH

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
• DNA Adducts analysis   chemistry   MeSH
• Nucleic Acid Denaturation MeSH
• DNA analysis   chemistry   MeSH
• Kinetics MeSH
• DNA Repair * MeSH
• Platinum analysis   chemistry   MeSH
• Energy Transfer MeSH
• Temperature MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• DNA MeSH
• Platinum 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
• DNA Adducts chemistry   metabolism   MeSH
• CHO Cells MeSH
• Cisplatin analogs & derivatives   chemistry   pharmacology   MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA chemistry   drug effects   metabolism   MeSH
• HMG-Box Domains MeSH
• HeLa Cells MeSH
• Nucleic Acid Conformation drug effects   MeSH
• Cricetinae MeSH
• Rats MeSH
• Humans MeSH
• Macromolecular Substances MeSH
• Oligonucleotides chemistry   metabolism   MeSH
• HMGB1 Protein chemistry   metabolism   MeSH
• Cross-Linking Reagents chemistry   pharmacology   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• Cisplatin MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH
• Macromolecular Substances MeSH
• Oligonucleotides MeSH
• HMGB1 Protein MeSH
• Cross-Linking Reagents MeSH