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Biomedical subjects

I H Goldberg

Publications and source records attributed to I H Goldberg.

At least 19 recordsLinked to original sources
PubMed1992

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)

Base Sequence↗
PubMed1992

Mismatch-induced switch of neocarzinostatin attack sites in the DNA minor groove.

Based on the finding that the wobble G.T mismatch 5' to the C of AGC.GCT results in switching of the attack chemistry by neocarzinostatin chromophore (NCS-Chrom) on the deoxyribose moiety of C from C-1' to C-4' [Kappen, L. S. & Goldberg, I. H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6706-6710], a series of mismatches has been explored for their effect on the chemistry of damage at the T of AGT.ACT in oligodeoxynucleotides, a site at which 4'-chemistry ordinarily occurs. Placement of a G.T mispair 5' to the T results in a marked increase in 4'-chemistry, as measured by the formation of breaks with 3'-phosphoglycolate ends and abasic sites due to 4'-hydroxylation. Strikingly, 4'-chemistry is induced at the T on the complementary strand, a site ordinarily restricted to 5'-chemistry. Substitution of dioxygen by the radiation sensitizer misonidazole exerts a pronounced effect on the partitioning of the 4'-chemistry in favor of the 3'-phosphoglycolate product. Both stable T.G and unstable T.C mismatches at the attack site itself are associated with marked inhibition of damage at this site. Whereas placement of the relatively stable G.A mismatch on the 5'-side of the T residue (AGT) results in substantial inhibition of damage at the T without shifting of chemistry, the same mismatch at the 3'-side of the attack site decreases damage only slightly but is associated with the appearance of significant 1'-chemistry. By contrast, no shift in chemistry is found with bleomycin, which attacks at C-4'.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Neocarzinostatin acts as a sensitive probe of DNA microheterogeneity: switching of chemistry from C-1' to C-4' by a G.T mismatch 5' to the site of DNA damage.

The diradical form of thiol-activated neocarzinostatin chromophore resides in the minor groove of DNA, where it has access to hydrogen atoms at the C-5', C-1', and C-4' positions of deoxyribose on each strand. In a dodecamer oligodeoxyribonucleotide containing the sequence AGC.GCT, a bistranded lesion staggered two nucleotides in the 3' direction, is generated that consists primarily of an abasic site (2'-deoxyribonolactone) at the C due to 1' chemistry and a direct strand break at the T due to 5' chemistry. Sequencing-gel analysis reveals that 72% of the damage at the C results from 1' chemistry with minor lesions consisting of a strand break due to 5' chemistry (15%) and 4' chemistry (less than 2%) and an abasic site (4'-hydroxylation product) (12%) due to 4' chemistry. Replacement of the G.C base pair 5' to the C by a G.T wobble mismatch results in a remarkable switching of the chemistry of damage at the C from C-1' to C-4'. The 1' chemistry is almost eliminated and replaced by 4' chemistry, so that the latter accounts for 64% of the damage, mainly in the form of the 4'-hydroxylation product (abasic site) and a smaller amount of the DNA fragment with a phosphoglycolate at the 3' end (strand break). Substitution of the radiation sensitizer misonidazole for dioxygen markedly enhances partitioning of the 4' chemistry in favor of the glycolate-containing product. On the complementary strand the G.T mismatch results in an increase in 4' chemistry at the T residue, but 5' chemistry remains the main mechanism. When a G.A mismatch is inserted 5' to the C, there is a marked decrease in all damage at this site without detectable switching of chemistry. These results show that the diradical form of thiol-activated neocarzinostatin chromophore acts as sensitive probe of DNA microheterogeneity.

Base Composition↗
PubMed1992

Influence of thiol structure on neocarzinostatin activation and expression of DNA damage.

Neocarzinostatin (NCS) is an enediyne antitumor antibiotic that cleaves DNA following a thiol-induced electronic rearrangement to a diradical form. Structure-function studies with 11 thiol-containing compounds were undertaken to clarify the role of the thiol in NCS-mediated DNA damage. The rates of activation of NCS in the presence of DNA with the various thiols approximated a Brønsted relation (beta = 0.43, r2 = 0.86), which suggests that the basicity/nucleophilicity of the thiol is important to NCS activation. However, an additional contribution to NCS activation may arise from the affinity of the thiol for DNA, since there is a correlation between the concentration of thiol producing maximal DNA damage, assessed by quantitating the topologic forms of plasmid pBR322 following treatment with NCS, and the apparent ability of the thiol to bind to DNA by hydrophobic or electrostatic interactions. The overall second-order rate constants for the activation of NCS were found to be inversely correlated with the thiol optima; a plot of the former versus the reciprocal of the optimal thiol concentration revealed a first-order rate constant of activation of 0.013 s-1 in the presence of DNA. This indicates that maximal DNA damage occurs when NCS is activated with a half-life of 52 s, a relatively slow rate of activation that suggests that NCS binds to DNA before undergoing activation by thiol. Finally, an analysis of strand breaks in pBR322 shows that thiols possessing a carboxylate moiety produce larger quantities of bistranded DNA lesions than their esterified or non-carboxylate-containing counterparts.

DNA Damage↗
PubMed1992

Neocarzinostatin-mediated DNA damage in a model AGT.ACT site: mechanistic studies of thiol-sensitive partitioning of C4' DNA damage products.

Double-strand (DS) DNA damage caused by neocarzinostatin (NCS) has been studied in the trinucleotide AGT-ACT sequence in an AP-1 transcription factor binding site. There are strong similarities between bistranded lesions produced at AGT.ACT and AGC-GCT, including the fact that DS lesions outnumber SS lesions on the AGT and AGC strands, while SS exceed DS on the ACT and GCT strands. Structure-function studies revealed that a variety of different thiols produced bistranded lesions in this model by predominantly C4'-hydrogen atom abstraction (84-93%) at the T of AGT and C5'-hydrogen atom abstraction (87-91%) at the T of ACT. Single-strand (SS) lesions were found to represent a variable mixture of C4' and C5' chemistry. The C4'-hydroxylated abasic site occurred in both SS and DS lesions at both sites and accounted for most of the DS damage at AGT (60-83%); the remaining damage consisted of 3'-phosphoglycolate- and 3'-phosphate-ended fragments. The nature of the thiol was found to affect the partitioning of the breakdown products arising from C4' and, to a lesser extent, C5' hydrogen atom abstraction. Production of 3'-phosphoglycolate residues, restricted mainly to the T of AGT in bistranded lesions, correlated with the incidence of direct DS breaks in the AGT.ACT model and in plasmid DNA and appeared to be influenced by the reducing power of the thiol activator. Furthermore, hydrazine and sodium borohydride both inhibited the formation of glycolate, an effect that was exploited to determine the rate constant for 3'-phosphoglycolate formation: 0.06 min-1 at 0 degree C, pH 7.4. Under anaerobic conditions, the nitroaromatic radiation sensitizer misonidazole caused a large increase in glycolate production in both SS and DS lesions formed by NCS, which suggests that the formation of 3'-phosphoglycolate, like 3'-formylphosphate generated by C5' chemistry, involves an oxyradical intermediate. The pathways for DNA damage involving C4' and C5' hydrogen atom abstraction thus share many common features, several of which are consistent with a mechanism for the production of NCS-mediated bistranded lesions at AGT.ACT that involves a tetraoxide bridge joining the lesions on opposite strands of DNA.

Base Sequence↗
PubMed1991

Selective abstraction of 2H from C-5' of thymidylate in an oligodeoxynucleotide by the radical center at C-6 of the diradical species of neocarzinostatin: chemical evidence for the structure of the activated drug-DNA complex.

Use has been made of the mechanism of DNA deoxyribose damage by the ene-diyne-containing chromophore of the antitumor antibiotic neocarzinostatin to provide chemical evidence for the structure of the activated drug-DNA complex. Radical centers at C-2 and C-6 of the diradical form of the glutathione-activated chromophore abstract hydrogen atoms from C-1' of the C residue and C-5' of the T residue in AGC.GCT to generate a bistranded lesion consisting of an abasic site at C and a strand break at T. This laboratory has proposed a molecular model for the drug-DNA interaction in which the naphthoate moiety of the chromophore intercalates between A.T and G.C, placing the diradical core in the minor groove, so that the radical centers at C-6 and C-2 are close to C-5' of T and C-1' of C, respectively. To determine which radical center abstracts one of the hydrogen atoms from C-5', the self-complementary oligodeoxynucleotide GCAGCGCTGC was synthesized with 2H at both 5' positions of the T residue and treated with glutathione-activated chromophore. Sequencing-gel electrophoresis showed that drug attack was limited to the T and C residues and that abstraction of 2H from C-5' exhibited a small isotope selection effect of 1.25. 1H NMR spectroscopic examination of the reacted chromophore, isolated by HPLC, indicated that 2H was selectively abstracted by C-6, providing experimental corroboration of the model and further elucidating the chemical mechanism. Since direct strand breakage at the T residue exceeds (44% more) abasic site formation at the C residue, other models of drug-DNA interaction leading to only single-strand breaks are also considered.

Base Sequence↗
PubMed1991

Neocarzinostatin-induced hydrogen atom abstraction from C-4' and C-5' of the T residue at a d(GT) step in oligonucleotides: shuttling between deoxyribose attack sites based on isotope selection effects.

The thiol-activated neocarzinostatin chromophore cleaves duplex oligonucleotides containing the sequence-TGTTTGA-, producing 3'-phosphoglycolate and 3'-phosphate fragments at T, indicating the involvement of 4'- as well as 5'-chemistry at this residue. Substitution of deuterium for hydrogen at the C-4' position of the affected T leads to a kinetic isotope effect (kH/kD) of 4.0 on the formation of the glycolate-ended product, whereas deuterium at C-5' of the same T reveals kH/kD of 1.6 in the formation of the phosphate-ended product. The proportion of the products representing 4'- and 5'-chemistry can be shifted on the basis of isotope selection effects. A second product resulting from 4'-chemistry, the abasic site associated with 4'-hydroxylation, has been identified as an alkali-labile site, and as a pyridazine derivative formed after cleavage by hydrazine. A comparable isotope effect on its production (kH/kD = 3.7) relative to that of 3'-phosphoglycolate production is consistent with a common intermediate, a putative 4'-peroxy radical, in their formation. The formation of both products of 4'-chemistry is oxygen-dependent, and the internal partitioning between them (3'-phosphate or 3'-phosphoglycolate) is influenced by thiols. Moreover, the nitroaromatic radiation sensitizer misonidazole can substitute for dioxygen, yielding 3'-phosphoglycolate and alkali-labile 3'-phosphate ends, indicative of 4'-chemistry. In addition to the internal partitioning of 4'-chemistry, thiols also affect the overall extent of cleavage (4' plus 5') and the relative partitioning between both sites of attack (4' or 5').

Adenosine Triphosphate↗
PubMed1990

Sequence-specific double-strand breakage of DNA by neocarzinostatin involves different chemical mechanisms within a staggered cleavage site.

Direct double-strand breaks in DNA have been implicated in cellular lethality of the antitumor antibiotic neocarzinostatin, but the mechanism of their formation has not been elucidated. Evidence is presented that neocarzinostatin causes sequence-specific direct double-strand breaks whose formation is strongly influenced by the activating thiol. Seven-fold more double-strand breaks result when glutathione rather than 2-mercaptoethanol is used to activate the drug to its putative diradical form, while the sequence specificity of cleavage remains the same. These data explain earlier inconsistencies in the ratios of double-strand to single-strand breaks obtained from in vitro and in vivo studies. Double-strand cleavage sites, occurring predominantly at GT steps, especially AGT.ACT, consist of trinucleotide sequences with a two-nucleotide 3'-stagger of the cleaved residues. The chemical structures of the cleavage sites suggest a model in which a neocarzinostatin-induced double-strand break results from abstraction of a C5' hydrogen atom from the T of ACT and the C4' hydrogen atom of the T of AGT by a single molecule of the diradical form of the drug. Single-strand breaks at these sites occur as separate events with attack at the C5' hydrogens. These findings permit the generalization that single-strand breaks produced by neocarzinostatin show a base preference but no clear sequence specificity, while bistranded lesions are sequence-specific in nature.

Antibiotics, Antineoplastic↗
PubMed1990

Molecular models of neocarzinostatin damage of DNA: analysis of sequence dependence in 5'GAGCG:5'CGCTC.

Model building and molecular mechanics and dynamics calculations have been performed on a number of complexes of the post-activated form of the neocarzinostatin chromophore (NCS) with the B-DNA oligomer 5'GAGCG:5'CGCTC. Stable structures with the naphthoic acid moiety intercalated at all base pairs can be constructed. The observed bistranded lesions consisting of an abasic site at the Cyt residue in AGC and a direct break at the Thy residue on the complementary strand can be explained by assuming that NCS in the (R,R) form intercalates between the Ade2-Thy9/Gua3-Cyt8 base step with its 'diradical' core oriented towards the 3'-end of the (+) strand. Sites at C5', C4' and C1' in the minor groove are within a short enough distance from the two radical centers on NCS to permit hydrogen atom abstraction and the formation of the bistranded lesions. Strand cleavage at Thy9 may occur as a single lesion if NCS is intercalated into the Gua3-Cyt8/Cyt4-Gua7 base step with its active core towards the 3'-end of the (-) strand. The results are analyzed, and the utility and limitations of this type of model building are discussed.

Antibiotics, Antineoplastic↗
PubMed1989

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.

Antibiotics, Antineoplastic↗
PubMed1989

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.

Antibiotics, Antineoplastic↗
PubMed1989

Sequence-specific, strand-selective, and directional binding of neocarzinostatin chromophore to oligodeoxyribonucleotides.

The sequence-specific interaction of neocarzinostatin chromophore (NCS-C) has been evaluated with a series of synthetic oligodeoxyribonucleotides of defined sequences containing the most preferred nucleotide cleavage site, T or A, or both. NCS-C preferentially cleaves T or A residues in the sequence GN1N2, where N2 is T or A. Greater cleavage occurs on the strand enriched with G residues, provided that they are adjacent to other G residues, but not at N1. These results are compatible with a model for drug binding in which the naphthoate moiety of NCS-C preferentially intercalates at GN1. This is accompanied by electrostatic binding interaction provided by the positively charged amino sugar moiety so as to place the reactive bicyclo[7.3.0]dodecadienediyne epoxide moiety in an appropriate orientation in the minor groove enabling, upon thiol activation, attack at C-5' of T or A. At certain sequences, such as GCT.AGC, a similar binding mode is also able to generate a basic lesions at the C residue on the opposite strand, forming a bistranded lesion. Although the reactions with glutathione generally show the same strand selectivity and sequence specificity as those with dithiothreitol, the former is usually more efficient than the latter.

Antibiotics, Antineoplastic↗
PubMed1989

Identification of 2-deoxyribonolactone at the site of neocarzinostatin-induced cytosine release in the sequence d(AGC).

Neocarzinostatin- (NCS) induced release of cytosine from the deoxycytidylate residues of d(AGC) sequences of duplex oligonucleotides leaves a damaged sugar residue with intact phosphodiester linkages [Kappen, L.S., Chen, C., & Goldberg, I.H. (1988) Biochemistry 27, 4331-4340]. In order to isolate and characterize the sugar damage product, drug-treated duplex d(AGCGAGC*G) (the single target C* residue has 3H in its 5- and 5'-positions) was enzymatically digested to mononucleosides. High-pressure liquid chromatographic analysis of the digest revealed drug-induced products which could be cleanly separated by thin-layer chromatography (TLC) into two components: product a (Rf0.47) and product 1 (Rf0.87). The more polar product a was further purified by adsorption onto DEAE-Sephadex A-25. After elution with HCl and lyophilization, this material behaved like product 1 on TLC. Readjustment to alkaline pH caused its quantitative conversion back to product a. On electrophoresis product 1 behaved like a neutral compound, and the negatively charged product a migrated just behind formate. On the basis of the various chemical and biochemical characteristics of the lesion and apparent 3H abstraction by NCS from the C-1' position, it appears that the two interconvertible products a and 1 are respectively the acid (carboxylate) and lactone forms of 2-deoxyribonic acid. The structure of the sugar damage product was confirmed by gas chromatography/mass spectrometry. The amount of 2-deoxyribonolactone recovered is about 60% of the cytosine released on a molar basis, showing that it is the major, if not the only, product associated with cytosine release.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiotics, Antineoplastic↗
PubMed1988

Specific binding of o-phenanthroline at a DNA structural lesion.

DNA intercalators are found to recognize a DNA lesion as a high affinity receptor site. This lesion-specific binding is observed when one strand of a DNA double helix contains an extra, unpaired nucleotide. Our assay for binding controls for the effects of sequence with a series of oligodeoxynucleotide duplexes which are identical except for the location of the lesion, an extra cytidine. Scission of the series of oligodeoxynucleotides by the cuprous complex of ortho-phenanthroline (OP-Cu) indicates that OP-Cu binds at the lesion-specific stable intercalation site, suggesting that OP-Cu intercalates into DNA. The dispersion of OP-Cu scission sites over three residues is consistent with scission via a diffusible intermediate. The location of the scission sites, directly on the 3' side of the lesion, is consistent with minor groove binding in B DNA.

Base Sequence↗
PubMed1988

Sites in the diyne-ene bicyclic core of neocarzinostatin chromophore responsible for hydrogen abstraction from DNA.

The antitumor antibiotic neocarzinostatin exhibits its main drug action by abstracting hydrogen from DNA deoxyribose with consequent strand breakage or related lesions. All biological activities of the drug derive solely from a nonprotein chromophoric substance (NCS-chrom) consisting of a novel epoxy-bicyclo-diyne-ene system. Thiol or sodium borohydride activates NCS-chrom into a labile, reactive species that induces DNA damage but causes inactivation of the drug in the absence of the target DNA. Mass spectrometric studies indicate that the isolated thiol-activated NCS-chrom product in the presence of DNA has the same molecular weight as the thiol-inactivated NCS-chrom product in the absence of DNA. No deuterium is incorporated into the chromophore from the deuterium-labeled sulfhydryl group. Since three deuterium atoms can be incorporated into the drug by treatment with sodium borodeuteride without DNA, adding an unlabeled DNA under parallel conditions permitted the ready identification of the activated NCS-chrom product that abstracted hydrogen from the DNA. Not only does the activated NCS-chrom product have the same structure as the inactivated drug without DNA, but two of the incorporated deuterium atoms have been substituted by hydrogen. With the aid of NMR spectrometry, the two replaced hydrogen atoms are found to be incorporated into the C-2 and C-6 positions of the bicyclo-diyne-ene ring of NCS-chrom and are derived neither from borodeuteride nor from the hydroxyl functions of the solvents. In accord with current proposals, the two hydrogens incorporated into the drug may come from closely opposed sites on the complementary strands of the DNA at which the drug is bound.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibiotics, Antineoplastic↗
PubMed1988

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.

Antibiotics, Antineoplastic↗