Activation of neocarzinostatin chromophore and formation of nascent DNA damage do not require molecular oxygen.

Thiol-activated neocarzinostatin chromophore abstracts tritium from the 5', but not from the 1' or 2' positions of deoxyribose in DNA and incorporates it into a stable, non-exchangeable form. The abstracted tritium remains covalently associated with the chromophore or its degradation product after treatment with acid or alkali, respectively. Drug activation and the consequent hydrogen abstraction reaction, presumably generating a carbon-centered radical at C-5', do not require molecular oxygen but have a dose-dependent relation with thiol. Under aerobic conditions, where base release and DNA strand breaks with nucleoside 5'-aldehyde at the 5'-ends are produced, hydrogen abstraction from C-5' parallels these parameters of DNA damage. It is possible to formulate a reaction scheme in which the carbon- centered radical at C-5' is an intermediate in the formation of the various DNA damage products found under both aerobic and anaerobic conditions.     

Free radical mechanisms in neocarzinostatin-induced DNA damage

The molecular mechanisms by which the antitumor protein antibiotic, neocarzinostatin, interacts with DNA and causes DNA sugar damage is discussed. Physical binding of the nonprotein chromophore of neocarzinostatin to DNA, involving an intercalative process and dependent on the microheterogeneity of DNA structure, is followed by thiol activation of the drug to a probable radical species. The latter attacks the deoxyribose, especially at thymidylate residues, by abstracting a hydrogen atom from C-5' to generate a carbon-centered radical on the DNA. This nascent form of DNA damage either reacts with dioxygen to form a peroxyl radical derivative, which eventuates in a strand break with a nucleoside 5'-aldehyde at the 5'-end or reacts with the bound drug to form a novel drug-deoxyribose covalent adduct. Nitroaromatic radiation sensitizers can substitute for dioxygen, but the DNA damage products are different. Similarities between the various biological effects of neocarzinostatin and ionizing radiation are reviewed.     

Neocarzinostatin chromophore-DNA adducts: evidence for a covalent linkage to the oxidized C-5'' of deoxyribose.

The nonprotein chromophore of neocarzinostatin forms a variety of adducts with DNA. The predominant adduct recovered from nuclease digests of chromophore-treated poly(dA-dT). poly(dA-dT) is a compound with structure chromophore-d(TpApT). Mild acid hydrolysis of this compound released free adenine, while snake venom exonuclease (pH 6.5) released 5'-dTMP leaving in both cases adducts of slightly altered chromatographic mobility. These results eliminate adenine and 5'-dTMP as possible sites of covalent chromophore attachment. Electrophoresis data suggest that the adduct is not a phosphotriester. At pH 8.6, chromophore-d(TpApT) spontaneously hydrolyzed, releasing chromophore and 3'-dTMP, leaving a modified d(ApT) which contained deoxyadenosine-5'-aldehyde. Deoxyadenosine-5'-aldehyde was released from the modified d(ApT) by snake venom exonuclease, and identified by a series of derivatizations including 1) mild oxidation to deoxyadenosine-5'-carboxylic acid, 2) NaBH4 reduction to deoxyadenosine, and 3) formation of a hydrazone with phenylhydrazine. Since deoxyadenosine-5'-aldehyde cannot exist as such in the chromophore-d(TpApT) adduct, we suggest that the chromophore may be covalently attached to the C-5' of deoxyadenosine as a phosphorylacetal or similar structure. Hydrolysis of the chromophore-acetal bond at pH 8.6 would leave a phosphorylhemiacetal on C-5', which would be expected to spontaneously decompose to yield the observed 3'-phosphate and 5'-aldehyde groups.     

Poly(deoxyadenylic-deoxythymidylic acid) damage by radiolytically activated neocarzinostatin

The anaerobic reaction of poly(deoxyadenylic-deoxythymidylic acid) with neocarzinostatin activated by the carboxyl radical CO2-, an electron donor generated from gamma-ray radiolysis of nitrous oxide saturated formate buffer, has been characterized. DNA damage includes base release and strand breaks. Few strand breaks are formed prior to alkaline treatment; they bear 3'-phosphoryl termini. In contrast, most (66%) of the base release occurs spontaneously. DNA damage is highly (95%) specific for thymidine sites. Neither DNA-drug covalent adduct nor nucleoside 5'-aldehyde, which are major products in the DNA-nicking reaction initiated by mercaptans and oxygen, is formed in this reaction. Data are presented to show that the CO2(-)-activated neocarzinostatin intermediate is a short-lived free radical able to abstract hydrogen atoms from the C-1' and C-5' positions of deoxyribose. Attack occurs mostly (68%) at the C-1' position, producing a lesion whose properties are consistent with those of (oxidized) apyrimidinic sites.     

Deoxyribonucleic acid damage by neocarzinostatin chromophore: strand breaks generated by selective oxidation of C-5' of deoxyribose

Among the lesions induced in DNA by neocarzinostatin chromophore are spontaneous and alkali-dependent base release, sugar damage, and single-strand breaks with phosphate (PO4) at their 3' ends and PO4 or nucleoside 5'-aldehyde at the 5' ends. By measuring alkali-dependent thymine release and decomposition of the 5'-terminal thymidine 5'-aldehyde in drug-cut DNA, we show that the kinetics are the same for each process and that the nucleoside aldehyde is the source of about 85% of alkali-dependent thymine release. Reduction of the 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase permits their selective quantitation. Nucleoside 5'-aldehyde so measured accounts for over 80% of the drug-generated 5' ends; the remainder have PO4 termini. Since these techniques also include the contribution of alkali-labile sites in the measurement of PO4 ends, DNA sequencing was used to measure the ends directly. Using 3'-32P end-labeled DNA restriction fragments as substrates for the drug, it was found that drug attack at a T results in mainly two bands--the stronger one represents oligonucleotide with 5'-terminal nucleoside 5'-aldehyde and may account for over 90% of a particular break. Its structure was verified by its isolation from the sequencing gel, followed by various chemical and enzymatic treatments. In each case, the mobility of the product on the gel was altered in a predictable manner. In addition to spontaneous breaks, neocarzinostatin also causes alkali-labile breaks preferentially at T residues. These sites are heterogeneous in their sensitivity to alkali and are protected by reduction.     

Selective abstraction of 2H from C-1' of the C residue in AGC.ICT by the radical center at C-2 of activated neocarzinostatin chromophore: structure of the drug/DNA complex responsible for bistranded lesion formation.

Glutathione-activated neocarzinostatin chromophore (NCS-Chrom) generates bistranded lesions at AGC.GCT sequences in DNA, consisting of an abasic site at the C residue and a strand break at the T residue on the complementary strand, due to hydrogen atom abstraction from C-1' and C-5', respectively. Earlier work showed that 2H from C-5' of T was selectively abstracted by the radical center at C-6 of activated NCS-Chrom, supporting a proposed model of the active-drug/DNA complex. However, since under the conditions used breaks at the T exceeded their inclusion in bistranded lesions, it was not clear what fraction of the hydrogen transfer represented bistranded lesions. Since virtually all abasic sites at the C are part of a bistranded lesions, hydrogen transfer from C-1' of C into the drug should reflect only the bistranded reaction. Accordingly, a self-complementary oligodeoxynucleotide 5'-GCAGCICTGC-3' was synthesized in which the C contained 2H at the C-1' position. In order to eliminate an 2H isotope effect on the transfer and to increase the extent of the bistranded reaction, an I residue was substituted for the G opposite the C residue. Sequencing gel electrophoretic analysis revealed that under one-hit kinetics, 37% of the damage reaction was associated with abasic site (alkali-labile break) formation at the C residue and 48% with direct strand breaks at the T residue. Thus, 74% of the damage involved a bistranded lesion. 1H NMR spectroscopic analysis of the reacted chromophore showed that 2H had been selectively transferred into the C-2 position to the extent of approximately 22%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neocarzinostatin-induced DNA base release accompanied by staggered oxidative cleavage of the complementary strand

Treatment of an end-labeled DNA restriction fragment with the nonprotein chromophore of neocarzinostatin induced lesions which, after treatment with endonuclease IV or putrescine, were expressed as site-specific double-strand breaks. Analysis of the termini at cleavage sites in each strand showed that the neocarzinostatin-induced lesions consisted of an apurinic/apyrimidinic site plus a closely opposed break in the complementary strand. The break always occurred opposite the base two positions upstream from the apurinic/apyrimidinic site and had the 3'-phosphate and 5'-aldehyde termini characteristic of neocarzinostatin-induced breaks. This positioning suggests that neocarzinostatin simultaneously attacks two DNA sugars on opposite edges of the minor groove. The sequence specificity for formation of apurinic/apyrimidinic sites with closely opposed breaks reflected that of neocarzinostatin-induced mutagenesis. The potent mutagenicity of these lesions may be attributable to the presence of closely opposed damage in both DNA strands.     

Oxygen transfer from the nitro group of a nitroaromatic radiosensitizer to a DNA sugar damage product

Mechanisms based on one-electron oxidation appear incomplete in explaining cellular radiosensitization by nitroaromatic compounds such as misonidazole. Evidence is presented for a novel mechanism that may be involved in enhancing DNA strand breakage due to a variety of agents, including ionizing radiation, that generate carbon-centered radicals on DNA deoxyribose. Under anaerobic conditions the carbon-centered radical generated selectively at C-5' of deoxyribose of thymidylate residues in DNA by the antitumor antibiotic neocarzinostatin reacts with misonidazole to produce a DNA damage product in the form of 3'-(formyl phosphate)-ended DNA. In an 18O-transfer experiment we find that the carbonyl oxygen of the activated formyl moiety (trapped as formyl-Tris) is derived from the nitro group oxygen of misonidazole. This result strongly supports a mechanism in which a nitroxide radical adduct, formed by the addition of misonidazole to the radical at C-5' of deoxyribose, cleaves between the N and O so as to form an oxy radical precursor of the formyl moiety and a two-electron reduction species of misonidazole.     

Generation of superoxide free radical by neocarzinostatin and its possible role in DNA damage

Spectroscopic analysis of the reduction of both nitro blue tetrazolium and ferricytochrome c induced by neocarzinostatin shows that superoxide free radical is produced during the spontaneous degradation of the antibiotic. The amount of superoxide free radical produced from neocarzinostatin is not affected by the presence of thiol, although earlier work has shown that DNA damage is stimulated at least 1000-fold by thiol. Transition metals are not involved in this reaction. Although superoxide dismutase inhibits the reduction of nitro blue tetrazolium and cytochrome c induced by neocarzinostatin, neither it nor catalase interferes with the action of neocarzinostatin on DNA, whether or not drug has been activated by thiol. The pH profiles for spontaneous base release and alkali-labile base release (a measure of nucleoside 5'-aldehyde formation at a strand break) do not correspond with that for the generation of superoxide free radical from neocarzinostatin. The same holds for supercoiled DNA cutting by neocarzinostatin chromophore in the absence of a thiol, which is an acid-favored reaction. These results indicate that the generation of superoxide free radical by the drug does not correlate with DNA damage activity, whether or not thiol is present. Furthermore, the failure of hydroxyl free-radical scavengers to inhibit drug-induced single-strand breaks in supercoiled DNA in the absence of thiol also indicates that a diffusible hydroxyl free radical is most probably not involved in this reaction.     

DNA microstructural requirements for neocarzinostatin chromophore-induced direct strand cleavage.

The microstructural requirements for optimal interaction of neocarzinostatin chromophore (NCS-C) with DNA have been investigated using a series of hexadeoxyribonucleotides with modified bases such as O6-methyl G (MeG), I, 5-methyl C (MeC), U, or 5-Bromo U (BrU) at specific sites in its preferred trinucleotide 5'GNaNb3':5'Na,Nb,C3' (Na = A, C, or T). Results show that MeG:C and G:MeC in place of G:C improve direct strand cleavage at the target Nb (Nb = T greater than A much greater than C greater than G), whereas MeC:G and C:MeG in place of Na:Nb, hinder cleavage. The optimal base target at Nb appears to be determined by its ability to form T:A type base pairing instead of C:G type. The observed differences in DNA strand cleavage patterns can be rationalized by induced changes in target site structure and are compatible with a model for NCS-C:DNA interaction in which the naphthoate moiety intercalates between 5'GNa3', and the activated tetrahydro-s-indacene, lying in the minor groove, abstracts a hydrogen atom from C-5' of Nb.     

Detection of neocarzinostatin chromophore-deoxyribose adducts as exonuclease-resistant sites in defined-sequence DNA

A 5'-end-labeled DNA restriction fragment was treated with the nonprotein chromophore of neocarzinostatin under anoxia in the presence of dithiothreitol, conditions known to maximize formation of chromophore-deoxyribose adducts. Under conditions where unmodified DNA was digested to completion, chromophore-treated DNA was highly resistant to digestion by exonuclease III plus the 3'----5' exonucleolytic activity of T4 DNA polymerase and partially resistant to digestion by exonuclease III plus snake venom exonuclease. The electrophoretic mobilities of the products of exonucleolytic digestion suggested that (i) digestion by exonuclease III or T4 polymerase terminated one nucleotide before the nucleotide containing the adduct, (ii) the remaining nucleotide directly adjacent to the adduct (3' side) could be removed by snake venom phosphodiesterase, but at a slow rate, (iii) the covalently linked chromophore decreased the electrophoretic mobilities of the digestion products by the equivalent of approximately three nucleotides, and (iv) adducts formed under anaerobic conditions occurred at the same nucleotide positions as the strand breaks formed under aerobic conditions (primarily at T and, to a lesser extent, A residues). The close similarity in sequence specificity of adducts and strand breaks suggests that a common form of nascent DNA damage may be a precursor to both lesions. A chromophore-induced free radical on C-5' of deoxyribose, subject to competitive fixation by addition reactions with either oxygen or chromophore, is the most likely candidate for such a precursor. The base specificity of adduct formation does not reflect the reported base specificity of neocarzinostatin-induced mutagenesis, suggesting that lesions other than adducts may be responsible for at least some neocarzinostatin-induced mutations, particularly those occurring at G X C base pairs.     

C-1' hydrogen abstraction of deoxyribose in DNA strand scission by dynemicin A.

Dynemicin A, which is a hybrid antitumor antibiotic containing anthraquinone and enediyne cores, abstracts the C-1' hydrogen of DNA deoxyribose and then the damaged DNA leads to strand breaks with the formation of 5'- and 3'-phosphate termini. The lesions of C-4' hydrogen also occur at 3' side of G.C base pairs (i. e., 5'-CT and 5'-GA), leading to 5'-phosphate and 3'-phosphoglycolate termini or 4'-hydroxylated abasic sites. The C-1' hydrogen abstraction by dynemicin A is distinct from the preferential C-5' hydrogen abstraction of calicheamicin and neocarzinostatin.     

Stoichiometric uptake of molecular oxygen and consumption of sulfhydryl groups by neocarzinostatin chromophore bound to DNA

In the presence of DNA, and under conditions which resulted in efficient DNA degradation, the reaction of the neocarzinostatin chromophore with sulfhydryl groups was accompanied by a rapid drop in the oxygen tension of the solution. The total extent of oxygen uptake indicated that, consistently, 1 mol of O2 was consumed/mol of chromophore. The rate of oxygen uptake, however, was strongly dependent on the sulfhydryl concentration, and uptake occurred within a few seconds of the sulfhydryl-induced increase in 420-nm fluorescence of the chromophore. Parallel experiments, in which the sulfhydryl concentration of the solution was monitored, showed that approximately 2 mol of sulfhydryl groups were consumed/mol of chromophore, with kinetics similar to those of O2 uptake. Under anaerobic conditions, only 1 mol of sulfhydryl was consumed, but the sulfhydryl-induced fluorescence increase was not inhibited. These results suggest that (i) a reaction with a single sulfhydryl group converts the chromophore to an activated form, (ii) in the presence of DNA this activated chromophore participates in a subsequent reaction which consumes 1 mol of O2 followed by an additional mole of sulfhydryl, and (iii) each chromophore molecule undergoes only one such reaction cycle. In the absence of sulfhydryl groups, the chromophore slowly degraded, giving a product with intense 490-nm fluorescence. This spontaneous degradation reaction, which does not result in DNA damage, was also accompanied by uptake of nearly 1 mol of O2/mol of chromophore.     

Degradation of HeLa cell chromatin by neocarzinostatin and its chromophore

Chromatin is the in vivo target site for neocarzinostatin, a DNA strand scission antitumor drug. The effect of neocarzinostatin and its active chromophore component on HeLa cell chromatin is described here. Chromatin consisting of a mixture of mono-, di-, tri- and larger nucleosome fragments is prepared by micrococcal nuclease digestion of HeLa cell nuclei. Drug-induced conversion of chromatin to smaller sized fragments is measured by electrophoresis of the DNA on non-denaturing 4% polyacrylamide gels. Chromatin breakdown measured under these conditions is double-stranded in nature. In the presence of 2 mM dithiothreitol, neocarzinostatin causes degradation of large chromatin fragments and a loss of distinct nucleosome peaks. Detection of chromatin breakdown by neocarzinostatin is dependent upon the concentration of chromatin in the assay. When chromatin is increased from 14 to 70 micrograms/ml, changes in the larger fragments caused by 100 micrograms/ml neocarzinostatin become less obvious are are almost undetectable at 140 micrograms/ml chromatin. No change is observed when chromatin is treated with either neocarzinostatin or its chromophore in the absence of dithiothreitol. For detectable levels of chromatin degradation, 10 micrograms/ml neocarzinostatin is required compared to only 2.5 microgram/ml chromosome (expressed in microgram equivalent neocarzinostatin). Such degradation also occurs more rapidly with chromophore than with neocarzinostatin. Digestion of chromatin with neocarzinostatin continues for at least 30 min at 37 degrees C, while similar degradation caused by chromophore is complete in 1 min. Neocarzinostatin levels which actively degrade isolated chromatin can also effect release of soluble chromatin from intact nuclei. The released chromatin can serve as a substrate for micrococcal nuclease digestion. Such chromatin studies should prove useful in characterizing the mechanism of action of DNA reactive drugs such as neocarzinostatin.     

A role of oxidative DNA sugar damage in mutagenesis by neocarzinostatin and bleomycin

The anti-tumor antibiotics neocarzinostatin and bleomycin specifically oxidize deoxyribose in DNA at the C-5' and C-4' positions, respectively. The resulting DNA lesions include strand breaks and apyrimidinic sites. Both agents are broad specificity mutagens, inducing, in various systems, base substitutions, frameshifts and deletions. Sequencing studies in bacterial systems have suggested that the base substitutions may result primarily from replicative bypass of the oxidized apyrimidinic sites.     

A tentative model of the intercalative binding of the neocarzinostatin chromophore to double-stranded tetranucleotides.

Theoretical computations are performed of the intercalative binding of the neocarzinostatin chromophore (NCS) with the double-stranded oligonucleotides d(CGCG)2, d(GCGC)2, d(TATA)2 and d(ATAT)2. Minor groove binding is preferred over major groove binding. It is found that the long axis of the stacked naphtoate ring lies approximately parallel to the long axis of the base pairs of the intercalation site. The galactosamine ammonium group interacts with specific sites of the groove (O2/N3 of bases 2 and O1' of sugar S3), whereas the dodecadyine ring system wraps around the groove towards the backbone. An overall AT versus GC preference is derived. Intercalation in a central purine-(3', 5')-pyrimidine sequence appears to be preferred over that in a central pyrimidine-(3', 5')-purine sequence.     

Modulation of neocarzinostatin-mediated DNA double strand damage by activating thiol: deuterium isotope effects.

The neocarzinostatin chromophore causes double-strand damage at AGC sequences on DNA by concomitant 1'-oxidation at C and 5'-oxidation at the T on the complementary strand. The extent of this damage is dependent upon the structure of the thiol used for activation. Deuterium isotope effects suggest that this dependence on thiol structure may be due to internal quenching of one radical site of the activated chromophore by the hydrogen atoms of the thiol sidechain. The 12-mer d[GCAAGCGCTTGC] is treated with the neocarzinostatin chromophore and either sodium thioglycolate or [2-2H2]-thioglycolate, and the distribution of strand breaks is determined by gel electrophoresis. Two isotope effects are noted: an overall sequence-independent effect in which deuterated thioglycolate increases total strand damage by a factor of 2, and a sequence-specific effect by which deuteration increases the proportion of alkali-sensitive strand damage at C6 by an additional factor of 1.5. Methyl thioglycolate shows essentially identical behavior to that of thioglycolate anion, ruling out electrostatic effects as major contributors to the effect of thiol structure on the mode of DNA damage observed. A model for NCSC action consistent with these results is discussed.     

Atypical abasic sites generated by neocarzinostatin at sequence-specific cytidylate residues in oligodeoxynucleotides

Neocarzinostatin chromophore produces alkali-labile, abasic sites at cytidylate residues in AGC sequences in oligonucleotides in their duplex form. Glutathione is the preferred thiol activator of the drug in the formation of these lesions. The phosphodiester linkages on each side of the abasic site are intact, but when treated with alkali, breaks are formed with phosphate moieties at each end. Similar properties are exhibited by the abasic lesions produced at the purine residue to which the C in AGC is base-paired on the complementary strand. The abasic sites at C residues differ from those produced by acid-induced depurination in the much greater lability of the phosphodiester linkages on both sides of the deoxyribose, in the inability of NaBH4 to prevent alkali-induced cleavage, and in the relative resistance to apurinic/apyrimidinic endonucleases. The importance of DNA microstructure in determining attack site specificity in abasic site formation at C residues is shown not only by the requirement for the sequence AGC but also by the findings that substitution of G by I 5' to the C decreases the attack at C, whereas placement of an I opposite the C markedly enhances the reaction. Quantitation of the abstraction of 3H into the drug from C residues in AGC specifically labeled in the deoxyribose at C-5' or C-1',2' suggests that, in contrast to the attack at C-5' in the induction of direct strand breaks at T residues, abasic site formation at C residues may involve attack at C-1'. Each type of lesion may exist on the complementary strands of the same DNA molecule, forming a double-stranded lesion.     

Neocarzinostatin abstracts a hydrogen during formation of nucleotide 5'-aldehyde on DNA

The oxidative reaction of polydeoxyadenylic-deoxythymidylic acid [poly(dA-dT)] with neocarzinostatin that produces 5'-thymidine aldehyde esterified to the 5'-end of strand breaks proceeds with hydrogen abstraction. The abstracted hydrogen is covalently bound to the non-protein component of neocarzinostatin; only a small amount (5%) is washed out into solvent. These data rule out a peroxyl radical as the primary DNA damaging species involved in the production of the 5'-aldehyde group. In contrast to earlier reports, it is demonstrated that alpha-tocopherol is not an inhibitor of the reaction.