Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes.

• MeSH
• DNA Adducts chemistry   metabolism   MeSH
• Cisplatin chemistry   metabolism   pharmacology   MeSH
• HEK293 Cells MeSH
• Kinetics MeSH
• Coordination Complexes chemistry   metabolism   pharmacology   MeSH
• Consensus Sequence MeSH
• Humans MeSH
• NF-kappa B chemistry   genetics   metabolism   MeSH
• Oligodeoxyribonucleotides chemistry   metabolism   MeSH
• Organoplatinum Compounds chemistry   toxicity   MeSH
• Platinum chemistry   metabolism   MeSH
• Surface Plasmon Resonance MeSH
• Antineoplastic Agents chemistry   metabolism   pharmacology   MeSH
• Recombinant Proteins biosynthesis   chemistry   isolation & purification   MeSH
• Electrophoretic Mobility Shift Assay MeSH
• Thermodynamics MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Cell Survival drug effects   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
• Names of Substances
• DNA Adducts MeSH
• BBR 3464 MeSH Browser
• Cisplatin MeSH
• Coordination Complexes MeSH
• NF-kappa B MeSH
• Oligodeoxyribonucleotides MeSH
• Organoplatinum Compounds MeSH
• Platinum MeSH
• Antineoplastic Agents MeSH
• Recombinant Proteins MeSH

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

The trinuclear BBR3464 ([{trans-PtCl(NH(3))(2)}(2)µ-(trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2))](4+)) belongs to the polynuclear class of platinum-based anticancer agents. DNA adducts of this complex differ significantly in structure and type from those of clinically used mononuclear platinum complexes, especially, long-range (Pt, Pt) intrastrand and interstrand cross-links are formed in both 5'-5' and 3'-3' orientations. We show employing short oligonucleotide duplexes containing single, site-specific cross-links of BBR3464 and gel electrophoresis that in contrast to major DNA adducts of clinically used platinum complexes, under physiological conditions the coordination bonds between platinum and N7 of G residues involved in the cross-links of BBR3464 can be cleaved. This cleavage may lead to the linkage isomerization reactions between this metallodrug and double-helical DNA. Differential scanning calorimetry of duplexes containing single, site-specific cross-links of BBR3464 reveals that one of the driving forces that leads to the lability of DNA cross-links of this metallodrug is a difference between the thermodynamic destabilization induced by the cross-link and by the adduct into which it could isomerize. The rearrangements may proceed in the way that cross-links originally formed in one strand of DNA can spontaneously translocate from one DNA strand to its complementary counterpart, which may evoke walking of the platinum complex on DNA molecule.

• MeSH
• DNA Adducts chemistry   MeSH
• Calorimetry, Differential Scanning MeSH
• DNA chemistry   MeSH
• Organoplatinum Compounds chemistry   MeSH
• Antineoplastic Agents chemistry   MeSH
• Cross-Linking Reagents chemistry   MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Adducts MeSH
• BBR 3464 MeSH Browser
• DNA MeSH
• Organoplatinum Compounds MeSH
• Antineoplastic Agents MeSH
• Cross-Linking Reagents MeSH

Reported herein is a detailed biochemical and molecular biophysics study of the molecular mechanism of action of antitumor dinuclear Pt(II) complex [{PtCl(DACH)}(2)-mu-Y](4+) [DACH=1,2-diaminocyclohexane, Y=H(2)N(CH(2))(6)NH(2)(CH(2))(2)NH(2)(CH(2))(6)NH(2)] (complex 1). This new, long-chain bifunctional dinuclear Pt(II) complex is resistant to metabolic decomposition by sulfur-containing nucleophiles. The results show that DNA adducts of 1 can largely escape repair and yet inhibit very effectively transcription so that they should persist longer than those of conventional cisplatin. Hence, they could trigger a number of downstream cellular effects different from those triggered in cancer cells by DNA adducts of cisplatin. This might lead to the therapeutic effects that could radically improve chemotherapy by platinum complexes. In addition, the findings of the present work make new insights into mechanisms associated with antitumor effects of dinuclear/trinuclear Pt(II) complexes possible.

• MeSH
• Cell-Free System MeSH
• DNA chemistry   MeSH
• Fluorescence MeSH
• Glutathione chemistry   MeSH
• Nucleic Acid Conformation * MeSH
• Molecular Sequence Data MeSH
• DNA Repair MeSH
• Organoplatinum Compounds chemistry   pharmacology   MeSH
• Antineoplastic Agents chemistry   pharmacology   MeSH
• Base Sequence MeSH
• Sulfur chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Glutathione MeSH
• Organoplatinum Compounds MeSH
• Antineoplastic Agents MeSH
• Sulfur 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

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

The trinuclear platinum agent BBR3464, a representative of a new class of anticancer drugs, is more potent than conventional mononuclear cisplatin [cis-diamminedichloroplatinum(II)]. BBR3464 retains significant activity in human tumor cell lines and xenografts that are refractory or poorly responsive to cisplatin, and displays a high activity in human tumor cell lines that are characterized by both wild-type and mutant p53 gene. In contrast, on average, cells with mutant p53 are more resistant to the effect of cisplatin. It has been hypothesized that the sensitivity or resistance of tumor cells to cisplatin might be also associated with cell cycle control and repair processes that involve p53. DNA is a major pharmacological target of platinum compounds and DNA binding activity of the p53 protein is crucial for its tumor suppressor function. This study, using gel-mobility-shift assays, was undertaken to examine the interactions of active and latent p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by BBR3464 in a cell free medium and to compare these results with those describing the interactions of these proteins with DNA modified by cisplatin. The results indicate that structurally different DNA adducts of BBR3464 and cisplatin exhibit a different efficiency to affect the binding affinity of the modified DNA to p53 protein. It has been suggested that different structural perturbations induced in DNA by the adducts of BBR3464 and cisplatin produce a differential response to p53 protein activation and recognition and that a 'molecular approach' to control of downstream effects such as protein recognition and pathways of apoptosis induction may consist in design of structurally unique DNA adducts as cell signals.

• MeSH
• DNA Adducts chemistry   drug effects   genetics   metabolism   MeSH
• Cisplatin chemistry   pharmacology   MeSH
• Consensus Sequence genetics   MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Organoplatinum Compounds chemistry   pharmacology   MeSH
• Antineoplastic Agents chemistry   pharmacology   MeSH
• Response Elements genetics   MeSH
• Base Sequence MeSH
• Substrate Specificity MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Names of Substances
• DNA Adducts MeSH
• BBR 3464 MeSH Browser
• Cisplatin MeSH
• Tumor Suppressor Protein p53 MeSH
• Organoplatinum Compounds MeSH
• Antineoplastic Agents 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