Specific recognition by the tripeptide lysyl-tryptophyl-α-lysine of structural damage induced in DNA by platinum derivatives

The antitumoral derivative cisPt binds to DNA, as do its inactive analogs, trans- and dienPt. Structural damage introduced into DNA after reaction with the Pt derivatives were probed by using the peptide LysTrpLys. This peptide was used for its preferential binding to single-stranded structures (Brun, F., Toulmé, J.J. and Hélène, C. (1975) Biochemistry 14, 558–563). Phosphorescence lifetime measurements show that the Pt-induced heavy atom effects are quite similar in the three peptide-DNA-Pt complexes whatever the nature of the Pt derivative used. In contrast, fluorescence quenching strongly depends on the nature of the Pt derivatives. This quenching was therefore attributed to the stacking interactions engaged by the tryptophan residue with nucleic acid bases. A comparison of fluorescence quenching data for native and modified DNAs demonstrates that modification by dienPt has no effect on stacking interactions and that high levels of modifications by transPt are required to observe a change in stacking efficiency. In contrast modification by cisPt induces the formation of strong stacking sites. The results strongly suggest the existence of locally opened regions in DNA modified by cisPt.     

DNA damage induced by catechol derivatives

We investigated the effect of catechol derivatives, including dopa, dopamine, adrenaline and noradrenaline, on DNA damage and the mechanisms of DNA strand breakage and formation of 8-hydroxyguanine (8HOG). The catechol derivatives caused strand breakage of plasmid DNA in the presence of ADP-Fe(3+). The DNA damage was prevented by catalase, mannitol and dimethylsulfoxide, suggesting hydroxyl radical (HO..)-like species are involved in the strand breakage of DNA. Iron chelators, such as desferrioxamine and bathophenanthroline, and reduced glutathione also inhibited the DNA damage. Deoxyribose, a molecule that is used to detect HO,, was not degraded by dopa in the presence of ADP-Fe(3+). By adding EDTA, however, dopa induced the marked deoxyribose degradation in the presence of ADP-Fe(3+), indicating that EDTA may extract iron from ADP-Fe(3+) to catalyze HO. formation by dopa. Thus, EDTA was a good catalyst for HO.-generation, whereas it did not promote the strand breakage of DNA. However, calf thymus DNA base damage, which was detected as 8-HOG formation, was caused by dopa in the presence of EDTA-Fe(3+), but not in the presence of ADP-Fe(3+). The 8HOG formation was also inhibited by catalase and HO. scavengers, indicating that HO&z.rad; was involved in the base damage. These results suggest that DNA strand breakage is due to ferryl species rather than HO., and that 8HOG formation is due to HO. rather than ferryl species.     

DNA damage induced by novel demethylcantharidin-integrated platinum anticancer complexes

Oxaliplatin is a third generation platinum (Pt) drug with a diaminocyclohexane (DACH) entity, which has recently obtained worldwide approval for the clinical treatment of colon cancer, and apparently operates by a different mechanism of action to the classical cisplatin or carboplatin. Introducing a novel dual mechanism of action is one approach in designing a new platinum-based anticancer agent, whereby an appropriate ligand, such as demethylcantharidin (DMC), is released from the parent compound to exert a cytotoxic effect, in addition to that of the DNA-alkylating function of the platinum moiety. To investigate the likelihood of a novel dual mechanism of anticancer action, demethylcantharidin-integrated Pt complexes: Pt(R,R-DACH)(DMC) with the same Pt-DACH moiety as oxaliplatin, and Pt(NH(3))(2)(DMC) akin to carboplatin; were studied for their ability to induce DNA damage in HCT116 colorectal cancer cells by an alkaline comet assay. The results showed that the DMC ligand released from the novel complexes caused additional DNA lesions when compared with oxaliplatin and carboplatin. The comet assay also revealed that the DNA-damaging behavior of cisplatin is characteristically different; and this study is the first to demonstrate the ability of DMC to induce DNA lesions, thus providing sufficient evidence to explain the superior antiproliferative effect of the novel DMC-integrated complexes.     

DNA damage induced in vivo in various tissues by nitrochlorobenzene derivatives

Mono-, di-, and trinitrochlorobenzenes were injected i.p. into albino Swiss CD1 mice. Their effects were evaluated, in brain, liver and kidney, as single-strand DNA breaks.DNA damage was recognizable 4 h after administration in vivo, and its increment seemed to be related to the number of nitro groups contained in the chlorobenzene molecule.The simple and accurate microfluorometric procedure for DNA assay associated to the alkaline elution technique improved the application in vivo, avoiding the radiolabeling of DNA.     

Specific damage induced by X-ray radiation and structural changes in the primary photoreaction of bacteriorhodopsin

Bacteriorhodopsin, the sole membrane protein of the purple membrane of Halobacterium salinarum, functions as a light-driven proton pump. A 3-D crystal of bacteriorhodopsin, which was prepared by the membrane fusion method, was used to investigate structural changes in the primary photoreaction. It was observed that when a frozen crystal was exposed to a low flux of X-ray radiation (5 x 10(14)photons mm(-2)), nearly half of the protein was converted into an orange species, exhibiting absorption peaks at 450 nm, 478 nm and 510 nm. The remainder retained the normal photochemical activity until Asp85 in the active site was decarboxlyated by a higher flux of X-ray radiation (10(16)photons mm(-2)). The procedure of diffraction measurement was improved so as to minimize the effects of the radiation damage and determine the true structural change associated with the primary photoreaction. Our structural model of the K intermediate indicates that the Schiff base linkage and the adjacent bonds in the polyene chain of retinal are largely twisted so that the Schiff base nitrogen atom still interacts with a water molecule located near Asp85. With respect to the other part of the protein, no appreciable displacement is induced in the primary photoreaction.     

DNA modified by platinum derivatives cannot adopt the A-form

DNA fragments from chicken erythrocytes were modified by cis-diamminedichloroplatinum(II), its trans-isomer and chlorodiethylenetriaminoplatinum(II) chloride. The conformation of the modified DNA fragments in ethanolic solutions was studied by circular dichroism spectroscopy. Non-modified DNA adopted the A-form in 81% ethanol. The modification of DNA by the three platinum compounds inhibited the B to A transition of DNA induced by high concentrations of ethanol roughly to the same extent. The results support the view that the binding of the platinum complexes to B-DNA lowers the conformational freedom of DNA so that it cannot acquire the A-conformation.     

Specific DNA structural attributes modulate platinum anticancer drug site selection and cross-link generation

Heavy metal compounds have toxic and medicinal potential through capacity to form strong specific bonds with macromolecules, and the interaction of platinum drugs at the major groove nitrogen atom of guanine bases primarily underlies their therapeutic activity. By crystallographic analysis of transition metal-and in particular platinum compound-DNA site selectivity in the nucleosome core, we establish that steric accessibility, which is controlled by specific structural parameters of the double helix, modulates initial guanine-metal bond formation. Moreover, DNA conformational features can be linked to both similarities and distinctions in platinum drug adduct formation between the naked and nucleosomal DNA states. Notably, structures that facilitate initial platinum-guanine bond formation can oppose cross-link generation, rationalizing the occurrence of long-lived therapeutically ineffective monofunctional adducts. These findings illuminate DNA structure-dependent reactivity and provide a novel framework for understanding metal-double helix interactions, which should facilitate the development of improved chromatin-targeting medicinal agents.     

Specific recognition of loop DNA by dicationic porphyrins

A series of dicationic porphyrins were found to specifically recognize loop structures of oligodeoxynucleotides, and to selectively oxidize guanine residues upon photo-irradiation at micromolar concentrations. These compounds could, thus, be employed as promising structural probes for specific secondary DNA structures. The production of singlet oxygen ((1)O2) is responsible for the DNA-modification profiles. Both UV-titration and thermal-melting experiments indicate that the strong affinity of the charged porphyrins towards DNA loops is responsible for these molecular-recognition phenomena.     

Persistent and heritable structural damage induced in heterochromatic DNA from rat liver by N-nitrosodimethylamine

Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine (10 mg/kg body weight). Either hepatic DNA was prelabeled with [3H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Following incubation of purified nuclei with micrococcal nuclease, 10-12% of labeled DNA was solubilized (eu-chromatin) by 1.0 unit of micrococcal nuclease (5 mg of DNA)-1 mL-1 after 9 min. In prelabeled animals, administration of N-nitrosodimethylamine caused a marked fall in the specific radioactivity of solubilized DNA, while that of sedimenting DNA was not affected. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular recognition of quadruplex DNA by quinacridine derivatives

The interaction of monomeric and dimeric quinacridines with quadruplex DNA has been investigated using a variety of biophysical methods. Both series of compounds were shown to exhibit a high affinity for the G4 conformation with two equivalent binding sites. As shown from the SPR and dialysis experiments the macrocyclic dimer appears more selective than its monomeric counterpart.     

DNA damage spectra induced by photosensitization

DNA damage induced by photosensitization is not only responsible for the genotoxic effects of various types of drugs in the presence of light, but is also relevant for some of the adverse effects of sunlight, in particular in the UVA and visible range of the spectrum. The types of DNA modifications induced are very diverse and include pyrimidine dimers, covalent adducts, various base modifications generated by oxidation, single-strand breaks and (regular and oxidized) sites of base loss. The ratios in which the various modifications are formed (damage spectra) can be regarded as a fingerprint of the damaging mechanism. Here, we describe the damage spectra of various classes of photosensitizers in relation to the underlying damaging mechanisms. In mammalian cells irradiated with solar radiation, damage at wavelengths <400 nm is characteristic for a (not yet identified) endogenous type-I or type-II photosensitizer. In the UVA range, however, both direct DNA excitation and photosensitized damage appear to be relevant, and there are indications that other chromophore(s) are involved than in the visible range.     

Sequence-dependent conformational changes in DNA induced by polynuclear platinum complexes

In this work, the B-->Z transition of poly(dG-dC).poly(dG-dC) and the B-->A transition of poly(dG).poly(dC) and of calf thymus (CT) DNA fragments modified by antitumor bifunctional polynuclear platinum complexes were investigated by circular dichroism (CD). The transition from the B- to Z-form of DNA was inducible with all three compounds studied, as indicated by an inversion of the B-form spectra. The B-->A transition in poly(dG).poly(dC) was induced easily by platinum complex binding alone, while the B-->A transition in CT DNA was induced by ethanol but inhibited by coordination of all polynuclear platinum compounds used here. It was shown that the compound [?cis-PtCl(NH3)2?2 mu-?H2N(CH2)6NH2?] (NO3)2 (1,1/c,c) was most effective at inhibiting the B-->A transition in CT DNA, and [?trans-PtCl(NH3)2?2 mu-?trans-Pt(NH3)2(H2N(CH2)6NH2)2?] (NO3)4 (1,0,1/t,t,t) was least effective, while the effectiveness of [?trans-PtCl(NH3)2?2 mu-?H2N(CH2)6NH2?] (NO3)2 (1,1/t,t) fell between the two. This corresponded to the relative amounts of interstrand crosslinks in double-stranded DNA caused by each compound.     

Differences in DNA damage induced by mutagenic and nonmutagenic 4-aminoazobenzene derivatives in Escherichia coli.

DNA lesions produced in Escherichia coli AB2500 (uvrA) exposed to the carcinogen N-hydroxy-3-methoxy-4-aminoazobenzene (N-OH-3-MeO-AAB) or the noncarcinogen N-hydroxy-2-methoxy-4-aminoazobenzene (N-OH-2-MeO-AAB) were investigated by alkaline sucrose gradient sedimentation and 32P-postlabeling analysis. Alkali-labile sites appeared to be formed equally in cells treated with both aminoazobenzene derivatives. 32P-Postlabeling analysis revealed that the 3-MeO-AAB-DNA adduct level was 25-fold higher than that for 2-MeO-AAB-DNA adducts. In addition to major adducts, 4 minor spots were detected in N-OH-3-MeO-AAB-treated cells, while only one major adduct was found in N-OH-2-MeO-AAB-treated cells. The mutagenicities and cytotoxicities were also determined with E. coli with different repair capacities; we found that repair of 3-MeO-AAB damages is strongly dependent on the UVR repair system. Moreover, N-OH-3-MeO-AAB, but not N-OH-2-MeO-AAB, could induce recA and umuC gene expression, which was higher in uvrA strains than in the wild type.     

Cytotoxicity and DNA damage induced by a new platinum(II) complex with pyridine and dithiocarbamate

A new platinum(II) complex containing a pyridine nucleus and a dithiocarbamate moiety as ligands ([Pt(ESDT)(Py)Cl]) was evaluated for in vitro cytotoxicity in the cisplatin-sensitive human ovarian 2008 and in the isogenic-resistant C13* cell lines. In both cell types, a tumor cell growth inhibition greater than cisplatin and a complete lack of cross-resistance in C13* cells were found. Despite its molecular size, [Pt(ESDT)(Py)Cl] accumulation was much higher than cisplatin both in parent and resistant cells. Studying the mechanism of action in cell-free media, we established that [Pt(ESDT)(Py)Cl] more efficiently interacts with DNA in vitro compared to cisplatin maintaining a binding preference for GG rich sequences of DNA. On the contrary, DNA platination in vivo by [Pt(ESDT)(Py)Cl] was found lower than cisplatin. An analysis of the type of DNA lesions induced by [Pt(ESDT)(Py)Cl] suggests that the cytotoxic efficacy and the ability to overcome cisplatin resistance seem to be related to a different mechanism of interaction with DNA and/or with other key cellular components.     

Interspecies variation in DNA damage induced by pollution

The choice of a suitable species to translate pollution signals into a quantitative monitor is a fundamental step in biomonitoring plans. Here we present the results of three years of biomonitoring at a new coal power plant in central Italy using three different aquatic and terrestrial wildlife species in order to compare their reliability as sentinel organisms for genotoxicity. The comet assay was applied to the common land snail Helix spp., the lagoon fish Aphaniusfasciatus, and the green frog Rana esculenta sampled in the area potentially exposed to the impact of the power station. The tissue concentration of some expected pollutants （As, Cd, Ni, Pb, Cr） was analysed in parallel samples collected in the same sampling sites. The three species showed different values in the comet assay （Tail Intensity） and different accumulation profiles of heavy metals. Aphanius fasciatus showed an increasing genotoxic effect over time that paralleled the temporal increase of the heavy metals, especially arsenic, and the highest correlation between heavy metals and DNA damage. Helix spp. showed levels of damage inversely related to the distance from the source of pollution and in partial accordance with the total accumulation of trace elements. On the contrary, Rana esculenta showed a low capability to accumulate metals and had inconsistent results in the comet test. The fish appeared to be the most efficient and sensitive species in detecting chemical pollution. Overall, both the fish and the snail reflected a trend of increasing pollution in the area surrounding the power plant across time and space [Current Zoology 60 （2）： 308-321, 2014].     

Toxicity and DNA damage induced by 1-nitropyrene and its derivatives in Chinese hamster lung fibroblasts

1-Nitropyrene and its chemically synthesised derivatives were investigated for their cytotoxicity and ability to induce DNA-strand breaks in Chinese hamster lung fibroblasts. Both 1-nitrosopyrene (0.25-60 micrograms/ml) and 1-aminopyrene (0.25-25 micrograms/ml) were cytotoxic, and induced the formation of DNA lesions, which were measured as DNA single-strand breaks after sedimentation in alkaline sucrose-density gradients. Higher doses of 1-aminopyrene (25-60 micrograms/ml) inhibited the formation of DNA single-strand breaks. 1-Nitropyrene was not toxic (0.25-60 micrograms/ml) and induced low levels of detectable DNA strand breaks, whilst N-acetyl-1-aminopyrene was inactive. The post-mitochondrial supernatant fraction of Aroclor-induced rat-liver containing 4 mM NADPH (S9 mix) did not promote the activation of 1-nitropyrene. In fact DNA strand breaks induced by either 1-nitropyrene or 1-nitrosopyrene was abolished in the presence of S9 mix. The 1-nitropyrene reduced intermediate, N-hydroxy-1-aminopyrene was synthesised by the reduction of 1-nitrosopyrene with ascorbic acid. In the presence of ascorbic acid, 1-nitrosopyrene caused a 5-fold increase in the number of DNA single-strand breaks when compared to cells treated with 1-nitrosopyrene alone. The results are discussed in terms of the metabolic activation of 1-nitropyrene and 1-aminopyrene in Chinese hamster lung cells.     

Specific recognition of a DNA immunogen by its elicited antibody

DNA recognition by antibodies is a key feature of autoimmune diseases, yet model systems with structural information are very limited. The monoclonal antibody ED-10 recognizes one of the strands of the DNA duplex used in the immunogenic complex. Modifications of the 5' end decrease the binding affinity and short oligonucleotides retain high binding affinity. We determined crystal structures for the Fab bound to a 6-mer oligonucleotide containing the specific sequence that raised the antibody and compared it with the unliganded Fab. Only the first two bases from the 5' end (dTdC) display electron density and we observe four key hydrogen bonds at the interface. The thymine ring is stacked between TrpH50 and TrpH95, and the cytosine ring is packed against TyrL32. Upon DNA binding, TyrH97 and TrpH95 rearrange to allow subnanomolar binding affinity, five orders of magnitude higher than other reported complexes, possibly because of having gone through affinity maturation. This structure represents the first bona fide antibody DNA immunogen complex described in atomic detail.     

DNA damage recognition and repair by the murine MutY homologue

E. coli MutY excises adenine from duplex DNA when it is mispaired with the mutagenic oxidative lesion 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG). While E. coli MutY has been extensively studied, a detailed kinetic analysis of a mammalian MutY homologue has been inhibited by poor overexpression in bacterial hosts. This current work is the first detailed study of substrate recognition and repair of mismatched DNA by a mammalian adenine glycosylase, the murine MutY homologue (mMYH). Similar to E. coli MutY, the processing of OG:A substrates by mMYH is biphasic, indicating that product release is rate-limiting. Surprisingly, the intrinsic rates of adenine removal from both OG:A and G:A substrates by mMYH are diminished ( approximately 10-fold) compared to E. coli MutY. However, similar to E. coli MutY, the rate of adenine removal is approximately nine-fold faster with an OG:A- than a G:A-containing substrate. Interestingly, the rate of removal of 2-hydroxyadenine mispaired with OG or G in duplex DNA by mMYH was similar to the rate of adenine removal from the analogous context. In contrast, 2-hydroxyadenine removal by E. coli MutY was significantly reduced compared to adenine removal opposite both OG and G. Furthermore, dissociation constant measurements with duplexes containing noncleavable 2'-deoxyadenosine analogues indicate that mMYH is less sensitive to the structure of the base mispaired with OG or G than MutY. Though in many respects the catalytic behavior of mMYH is similar to E. coli MutY, the subtle differences may translate into differences in their in vivo functions.     

Mechanism of damage recognition by Escherichia coli DNA photolyase

Escherichia coli DNA photolyase binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light- (300-500 nm) dependent reaction. In order to determine the structural features important for this level of specificity, we have constructed a 43 base pair (bp) long DNA substrate that contains a thymine dimer at a unique location and studied its interaction with photolyase. We find that the enzyme protects a 12-16-bp region around the dimer from DNase I digestion and only a 6-bp region from methidium propyl-EDTA-Fe (II) digestion. Chemical footprinting experiments reveal that photolyase contacts the phosphodiester bond immediately 5' and the 3 phosphodiester bonds immediately 3' to the dimer but not the phosphodiester bond between the two thymines that make up the dimer. Methylation protection and interference experiments indicate that the enzyme makes major groove contacts with the first base 5' and the second base 3' to the dimer. These data are consistent with photolyase binding in the major groove over a 4-6-bp region. However, major groove contacts cannot be of major significance in substrate recognition as the enzyme binds equally well to a thymine dimer in a 44-base long single strand DNA and protects a 10-nucleotide long region around the dimer from DNase I digestion. It is therefore concluded that the unique configuration of the phosphodiester backbone in the strand containing the pyrimidine dimer, as well as the cyclobutane ring of the dimer itself are the important structural determinants of the substrate for recognition by photolyase.     

Apoptosis and growth arrest induced by platinum compounds in U2-OS cells reflect a specific DNA damage recognition associated with a different p53-mediated response

Mononuclear and multinuclear platinum complexes are known to induce distinct types of DNA lesions and exhibit different profiles of antitumor activity, in relation to p53 mutational status. In this study, we investigated the cellular effects of exposure to two platinum compounds (cisplatin and the multinuclear platinum complex BBR 3464), in the osteosarcoma cell line, U2-OS, carrying the wild-type p53 gene and capable of undergoing apoptosis or cell cycle arrest in response to diverse genotoxic stresses. In spite of the ability of both compounds to up-regulate p53 at cytotoxic concentrations, exposure to BBR 3464 resulted in cell cycle arrest but only cisplatin was capable of inducing significant levels of apoptosis and phosphorylation at the Ser15 residue of p53. The cisplatin-induced protein phosphorylation, not detectable in cells treated with BBR 3464, was associated with RPA phosphorylation, a specific up-regulation of Bax and down-regulation of p21(WAF1). Cells treated with BBR 3464 displayed a different cellular response with evidence of cytostasis associated with a high induction of p21(WAF1). The regulation of p21(WAF1) after cisplatin or BBR 3464 exposure required a p53 signal, as documented using stable transfectants expressing a dominant-negative form of p53 (175(his)). Taken together, these results indicate that cellular response to different genotoxic lesions (i.e. apoptosis or growth arrest) is associated with a specific recognition of DNA damage and a different p53-mediated signaling pathway. Multinuclear platinum complexes could be regarded as useful tools for investigating the p53-mediated process of cell cycle arrest in response to DNA damage.