Copper(I)-bleomycin: structurally unique complex that mediates oxidative DNA strand scission

Copper(I)-bleomycin [Cu(I) X BLM] was characterized in detail by 13C and 1H NMR. Unequivocal chemical shift assignments for Cu(I) X BLM and Cu(I) X BLM X CO were made by two-dimensional 1H-13C correlated spectroscopy and by utilizing the observation that Cu(I) X BLM was in rapid equilibrium with Cu(I) and metal-free bleomycin, such that individual resonances in the spectra of BLM and Cu(I) X BLM could be correlated. The binding of Cu(I) by bleomycin involves the beta-aminoalaninamide and pyrimidinyl moieties, and possibly the imidazole, but not N alpha of beta-hydroxyhistidine. Although no DNA strand scission by Cu(II) X BLM could be demonstrated in the absence of dithiothreitol, in the presence of this reducing agent substantial degradation of [3H]DNA was observed, as was strand scission of cccDNA. DNA degradation by Cu(I) X BLM was shown not to depend on contaminating Fe(II) and not to result in the formation of thymine propenal; the probable reason(s) for the lack of observed DNA degradation in earlier studies employing Cu(II) X BLM and dithiothreitol was (were) also identified. DNA strand scission was also noted under anaerobic conditions when Cu(II) X BLM and iodosobenzene were employed. If it is assumed that the mechanism of DNA degradation in this case is the same as that under aerobic conditions (i.e., with Cu(I) X BLM + O2 in the presence of dithiothreitol), then Cu X BLM must be capable of functioning as a monooxygenase in its degradation of DNA.     

Bleomycin may be activated for DNA cleavage by NADPH-cytochrome P-450 reductase

In the presence of NADPH and O2, NADPH-cytochrome P-450 reductase was found to activate Fe(III)-bleomycin A2 for DNA strand scission. Consistent with observations made previously when cccDNA was incubated in the presence of bleomycin and Fe(II) + O2 or Fe(III) + C6H5IO, degradation of DNA by NADPH-cytochrome P-450 reductase activated Fe(III)-bleomycin A2 produced both single- and double-strand nicks with concomitant formation of malondialdehyde (precursors). Cu(II)-bleomycin A2 also produced nicks in SV40 DNA following activation with NADPH-cytochrome P-450 reductase, but these were not accompanied by the formation of malondialdehyde (precursors). These findings confirm the activity of copper bleomycin in DNA strand scission and indicate that it degrades DNA in a fashion that differs mechanistically from that of iron bleomycin. The present findings also-establish the most facile pathways for enzymatic activation of Fe(III)-bleomycin and Cu(II)-bleomycin, provide data concerning the nature of the activated metallobleomycins, and extend the analogy between the chemistry of cytochrome P-450 and bleomycin.     

Studies on the chemical reactivity of copper bleomycin

The copper(II) complex of the clinically used antitumor agent bleomycin (Blm) has cytotoxic as well as antitumor properties. To understand the relationship of the bleomycin ligand, copper bleomycin, and other possible metal complexes of this agent, kinetic studies of the formation of Cu(II)Blm, ligand substitution reactions of CuBlm with ethylenediaminetetraaletic acid, and the redox reaction of CuBlm with thiols have been completed and interpreted along with previous studies of the thermodynamic stability of Cu2+ with bleomycin. Cu(II)Bm is found to be kinetically and thermodynamically stable in ligand substitution processes and is only slowly reduced and dissociated by sulfhydryl reagents. The rate constant of reduction of the complex by 2-mercaptoethanol (2-ME) at pH 7.4 and 25 degrees C is 9.5 X 10(-3) M-1 sec-1, explaining the inhibition of Fe2+-dependent strand scission of DNA by Cu2+ in the presence of 2-ME. CuBlm forms in preference to Fe(II)Blm and cannot be reduced and dissociated rapidly enough by thiols to liberate Blm and form the reactive iron complex. In agreement with the observed chemical stability of CuBlm, it is also shown that the complex is stable in human plasma and in the presence of Ehrlich cells suspended in ascites fluid. Interestingly, little CuBlm enters these cells to carry out cytotoxic reactions. Finally, it is shown that both Cu2+ and Zn2+, at equivalent concentrations to Fe2+, effectively inhibit the strand scission of DNA by Fe(II)Blm plus oxygen. However, at substoichiometric amounts of Cu2+, the ferroxidase activity of Blm enables the drug to remain effective in the strand-scission reaction, despite the lowered Cu-free Blm/Fe2+ ratio. These results are discussed in light of the proposed mechanism of action of bleomycin.     

Copper-bleomycin has no significant DNA cleavage activity

In contrast to a very recent report [Ehrenfeld, G. M., Rodriguez, L. O., Hecht, S. M., Chang, C., Basus, V. J., & Oppenheimer, N. J. (1985) Biochemistry 24, 81-92], the present careful reexamination demonstrated that copper-bleomycin systems have no significant DNA cleavage activity. In the presence of dithiothreitol, the bleomycin-Cu(II) complex showed little activity for DNA degradation. The DNA strand scission by the Cu(I)-bleomycin-dithiothreitol system was remarkably depressed by deferoxamine rather than by bathocuproine, suggesting the effect of trace amounts of contaminating iron in the experiments. It seems highly unlikely that the DNA breakage activity due specifically to the Cu(I)-bleomycin complex system is substantially strong. Our results indicate that the metal really relevant to the DNA cleavage by bleomycin is iron not copper.     

DNA strand scission by iron complexes of meso-tetra(N-methylpyridyl)porphines

The DNA strand scission activities of three positional isomers of Fe(III) meso-tetra(N-methylpyridyl)porphine (Fe(III)TnMPyP, where n = 2, 3 or 4) have been investigated using PM2 DNA as a substrate. A significant degree of strand scission activity was noted in the presence of oxygen without the addition of a reducing agent. This activity was probably due to the presence of reducing agents in the agarose gels used to separate the DNA forms, as higher levels were recorded with reducing agents added to the strand scission mixture. The relative order of strand scission activity in the absence of added reducing agents was found to be Fe(III)T2MPyP greater than Fe(III)T4MPyP greater than Fe(III)T3MPyP. Comparative studies were also made with Fe(II)bleomycin. High concentrations of some reducing agents inhibited strand scission. Oxygen was required to produce optimal strand scission activity for all three porphyrins. It was also noted from spectroscopic measurements that the reduced porphyrins were degraded in the presence of oxygen. Studies with a series of potential strand scission inhibitors suggest that hydrogen peroxide and possibly peroxy radicals are intermediates in the reaction mechanism, while diffusible hydroxyl radicals appear to be excluded. However, superoxide radicals cannot be ruled out.     

Degradation of structurally modified DNAs by bleomycin group antibiotics

Bleomycin-mediated DNA strand scission has been shown to be diminished at certain sequences in proximity to 5-methylcytidines. We have investigated the molecular basis of this observed diminution using selective bleomycin (BLM) modifications at the C-terminus. Of the four different bleomycin congeners investigated, only bleomycin A2 and bleomycin BAPP were substantially affected by cytidine methylation. We have also examined the effect of other DNA modifications on bleomycin-mediated strand scission. Methylation at the N6 position of adenosine resulted in diminution of DNA cleavage by all four bleomycin congeners. The presence of bulky 5-(glucosyloxy)methyl groups in the major groove of T4 DNA had little effect on the efficiency of DNA strand scission mediated by bleomycin A2 or B2, suggesting the absence of important steric interactions between Fe(II).BLM and DNA in the major groove. In contrast, DNA cleavage mediated by bleomycin congeners was very sensitive to a major DNA conformational change, the B----Z transition. Salt and MgCl2 titrations of the DNA copolymers poly(dG-dC).poly(dG-dC) and poly(dG-MedC).poly(dG-MedC) demonstrated that bleomycin A2 and B2 did not cleave Z-DNA efficiently. In addition, circular dichroism titrations of these copolymers revealed that both bleomycin congeners increased the cation concentration necessary to induce the B----Z transition, implying that bleomycin preferentially binds to and stabilizes B-form DNA. These results are consistent with a model in which cytidine methylation at appropriate sequences of DNA is sufficient to induce subtle conformational changes that render the helix unreceptive to cleavage by some bleomycin congeners.     

Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses

The synthesis of methidiumpropyl-EDTA (MPE) is described. The binding affinities of MPE, MPE.Ni(II), and MPE.Mg(II) to calf thymus DNA are 2.4 X 10(4) M-1, 1.5 X 10(5) M-1, and 1.2 X 10(5) M-1, respectively, in 50 mM NaCl, pH 7.4. The binding site size is two base pairs. MPE.Mg(II) unwinds PM2 DNA 11 +/- 3 degrees per bound molecule. MPE.Fe(II) in the presence of O2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate greater than dithiothreitol greater than NADPH. At concentrations of 0.1-0.01 microM in MPE.Fe(II) and 10 microM in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 microM in base pairs) with MPE.Fe(II) (0.1-0.01 microM) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HCl buffer). The optimum cleavage time is 3.5 h (22 degrees C). DNA cleavage is efficient in a Na+ ion concentration range of 5 mM to 1 M, with the optimum at 5 mM NaCl. The number of single-strand scissions on supercoiled DNA per MPE.Fe(II) under optimum conditions is 1.4. Metals such as Co(II), Mg(II), Ni(II), and Zn(II) inhibit strand scission by MPE. The released products from DNA cleavage by MPE.Fe(II) are the four nucleotide bases. The DNA termini at the cleavage site are 5'-phosphate and roughly equal proportions of 3'-phosphate and 3'-(phosphoglycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone, most likely by hydroxy radical.     

Effect of chelating agents and metal ions on the degradation of DNA by bleomycin.

The degradation of DNA by bleomycin was studied in the absence and in the presence of added reducing agents, including 2-mercaptoethanol, dithiothreitol, reduced nicotinamide adenine dinucleotide phosphate, H2O2, and ascorbate, and in the presence of a superoxide anion generating system consisting of xanthine oxidase and hypoxanthine. In all cases, breakage of DNA was inhibited by low concentrations of chelators; where examined in detail, deferoxamine mesylate was considerably more potent than (ethylenedinitrilo)tetraacetic acid. Iron was found to be present in significant quantities in all reaction mixtures. Thus, the pattern of inhibition observed is attributed to the involvement of contaminating iron in the degradation of DNA by bleomycin. Cu(II), Zn(II), and Co(II) inhibit degradation of DNA by bleomycin and Fe(II) in the absence of added reducing agents. A model is proposed in which the degradation of DNA in these systems is dependent on the oxidation of an Fe(II)-bleomycin-DNA complex.     

Synthetic analogues and biosynthetic intermediates of bleomycin. Metal-binding, dioxygen interaction, and implication for the role of functional groups in bleomycin action mechanism

In order to clarify the role of bleomycin functional groups in action mechanism, the metal-binding, dioxygen activation, and DNA cleavage of several synthetic analogues and biosynthetic intermediates of bleomycin have been investigated. The present results support that 1) the beta-aminoalaninepyrimidine-beta-hydroxyhistidine portion of the bleomycin molecule substantially participates in the Fe(II) and dioxygen interactions, 2) the transposition of the pyrimidine (or pyridine) and imidazole groups in the Fe(II)-coordination is essential for the effective binding and activation of molecular oxygen by the bleomycin ligands, and 3) the gulose-mannose moiety plays an important role as an environmental factor for the efficient dioxygen reduction and DNA cleavage, although the sugar portion does not contribute significantly to the nucleotide specificity in the DNA strand scission. Certain oligopeptides are able to mimic the metal-binding and dioxygen activation by bleomycin, but not induce the effective DNA cleavage. Probably, the bithiazole DNA interaction site of bleomycin delivers the iron/dioxygen chemistry to particularly the DNA (formula, see text) nucleotide sequences.     

Analysis of products formed during bleomycin-mediated DNA degradation

By the use of DNA, copolymers of defined nucleotide composition, and a synthetic dodecanucleotide having putative bleomycin cleavage sites in proximity to the 5'- and 3'-termini, the products formed concomitant with DNA strand scission have been isolated and subjected to structural identification and quantitation via direct comparison with authentic synthetic samples. The products of DNA strand scission by Fe(II)-bleomycin include oligonucleotides having each of the four possible nucleoside 3'-(phosphoro-2'-O-glycolates) at their 3'-termini, as well as the four possible base propenals. At least for 3-(adenin-9'-yl)propenal and 3-(thymin-1'-yl)propenal, the products formed were exclusively of the trans configuration.     

Copper-mediated nuclease activity of jadomycin B

The natural product jadomycin B, isolated from Streptomyces venezeulae ISP5230, has been found to cleave DNA in the presence of Cu(II) ions without the requirement for an external reducing agent. The efficiency of DNA cleavage was probed using supercoiled plasmid DNA in buffered solution as a model environment. EC?? and t(?) values for cleavage were 1.7 μM and 0.75 h, respectively, and varied ± 5% with the particular batch of plasmid and jadomycin employed. While UV-vis spectroscopy indicates that the cleavage event does not involve direct binding of jadomycin B to DNA, a stoichiometric Cu(II) preference for optimum cleavage suggests a weak binding interaction between jadomycin B and Cu(II) in the presence of DNA. The Cu(II)-mediated cleavage is greatly enhanced by UV light, which implicates the jadomycin B radical cation and Cu(I) as potential intermediates in DNA cleavage. Evidence in favor of this hypothesis was derived from a mechanistic assay which showed reduced cleavage as a function of added catalase and EDTA, scavengers of H?O? and Cu(II), respectively. Thus, jadomycin B may serve as a source of electrons for Cu(II) reduction, producing Cu(I) which reacts with H?O? to form hydroxyl radicals that cause DNA strand scission. In addition, scavengers of hydroxyl radicals and superoxide also display inhibitory effects, underscoring the ability of jadomycin B to produce a powerful arsenal of deleterious oxygen species when copper is present.     

Structural basis for DNA-cleaving activity of resveratrol in the presence of Cu(II)

Resveratrol (1, 3,5,4'-trihydroxy-trans-stilbene), a polyphenol found in grapes and other food products, is known as an antioxidant and cancer chemopreventive agent. However, 1 was shown to induce genotoxicity through a high frequency of micronucleus and sister chromatid exchange in vitro and DNA-cleaving activity in the presence of Cu(II). The present study was designed to explore the structure-activity relationship of 1 in DNA strand scission and to characterize the substrate specificity for Cu(II) and DNA binding. When pBR322DNA was incubated with 1 or its analogues differing in the number and positions of hydroxyl groups in the presence of Cu(II), the ability of 4-hydroxystilbene analogues to induce DNA strand scission is much stronger than that of 3-hydroxy analogues. The high binding affinity with both Cu(II) and DNA was also observed by 4-hydroxystilbene analogues. The reduction of Cu(II) which is essential for activation of molecular oxygen proceeded by addition of 1 to the solution of the Cu(II)-DNA complex, while such reduction was not observed with the addition of isoresveratrol, in which the 4-hydroxy group of 1 is changed to the 3-position. The results show that the 4-hydroxystilbene structure of 1 is a major determinant of generation of reactive oxygen species that was responsible for DNA strand scission.     

Charge dependence of Fe(II)-catalyzed DNA cleavage.

The effect of charge of the Fe(II) reagent used to induce DNA strand cleavage reactions in the presence of a source of reducing equivalents is investigated using two oligonucleotide models. The first consists of the two strands dA20 and dT20, and an equimolar complex between them. The second is a short four-arm branched DNA complex composed of four 16-mer strands. In the former case, cleavage of the 1:1 complex by three reagents with different formal charge, Fe(II).EDTA2-, Fe(II).EDDA and Fe2+, is comparable in rate to that of the individual dT20 and the dA20 strands. While the three reagents show similar cleavage rates for the duplex and single stranded molecules, they give distinctive cutting patterns in the DNA tetramer, consistent with the presence of a site of excess negative charge at the branch point. Scission induced by Fe(II).EDTA2- shows lower reactivity at the branch site relative to duplex controls, whereas Fe(II)2+ shows enhanced reactivity. Formally neutral Fe(II).EDDA shows weak loss of cutting reactivity at the branch. The position of attack by Fe(II)2+ in the branched tetramer is shifted with respect to those of Fe(II).EDTA2- or Fe(II).EDDA; a slower migrating species is also detected in the scission of dA20.dT20 duplex by Fe(II) reaction. These results suggest that the Fe(II)2+ reaction proceeds by a different mechanism from the other agents. The difference in cutting profiles induced by the neutral and negatively charged chelated complexes is consistent with a local electrostatic repulsion of a negatively charged source of radicals, not a positively charged one.     

Enhanced bleomycin-mediated damage of DNA opposite charged nicks. A model for bleomycin-directed double strand scission of DNA

The anticancer drug, bleomycin, causes both single and double strand scission of duplex DNA in vitro, with double strand scission occurring in excess of that expected from the random accumulation of single strand nicks. The mechanism of the preferential double strand scission of DNA by bleomycin has been investigated through the synthesis of a series of double hairpin and linear oligonucleotides designed to contain a single nick-like structure at a defined site to serve as models of bleomycin-damaged duplex DNA. The 3' and/or 5' hydroxyls flanking the nick have been phosphorylated to model the increased negative charge at a bleomycin-generated nick. The ability of bleomycin to cleave the intact strand opposite the nick was then determined by autoradiography. The results demonstrate that phosphorylation at either the 3' or 5' hydroxyl, and especially when both sites are phosphorylated, strongly enhances selective cleavage by bleomycin of the opposite strand. These experiments indicate that bleomycin-mediated double strand scission is a form of self-potentiation in which the high affinity of bleomycin for the initially generated nicked sites leads to a greatly enhanced probability of scission of the strand opposite those sites.     

DNA cleavage specificity of a group of cationic metalloporphyrins

The ability of a group of water-soluble metalloporphyrins to cleave DNA has been investigated. Incubation of Mn3+, Fe3+, or Co3+ complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2T4MPyP) with DNA in the presence of ascorbate, superoxide ion, or iodosobenzene results in DNA breakage. Comparisons between the rates of porphyrin autodestruction with the rates of strand scission of covalently closed circular PM2 DNA indicate that the porphyrins remain intact during the cleavage process. Analysis of the porphyrin-mediated strand scissions on a 139-base-pair restriction fragment of pBR322 DNA using gel electrophoresis/autoradiography/microdensitometry reveals that the minimum porphyrin cleavage site is (A X T)3. The cleavage pattern within a given site was found to be asymmetric, indicating that porphyrin binding and the strand scission process are highly directional in nature. In addition to an analysis of the mechanism of porphyrin-mediated strand breakage in terms of the DNA cleavage mechanism of methidium-propyl-iron-EDTA and Fe-bleomycin, the potential of the cationic metalloporphyrins as footprinting probes and as new "reporter ligands" for DNA is presented and discussed.     

Copper(II).bleomycin, iron(III).bleomycin, and copper(II).phleomycin: comparative study of deoxyribonucleic acid binding

The kinetics and mechanism of binding of Cu-(II).bleomycin, Fe(III).bleomycin, and Cu(II).phleomycin to DNA were studied by using fluorometry, equilibrium dialysis, electric dichroism, and temperature-jump and stopped-flow spectrophotometry. The affinity of Cu(II).bleomycin for DNA was greater than that of metal-free bleomycin but less than that of Fe(III).bleomycin. Cu(II).bleomycin exhibited a two-step binding process, with the slow step indicating a lifetime of 0.1 s for the Cu(II).bleomycin.DNA complex. Fe(III).bleomycin binding kinetics indicated the presence of complexes having lifetimes of up to 22 s. DNA was lengthened by 4.6 A/molecule of bound Cu(II).bleomycin and by 3.2 A/bound Fe(III).bleomycin but not at all by Cu(II).phleomycin, suggesting that both bleomycin complexes intercalate while the phleomycin complex does not. However, phleomycin exhibited nearly the same specificity of DNA base release as bleomycin. These results suggest that the coordinated metal ion plays a major role in the binding of metal-bleomycin complexes to DNA but that intercalation is neither essential for DNA binding and degradation nor primarily responsible for the specificity of DNA base release by these drugs.     

Oxidative cell wall damage mediated by bleomycin-Fe(II) in Saccharomyces cerevisiae.

Bleomycin mediates cell wall damage in the yeast Saccharomyces cerevisiae. Bleomycin treatments in the presence of Fe(II) increased the rate of spheroplast formation by lytic enzymes by 5- to 40-fold. Neither Fe(III) nor other tested ions caused significant cell wall damage in the presence of bleomycin. The effect of bleomycin-Fe(II) on the cell wall mimicked the characteristics of bleomycin-Fe(II)-mediated DNA damage in dependence on aeration, inhibition by ascorbate, and potentiation by submillimolar concentrations of sodium phosphate. Bleomycin-mediated cell wall damage was time and dose dependent, with incubations as short as 20 min and drug concentrations as low as 3.3 x 10(-7)M causing measurable cell wall damage in strain CM1069-40. These times and concentrations are within the range of effectiveness for bleomycin-mediated DNA damage and for the cytotoxicity of the drug. Although Fe(III) was inactive with bleomycin and O2, the bleomycin-Fe(III) complex damaged walls and lysed cells in the presence of H2O2. H2O2 causes similar activation of bleomycin-Fe(III) in assays of DNA scission. These results suggest that an activated bleomycin-Fe-O2 complex disrupts essential cell wall polymers in a manner analogous to bleomycin-mediated cleavage of DNA.     

Cleavage of stem-and-loop structure DNA by bleomycin. Reaction on the bacteriophage G4 origin of complementary strand synthesis

The cleavage by bleomycin-Fe(II) complex in the presence of dithiothreitol of 3'-or 5'-end-labeled DNA from the region of the bacteriophage G4 origin of complementary strand synthesis was investigated by using the DNA-sequencing technique. Bleomycin cleaved a single-stranded DNA substrate preferentially at inverted repeat sequences, which potentially form stem-and-loop structures, while it cleaved double-stranded DNA substrates with different specificity. The results support the formation of three adjoining stem-and-loop structures in the region of the phage G4 origin of complementary strand synthesis under the low-salt conditions used and suggest a difference in the form of the double helix between the stem and the double-stranded DNA fragment. Bleomycin appears to be a useful reagent for searching stem-and-loop structures. The results may also contribute to the understanding of the mode of action of bleomycin as an antitumor antibiotic.     

Chemical cleavage of plasmid DNA by Cu(II), Ni(II) and Co(III) desferal complexes.

It is demonstrated that the Cu(II), Co(III) and Ni(II) complexes of a siderophore chelating drug desferal cleave DNA, in contrast to the corresponding Fe(II) complex which does not bring about DNA scission. Hydroxy radical scavengers inhibit the cleavage reaction.     

Light-induced nicking of deoxyribonucleic acid by cobalt(III) bleomycins

The anticancer drug bleomycin is a glycopeptide that causes strand scission of DNA both in vivo and in vitro. Cleavage of DNA by bleomycin has been studied extensively in vitro, with the findings that ferrous ion and molecular oxygen must be present and that addition of reducing agents greatly enhances the reaction. To date, only iron has been shown to be an effective metal cofactor for the cleavage of DNA by bleomycin. Here it is reported that two stable cobalt(III) complexes of bleomycin are strikingly effective in causing single-strand breaks (nicks) in supercoiled DNA in the presence of ultraviolet or visible radiation. For example, 366-nm light from an 18-W long-wavelength mercury lamp for 1 h causes 10(-6) M cobalt(III) bleomycin to completely convert supercoiled phi X174 DNA (10(-8) M DNA, 10(-4) M phosphate) into the nicked circular form. Furthermore, numerous alkali-labile sites are produced on the DNA during this treatment. The observed reactions are not caused by adventitious iron, and they occur only in the presence of cobalt(III) bleomycin and light.