Specificity of platinum-DNA adduct repair

Cell lines with resistance to cisplatin and carboplatin often retain sensitivity to platinum complexes with different carrier ligands (e.g., oxaliplatin and JM216). HeLa cell extracts were shown to excise cisplatin, oxaliplatin, and JM216 adducts with equal efficiency, suggesting that nucleotide excision repair does not contribute to the carrier-ligand specificity of platinum resistance. We have shown previously that the extent of replicative bypass in vivo is influenced by the carrier ligand of the platinum adducts. The specificity of replicative bypass may be determined by the DNA polymerase complexes that catalyze translesion synthesis past Pt-DNA adducts, by the mismatch-repair system that removes newly synthesized DNA opposite Pt-DNA adducts, and/or by DNA damage-recognition proteins that bind to the Pt-DNA adducts and block translesion synthesis. Primer extension on DNA templates containing site-specifically placed cisplatin, oxaliplatin, or JM216 Pt-GG adducts revealed that the eukaryotic DNA polymerases beta, zeta, gamma and HIV-1 RT had a similar specificity for translesion synthesis past Pt-DNA adducts (oxaliplatin > or = cisplatin > JM216). In addition, defects in the mismatch-repair proteins hMSH6 and hMLH1 led to increased replicative bypass of cisplatin adducts, but not of oxaliplatin adducts. Finally, primer extension assays performed in the presence of HMG1, which is known to recognize cisplatin-damaged DNA, revealed that inhibition of translesion synthesis by HMG1 also depended on the carrier ligand of the Pt-DNA adduct (cisplatin > oxaliplatin = JM216). These studies show that DNA polymerases, the mismatch-repair system and damage-recognition proteins can all impart specificity to replicative bypass of Pt-DNA adducts. Replicative bypass, in turn, may influence the carrier-ligand specificity of resistance.     

Differential human nucleotide excision repair of paired and mispaired cisplatin-DNA adducts.

In order to understand the action of the chemotherapeutic drug cisplatin, it is necessary to determine why some types of cisplatin-DNA intrastrand crosslinks are repaired better than others. Using cell extracts and circular duplex DNA, we compared nucleotide excision repair of uniquely placed 1,2-GG, 1,2-AG, and 1,3-GTG cisplatin-crosslinks, and a 2-acetylaminofluorene lesion. The 1,3 crosslink and the acetylaminofluorene lesion were repaired by normal cell extracts approximately 15-20 fold better than the 1,2 crosslinks. No evidence was found for selective shielding of 1,2 cisplatin crosslinks from repair by cellular proteins. Fractionation of cell extracts to remove putative shielding proteins did not improve repair of the 1,2-GG crosslink, and cell extracts did not selectively inhibit access of UvrABC incision nuclease to 1,2-GG crosslinks. The poorer repair of 1,2 crosslinks in comparison to the 1,3 crosslink is more likely a consequence of different structural alterations of the DNA helix. In support of this, a 1,2-GG-cisplatin crosslink was much better repaired when it was opposite one or two non-complementary thymines. Extracts from cells defective in the hMutSalpha mismatch binding activity also showed preferential repair of the 1,3 crosslink over the 1,2 crosslink, and increased repair of the 1,2 adduct when opposite thymines, showing that hMutSalphais not involved in the differential NER of these substrates in vitro. Mismatched cisplatin adducts could arise by translesion DNA synthesis, and improved repair of such adducts could promote cisplatin-induced mutagenesis in some cases.     

Recognition of cisplatin adducts by cellular proteins

Cisplatin is a widely used chemotherapeutic agent. It reacts with nucleophilic bases in DNA and forms 1,2-d(ApG), 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand crosslinks, interstrand crosslinks and monofunctional adducts. The presence of these adducts in DNA is through to be responsible for the therapeutic efficacy of cisplatin. The exact signal transduction pathway that leads to cell cycle arrest and cell death following treatment with the drug is not known but cell death is believed to be mediated by the recognition of the adducts by cellular proteins. Here we describe the structural information available for cisplatin and related platinum adducts, the interactions of the adducts with cellular proteins and the implications of these interactions for cell survival.     

Stopped-flow fluorescence studies of HMG-domain protein binding to cisplatin-modified DNA

High-mobility group (HMG) domain proteins bind specifically to the major DNA adducts formed by the anticancer drug cisplatin and can modulate the biological response to this inorganic compound. Stopped-flow fluorescence studies were performed to investigate the kinetics of formation and dissociation of complexes between HMG-domain proteins and a series of 16-mer oligonucleotide probes containing both a 1,2-intrastrand d(GpG) cisplatin cross-link and a fluorescein-modified deoxyuridine residue. Rate constants, activation parameters, and dissociation constants were determined for complexes formed by HMG1 domain A and the platinated DNA probes. The sequence context of the cisplatin adduct modulates the value of the associative rate constant for HMG1 domain A by a factor of 2-4, contributing significantly to differences in binding affinity. The rates of association or dissociation of the protein-DNA complex were similar for a 71 bp platinated DNA analogue. Additional kinetic studies performed with HMG1 domain B, an F37A domain A mutant, and the full-length HMG1 protein highlight differences in the binding properties of the HMG domains. The stopped-flow studies demonstrate the utility of the fluorescein-dU probe in studying protein-DNA complexes. The kinetic data will assist in determining what role these proteins might play in the cisplatin mechanism of action.     

Interaction of novel bis(platinum) complexes with DNA.

Bis(platinum) complexes [[cis-PtCl2(NH3)]2H2N(CH2)nNH2] are a novel series of potential anticancer agents in which two cis-diamine(platinum) groups are linked by an alkyldiamine of variable length. These complexes are potentially tetrafunctional, a unique feature in comparison with known anticancer agents. Studies of DNA interactions of bis(platinum) complexes in comparison with cisplatin demonstrate significant differences. Investigations of interstrand crosslink formation in which crosslinking of a short DNA fragment is detected by gel electrophoresis under denaturing conditions demonstrate that interstrand crosslinks are 250 fold more frequent among bis(platinum) adducts than among cisplatin-derived adducts under the conditions examined. These investigations indicate that bis(platinum) adducts contain a high frequency of structurally novel interstrand crosslinks formed through binding of the two platinum centers to opposite DNA strands. Unlike cisplatin, bis(platinum) complex binding does not unwind supercoiled DNA. Studies with the E. coli UvrABC nuclease complex demonstrate that both linear and supercoiled DNA containing bis(platinum) adducts are subject to incision by the repair enzyme complex. Initial studies using UvrABC nuclease as a probe to define the base and sequence specificity for bis(platinum) complex binding suggest that the specificity of the bis(platinum)s is similar, but not identical, to that of cisplatin.     

Mutagenic potential of DNA-peptide crosslinks mediated by acrolein-derived DNA adducts

Current data suggest that DNA-peptide crosslinks are formed in cellular DNA as likely intermediates in the repair of DNA-protein crosslinks. In addition, a number of naturally occurring peptides are known to efficiently conjugate with DNA, particularly through the formation of Schiff-base complexes at aldehydic DNA adducts and abasic DNA sites. Since the potential role of DNA-peptide crosslinks in promoting mutagenesis is not well elucidated, here we report on the mutagenic properties of Schiff-base-mediated DNA-peptide crosslinks in mammalian cells. Site-specific DNA-peptide crosslinks were generated by covalently trapping a lysine-tryptophan-lysine-lysine peptide to the N(6) position of deoxyadenosine (dA) or the N(2) position of deoxyguanosine (dG) via the aldehydic forms of acrolein-derived DNA adducts (gamma-hydroxypropano-dA or gamma-hydroxypropano-dG, respectively). In order to evaluate the potential of DNA-peptide crosslinks to promote mutagenesis, we inserted the modified oligodeoxynucleotides into a single-stranded pMS2 shuttle vector, replicated these vectors in simian kidney (COS-7) cells and tested the progeny DNAs for mutations. Mutagenic analyses revealed that at the site of modification, the gamma-hydroxypropano-dA-mediated crosslink induced mutations at only approximately 0.4%. In contrast, replication bypass of the gamma-hydroxypropano-dG-mediated crosslink resulted in mutations at the site of modification at an overall frequency of approximately 8.4%. Among the types of mutations observed, single base substitutions were most common, with a prevalence of G to T transversions. Interestingly, while covalent attachment of lysine-tryptophan-lysine-lysine at gamma-hydroxypropano-dG caused an increase in mutation frequencies relative to gamma-hydroxypropano-dG, similar modification of gamma-hydroxypropano-dA resulted in decreased levels of mutations. Thus, certain DNA-peptide crosslinks can be mutagenic, and their potential to cause mutations depends on the site of peptide attachment. We propose that in order to avoid error-prone replication, proteolytic degradation of proteins covalently attached to DNA and subsequent steps of DNA repair should be tightly coordinated.     

Removal of psoralen monoadducts and crosslinks by human cell free extracts.

Human cell free extracts are capable of carrying out damage-induced DNA synthesis in response to DNA damage by UV, psoralen, and cisplatin. We show that this damage-induced DNA synthesis is associated with removal of psoralen adducts and therefore is 'repair synthesis' and not an aberrant DNA synthesis reaction potentiated by DNA deformed by adducts. By comparing the denaturable fraction of psoralen adducted DNA which becomes labeled in the repair reaction to that of terminally labeled DNA (without repair) we have found that all DNA synthesis induced by psoralen monoadducts is the consequence of removal of these adducts. By the same approach we have obtained preliminary evidence that this in vitro system is capable of removing psoralen crosslinks as well.     

Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin

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.     

Energetics,conformation, and recognition of DNA duplexes containing a major adduct of an anticancer azolato-bridged dinuclear Pt complex

Background

The design of anticancer metallodrugs is currently focused on platinum complexes which form on DNA major adducts that cannot readily be removed by DNA repair systems. Hence, antitumor azolato-bridged dinuclear Pt^II complexes, such as [{cis-Pt(NH₃)₂}₂(μ‐OH)(μ-pyrazolate)]²⁺ (AMPZ), have been designed and synthesized. These complexes exhibit markedly higher toxic effects in tumor cell lines than mononuclear conventional cisplatin.

Methods

Biophysical and biochemical aspects of the alterations induced in short DNA duplexes uniquely and site-specifically modified by the major DNA adduct of AMPZ, namely 1,2-GG intrastrand cross-links, were examined. Attention was also paid to conformational distortions induced in DNA by the adducts of AMPZ and cisplatin, associated alterations in the thermodynamic stability of the duplexes, and recognition of these adducts by high-mobility-group (HMG) domain proteins.

Results

Chemical probing of DNA conformation, DNA bending studies and translesion synthesis by DNA polymerase across the platinum adduct revealed that the distortion induced in DNA by the major adduct of AMPZ was significantly less pronounced than that induced by similar cross-links from cisplatin. Concomitantly, the cross-link from AMPZ reduced the thermodynamic stability of the modified duplex considerably less. In addition, HMGB1 protein recognizes major DNA adducts of AMPZ markedly less than those of cisplatin.

General significance

The experimental evidence demonstrates why the major DNA adducts of the new anticancer azolato-bridged dinuclear Pt^II complexes are poor substrates for DNA repair observed in a previously published report. The relative resistance to DNA repair explains why these platinum complexes show major pharmacological advantages over cisplatin in tumor cells.     

Cisplatin adducts inhibit 1,N(6)-ethenoadenine repair by interacting with the human 3-methyladenine DNA glycosylase

The human 3-methyladenine DNA glycosylase (AAG) is a repair enzyme that removes a number of damaged bases from DNA, including adducts formed by some chemotherapeutic agents. Cisplatin is one of the most widely used anticancer drugs. Its success in killing tumor cells results from its ability to form DNA adducts and the cellular processes triggered by the presence of those adducts in DNA. Variations in tumor response to cisplatin may result from altered expression of cellular proteins that recognize cisplatin adducts. The present study focuses on the interaction between the cisplatin intrastrand cross-links and human AAG. Using site-specifically modified oligonucleotides containing each of the cisplatin intrastrand cross-links, we found that AAG readily recognized cisplatin adducts. The apparent dissociation constants for the 1, 2-d(GpG), the 1,2-d(ApG), and the 1,3-d(GpTpG) oligonucleotides were 115 nM, 71 nM, and 144 nM, respectively. For comparison, the apparent dissociation constant for an oligonucleotide containing a single 1,N(6)-ethenoadenine (epsilonA), which is repaired efficiently by AAG, was 26 nM. Despite the affinity of AAG for cisplatin adducts, AAG was not able to release any of these adducts from DNA. Furthermore, it was demonstrated that the presence of cisplatin adducts in the reactions inhibited the excision of epsilonA by AAG. These data suggest a previously unexplored dimension to the toxicological response of cells to cisplatin. We suggest that cisplatin adducts could titrate AAG away from its natural substrates, resulting in higher mutagenesis and/or cell death because of the persistence of AAG substrates in DNA.     

Mechanisms of resistance to cisplatin

The use of cisplatin in cancer chemotherapy is limited by acquired or intrinsic resistance of cells to the drug. Cisplatin enters the cells and its chloride ligands are replaced by water, forming aquated species that react with nucleophilic sites in cellular macromolecules. The presence of the cisplatin adducts in DNA is thought to trigger cell cycle arrest and apoptosis. Knowledge of the mechanism of action of cisplatin has improved our understanding of resistance. Decreased intracellular concentration due to decreased drug uptake, increased reflux or increased inactivation by sulfhydryl molecules such as glutathione can cause resistance to cisplatin. Increased excision of the adducts from DNA by repair pathways or increased lesion bypass can also result in resistance. Finally, altered expression of regulatory proteins involved in signal transduction pathways that control the apoptotic pathway can also affect sensitivity to the drug. An improved understanding of the mechanisms of resistance operative in vivo has identified targets for intervention and may increase the utility of cisplatin for the treatment of cancer.     

Characterization of a DNA damage-recognition protein from mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, Kd, of the protein from cisplatin-modified DNA was estimated to be (1-20) X 10(-10) M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl2] (en, ethylenediamine), and [Pt(dach)Cl2] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein also does not bind to DNA containing UV-induced photoproducts. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin, as determined by gel mobility shifts with synthetic 110-bp duplex oligonucleotides; these modified oligomers contained five equally spaced adducts of either cis-[Pt(NH3)2d(GpG) or cis-[Pt(NH3)2d(ApG)]. Oligonucleotides containing the specific adducts cis-[Pt(NH3)2d(GpTpG)], trans-[Pt(NH3)2d(GpTpG)], or cis-[Pt(NH3)2(N3-cytosine)d(G)] were not recognized by the protein. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl2, CdCl2, CoCl2, or ZnCl2 and with 1 mM HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamic simulations of cisplatin- and oxaliplatin-d(GG) intrastand cross-links reveal differences in their conformational dynamics

Mismatch repair proteins, DNA damage-recognition proteins and translesion DNA polymerases discriminate between Pt-GG adducts containing cis-diammine ligands (formed by cisplatin (CP) and carboplatin) and trans-RR-diaminocyclohexane ligands (formed by oxaliplatin (OX)) and this discrimination is thought to be important in determining differences in the efficacy, toxicity and mutagenicity of these platinum anticancer agents. We have postulated that these proteins recognize differences in conformation and/or conformational dynamics of the DNA containing the adducts. We have previously determined the NMR solution structure of OX-DNA, CP-DNA and undamaged duplex DNA in the 5'-d(CCTCAGGCCTCC)-3' sequence context and have shown the existence of several conformational differences in the vicinity of the Pt-GG adduct. Here we have used molecular dynamics simulations to explore differences in the conformational dynamics between OX-DNA, CP-DNA and undamaged DNA in the same sequence context. Twenty-five 10 ns unrestrained fully solvated molecular dynamics simulations were performed starting from two different DNA conformations using AMBER v8.0. All 25 simulations reached equilibrium within 4 ns, were independent of the starting structure and were in close agreement with previous crystal and NMR structures. Our data show that the cis-diammine (CP) ligand preferentially forms hydrogen bonds on the 5' side of the Pt-GG adduct, while the trans-RR-diaminocyclohexane (OX) ligand preferentially forms hydrogen bonds on the 3' side of the adduct. In addition, our data show that these differences in hydrogen bond formation are strongly correlated with differences in conformational dynamics, specifically the fraction of time spent in different DNA conformations in the vicinity of the adduct, for CP- and OX-DNA adducts. We postulate that differential recognition of CP- and OX-GG adducts by mismatch repair proteins, DNA damage-recognition proteins and DNA polymerases may be due, in part, to differences in the fraction of time that the adducts spend in a conformation favorable for protein binding.