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.     

Tallimustine lesions in cellular DNA are AT sequence-specific but not region-specific.

Tallimustine (FCE 24517) is an AT-specific alkylating antitumor derivative of distamycin. This study examined levels of tallimustine lesions in intracellular DNA, their sequence- and region-specificity, and the long-range distribution of the drug binding motif. Tallimustine adducts in DNA converted to strand breaks by heating allowed the quantitation of drug lesions. In bulk DNA of intact human leukemia CEM cells, tallimustine formed 0.15 +/- 0.04 and 0.64 +/- 0.18 lesions/kbp at 5 and 50 microM, respectively. These lesions represent monoadducts as no interstrand cross-links or DNA-protein cross-links were detected. Tallimustine adducts in intracellularly treated DNA showed a general preference for sequences with T-tracts, suggesting a propensity for intrinsically bent motifs. Major drug-adducted sites identified by repetitive primer extension, included 5'-TTTTGPu-3' and 5'-TTTTGC-3' motif. Despite the high specificity at the nucleotide level, tallimustine did not differentiate among bulk DNA and three discrete AT-rich regions of genomic DNA examined by quantitative PCR stop assay with lesion frequencies ranging from 0.23 to 0.39 lesions/kbp at 25 microM drug. In comparisons of lesion frequencies and cytotoxicity, tallimustine adducts are approximately 50 times more lethal than relatively nonsequence specific cisplatin adducts but are >100 times less lethal than lesions by an unrelated AT-specific drug, bizelesin. However, the 5'-TTTTGPu-3' motifs targeted by tallimustine are relatively infrequent and scattered throughout the genome. In contrast, the motifs 5'-T(A/T)(4)A-3' motifs targeted by bizelesin, while also infrequent, cluster in defined AT-rich islands. The lack of region-specificity may be the reason tallimustine adducts, despite high AT-specificity at the nucleotide level, are less lethal than region-specific bizelesin adducts.     

The cellular response to DNA damage induced by the enediynes C-1027 and neocarzinostatin includes hyperphosphorylation and increased nuclear retention of replication protein a (RPA) and trans inhibition of DNA replication

This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.     

Repair synthesis by human cell extracts in cisplatin-damaged DNA is preferentially determined by minor adducts.

During reaction of cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, a number of adducts are formed which may be discriminated by the excision-repair system. An in vitro excision-repair assay with human cell-free extracts has been used to assess the relative repair extent of monofunctional adducts, intrastrand and interstrand cross-links of cis-DDP on plasmid DNA. Preferential removal of cis-DDP 1,2-intrastrand diadducts occurred in the presence of cyanide ions. In conditions where cyanide treatment removed 85% of total platinum adducts while approximately 70% of interstrand cross-links remained in plasmid DNA, no significant variation in repair synthesis by human cell extracts was observed. Then, we constructed three types of plasmid DNA substrates containing mainly either monoadducts, 1,2-intrastrand cross-links or interstrand cross-links lesions. The three plasmid species were modified in order to obtain the same extent of total platinum DNA adducts per plasmid. No DNA repair synthesis was detected with monofunctional adducts during incubation with human whole cell extracts. However, a two-fold increase in repair synthesis was found when the proportion of interstrand cross-links in plasmid DNA was increased by 2-3 fold. These findings suggest that (i) cis-DDP 1,2-intrastrand diadducts are poorly repaired by human cell extracts in vitro, (ii) among other minor lesions potentially cyanide-resistant, cis-DDP interstrand cross-links represent a major lesion contributing to the repair synthesis signal in the in vitro assay. These results could account for the drug efficiency in vivo.     

Amplification of C1027-induced DNA cleavage and apoptosis by a quinacrine-netropsin hybrid molecule in tumor cell lines

We examined the effect of a newly synthesized DNA-binding ligand, quinacrine-netropsin hybrid molecule (QN), on cytotoxicity, apoptosis, and DNA strand breaks induced by an enediyne antitumor antibiotic, C1027. QN significantly enhanced C1027-induced cellular DNA strand breaks, caspase-3 activation, and DNA ladder formation, characteristic of apoptosis, in human HL-60 cells. Flow cytometry revealed that C1027-induced intracellular H(2)O(2) generation was enhanced by QN, suggesting that QN enhances C1027-induced cytotoxic effect through H(2)O(2)-mediated apoptosis. QN also significantly enhanced C1027-induced apoptosis in BJAB cells, and the inhibition of apoptosis was observed in BJAB cells transfected with Bcl-2 gene. The experiment using (32)P-labeled DNA fragments showed that the addition of QN enhanced C1027-induced double-stranded DNA cleavage at the 5'-AGG-3'/3'-TCC-5' sequence (cutting sites are underlined). These results suggest that QN enhances C1027-induced antitumor effect via DNA cleavage and apoptosis. The present study shows a novel approach to the potentially effective anticancer therapy.     

DNA damage by C1027 involves hydrogen atom abstraction and addition to nucleobases

C1027 is a potent antitumor agent that damages DNA. It has the unusual ability to produce double strand breaks and interstrand cross-links (ICLs) intracellularly, which enable it to initiate concurrent ataxia-telangiestasia mutated (ATM) and Rad-3 related (ATR) independent damage responses. The latter form of damage is not well characterized. We have examined the effect of DNA sequence on C1027 reactivity and found it to be more diverse than previously thought. In addition, analysis of the chemical stability of ICLs suggests that they result from reaction with the deoxyribose ring on one strand but direct addition to a nucleobase on the opposite strand.     

ESR studies on DNA cleavage induced by enediyne C-1027 chromophore

C-1027 belongs to the family of chromoprotein antitumor antibiotics, which contain a carrier apoprotein and a highly unstable enediyne chromophore. The enediyne spontaneously aromatizes to generate p-benzyne biradical, and subsequently abstracts hydrogens from the DNA sugar backbone, resulting in cleavage of the double strand. Using spin-trapping methods, we obtained direct proof of radical intermediates during an DNA cleavage, and found intriguing difference in behavior between the trapping agents 2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO): MNP added to the sugar radicals of the DNA, whereas DMPO directly trapped a phenyl radical or p-benzyne biradical derived from the C-1027 chromophore.     

Monoalkylation of DNA by reductively activated FR66979

The antitumor antibiotic FR66979 has previously been shown to form interstrand cross-links in duplex DNA at the sequence [5'-d(CG)]2, linking the exocyclic amino groups (N2) of deoxyguanosine (dG) residues. During the reaction of reductively activated FR66979 with DNA. products are formed which have electrophoretic mobility in denaturing polyacrylamide gels which is intermediate between that of unmodified and interstrand cross-linked DNA. We show here that these products are monoadducts between FR66979 and DNA and provide strong evidence for the site of alkylation being N2 of dG. Moreover, the sequence selectivity of monoalkylation reactions between FR66979 and DNA containing either 5'-d(CG).5'-d(CI) or [5'-d(CG)]2 was observed to be ca. 5-fold less than for the related antitumor antibiotic mitomycin C (MC). The mechanistic implications of this result are discussed. Furthermore, it was demonstrated that contrary to a previous report, FR66979 requires DNA to be in duplex form for efficient monoadduct formation.     

Induction of AT-specific DNA-interstrand crosslinks by bizelesin in genomic and simian virus 40 DNA

Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal lesion. In genomic DNA of BSC-1 cells, bizelesin formed from approx. 0.3 to 6.03+/-0.85 interstrand crosslinks per 106 base pairs, at 5-100 nM drug concentration, respectively, comparable to the number of total adducts previously determined in the same system (J.M. Woynarowski, M.M. McHugh, L.S. Gawron, T.A. Beerman, Biochemistry 34 (1995) 13042-13050). Bizelesin did not induce DNA-protein crosslinks or strand breaks. A model defined target, intracellular simian virus 40 (SV40) DNA, was employed to map at the nucleotide level sites of bizelesin adducts, including potential interstrand crosslinks. Preferential adduct formation was observed at AT tracts which are abundant in the SV40 matrix associated region and the origin of replication. Many sites, including each occurrence of 5'-T(A/T)4A-3', co-mapped on both DNA strands suggesting interstrand crosslinks, although monoadducts were also formed. Bizelesin adducts in naked SV40 DNA were found at similar sites. The localization of bizelesin-induced crosslinks in AT-rich tracts of replication-related regions is consistent with the potent anti-replicative properties of bizelesin. Given the apparent lack of other types of lesions in genomic DNA, interstrand crosslinks localized in AT-rich tracts, and to some extent perhaps also monoadducts, are likely to be lethal effects of bizelesin.     

Deoxyribonucleic acid-protein and deoxyribonucleic acid interstrand cross-links induced in isolated chromatin by hydrogen peroxide and ferrous ethylenediaminetetraacetate chelates

DNA-protein and DNA interstrand cross-links were induced in isolated chromatin after treatment with H2O2 and ferrous ethylenediaminetetraacetate (EDTA). Retention of DNA on membrane filters after heating of chromatin in a dissociating solvent indicated the presence of a stable linkage between DNA and protein. Treatment of protein-free DNA with H2O2/Fe2+-EDTA did not result in enhanced filter retention. Incubation of cross-linked chromatin with proteinase K completely eliminated filter retention. Resistance to S1 nuclease after a denaturation-renaturation cycle was used to detect DNA interstrand cross-links. Heating the treated chromatin at 45 degrees C for 16 h and NaBH4 reduction enhanced the extent of interstrand cross-linking. The following data are consistent with, but do not totally prove, the hypothesis that cross-links are induced by hydroxyl radicals generated in Fenton-type reactions: (1) cross-linking was inhibited by hydroxyl radical scavengers; (2) the degree of inhibition of DNA interstrand cross-links correlated very closely with the rate constants of the scavengers for reaction with hydroxyl radicals; (3) cross-linking was eliminated or greatly reduced by catalase; (4) the extent of cross-linking was directly related to the concentration of Fe2+-EDTA. Partial inhibition of cross-linking by superoxide dismutase indicates that superoxide-driven Fenton chemistry is involved. The data indicate that DNA cross-linking may play a role in the manifestation of the biological activity of agents or systems that generate reactive hydroxyl radicals.     

DNA interstrand cross-links of the novel antitumor trinuclear platinum complex BBR3464. Conformation,recognition by high mobility group domain proteins,and nucleotide excision repair

The novel phase II antitumor polynuclear platinum drug BBR3464 ([(trans-PtCl(NH(3))(2))(2)(mu-trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2))](NO(3))(4)) forms intra- and interstrand cross-links (CLs) on DNA (which is the pharmacological target of platinum drugs). We examined first in our recent work how various intrastrand CLs of BBR3464 affect the conformation of DNA and its recognition by cellular components (Zehnulova, J., Kasparkova, J., Farrell, N., and Brabec, V. (2001) J. Biol. Chem. 276, 22191-22199). In the present work, we have extended the studies on the DNA interstrand CLs of this drug. The results have revealed that the interstrand CLs are preferentially formed between guanine residues separated by 2 base pairs in both the 3' --> 3' and 5' --> 5' directions. The major 1,4-interstrand CLs distort DNA, inducing a directional bending of the helix axis and local unwinding of the duplex. Although such distortions represent a potential structural motif for recognition by high mobility group proteins, these proteins do not recognize 1,4-interstrand CLs of BBR3464. On the other hand, in contrast to intrastrand adducts of BBR3464, 1,4-interstrand CLs are not removed from DNA by nucleotide excision repair. It has been suggested that interstrand CLs of BBR3464 could persist considerably longer in cells compared with intrastrand adducts, which would potentiate the toxicity of the interstrand lesions to tumors sensitive to this polynuclear drug.     

UvrABC incision of N-methylmitomycin A-DNA monoadducts and cross-links

The Escherichia coli UvrABC endonuclease is a multisubunit enzyme that initiates the repair of a wide variety of DNA lesions in vivo by making dual incisions on a damaged strand at the eighth or ninth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified base. It has been hypothesized that UvrABC is able to recognize a broad spectrum of lesions because it does not recognize the lesion per se but rather gross helical distortions that the lesion induces in the DNA. Several lesions have recently been studied which are thermal stabilizing and are not believed to distort the DNA grossly, including the CC-1065-N-3-adenine and anthramycin-N-2-guanine adducts. We have studied the activity of UvrABC in vitro on another thermal stabilizing and nondistortive adduct, N-methylmitomycin A (NMA), a bifunctional DNA-alkylating agent that reacts with guanine on the side facing the minor groove, yielding either monoadducts or interstrand cross-links. NMA adducts increase the thermal stability of DNA, and theoretical calculations indicate that NMA adducts do not grossly distort the DNA helix. Our results show that UvrABC makes incisions at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to an NMA monoadduct, consistent with the incision pattern observed for the majority of other lesions that are also recognized by UvrABC. DNA containing a site-specific NMA cross-link was also recognized and incised by UvrABC. The rate of incision of NMA cross-linked DNA was about 200-fold higher in supercoiled molecules than in relaxed molecules, whereas the rate of incision of DNA containing NMA monoadducts was stimulated approximately 2-fold by supercoiling. The signal for UvrABC recognition and incision of damaged DNA is discussed in relation to the ability of UvrABC to incise NMA adducts as well as other nondistortive lesions.     

Cross-linking of DNA induced by chloroethylnitrosourea is presented by O6-methylguanine-DNA methyltransferase.

The DNA repair enzyme O6-methylguanine-DNA methyltransferase has been used as a reagent to analyse the initial reaction sites of alkylating agents such as chloroethylnitrosourea that cross-link DNA. The transferase can be employed for this purpose because it removes substituted ethyl groups from DNA, as shown by its ability to act on O6-hydroxyethylguanine residues in DNA. The enzyme counteracts the formation of interstrand cross-links induced by bis-chloroethylnitrosourea, but not those induced by nitrogen mustard. Once formed, chloroethylnitrosourea-induced cross-links are not broken by the enzyme. In agreement with deductions from experiments with living cells, it is concluded that chloroethylnitrosourea act by forming reactive monoadducts at the O6 position of guanine and/or the O4 position of thymine, which subsequently generate -CH2CH2- bridges to the complementary DNA strand. A new method for quantitating interstrand cross-links in DNA has been employed.     

Unique cross-link and monoadduct repair characteristics of a xeroderma pigmentosum revertant cell line

Monoadducts and cross-links formed in DNA of human cells by a psoralen derivative, 4'-hydroxy-methyl-4,5',8-trimethylpsoralen (HMT), have been measured by a new, simple method, based on S1 nuclease digestion of 3H-labeled adducts in DNA, that provides rapid information on the repair of both classes of lesions. Normal human fibroblasts and cells from patients with dyskeratosis congenita and xeroderma pigmentosum (XP) group C were capable of removing both monoadducts and cross-links, whereas XP groups A and D failed to remove either. An XP revertant, isolated from a group A cell line on the basis of an acquired mutagen-induced resistance to ultraviolet light, has the unique property of being capable of removing cross-links but not monoadducts. Consistent with this property, the XP revertant was found to be resistant to cell killing by the cross-linking psoralen derivative, HMT, but as sensitive as its parental cell line to a monofunctional psoralen derivative, 5-methylisopsoralen.     

Sequence preferences of DNA interstrand cross-linking agents: dG-to-dG cross-linking at 5'-CG by structurally simplified analogues of mitomycin C

The nucleotide sequence preferences of the DNA interstrand cross-linking agents dehydroretronecine diacetate (DHRA), 2,3-bis(acetoxymethyl)-1-methylpyrrole (BAMP), dehydromonocrotaline, and dehydroretrorsine were studied by using synthetic DNA duplex fragments and polyacrylamide gel electrophoresis (PAGE). These agents have structural features in common with the reductively activated aziridinomitosene of mitomycin C (MC). Like MC, they preferentially cross-linked DNA duplexes containing the duplex sequence 5'-CG. For DHRA and BAMP interstrand cross-linked DNA duplexes, PAGE analysis of iron(II)-EDTA fragmentation reactions revealed the interstrand cross-links to be deoxyguanosine to deoxyguanosine (dG-to-dG), again analogous to DNA cross-links caused by MC. Unlike MC, DHRA could be shown to dG-to-dG cross-link a 5'-GC sequence. Furthermore, the impact of flanking sequence on the efficiency of interstrand cross-linking at 5'-CG was reduced for BAMP, with 5'-TCGA and 5'-GCGC being equally efficiently cross-linked. Possible origins of the 5'-CG sequence recognition common to all of the agents are discussed. A model is presented in which the transition state for the conversion of monoadducts to cross-links more closely resembles ground-state DNA at 5'-CG sequences.     

A filter incubation method for the determination of potentially crosslinkable sites in DNA in mammalian cells

A method for the determination of DNA monoadducts capable of forming interstrand crosslinks in mammalian cells is described. Such monoadducts were produced by brief treatment of cells with cis-diamminedichloro-Pt(II) (cis-DDP), 1-(2-chloroethyl)-1-nitrosourea (ClEtNU), L-phenylalanine mustard (L-PAM), or diaziridinylbenzoquinone (AZQ). The method is an alkaline elution procedure in which the DNA from lysed cells is incubated on polycarbonate filters at pH 10 and 37 degrees C. During this incubation, the progressive formation of interstrand crosslinking was observed in drug-treated cells. In the case of ClEtNU and AZQ, DNA strand breaks also formed, due to the presence of labile lesions in the DNA. This made quantitation of interstrand crosslinks difficult for these drugs. For cis-DDP and L-PAM, however, there was no significant production of strand breaks and the assay for interstrand crosslinks was quantifiable.     

Interstrand cross-links and sequence specificity in the reaction of cis-dichloro(ethylenediamine)platinum(II) with DNA

Characterization of the adducts produced in DNA by the cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) and a radiolabeled analogue, [3H]-cis-dichloro(ethylenediamine)platinum(II) ([3H]-cis-DEP) was recently reported [Eastman, A. (1983) Biochemistry 22, 3927]. Both drugs reacted at identical sites in DNA, most of which produced intrastrand cross-links. DNA-interstrand cross-links, which represent less than 1% of total platination, have now been characterized. DNA containing interstrand cross-links was enriched for on the basis of its renaturability after boiling. This DNA was digested to deoxyribonucleosides, and the adducts were separated by high-pressure liquid chromatography. A cross-link between two deoxyguanosines was observed to be the most prominent adduct. It is proposed that the major sequence in which this cross-link occurs is 5'-CG-3'. DNA that was incubated with [3H]-cis-DEP for 1 h showed low levels of interstrand cross-links. After removal of unreacted drug, their frequency increased significantly over 6 h with a maximum occurring at about 12 h. A similar phenomenon was seen in the case of intrastrand cross-links that contained adenine, in particular when the cross-link was between the terminal bases in an ANG trinucleotide sequence (N is any nucleotide). The primary site of reaction is at guanine, with a slow subsequent cross-link to the adenine. A model is presented that is consistent with the observation that adenine is always at the 5' terminus of these adducts. The proportion of adducts at ANG sequences also increased at elevated temperatures. This is discussed with regard to potential significance during hyperthermia treatment of patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delivery of photoreactive psoralen derivatives to specific biological targets

Psoralens are bifunctional molecules which photoreact with the pyrimidine bases of nucleic acids to form monoadducts and diadducts, or interstrand cross-links. We have prepared psoralen derivatives with additional functional groups which can be specifically directed to chosen biological targets. A sulfhydryl-containing psoralen which can form site-specific cross-links in plasmid DNA has been used to study psoralen repair and mutagenesis. Cloned DNA containing psoralen monoadducts has been cross-linked to specific regions of viral RNA and used to probe virus assembly. A biotinylated psoralen derivative which binds specifically to avidin has been used to detect small amounts of DNA. Finally, a psoralen derivative of insulin has been used to deliver psoralen specifically to activated lymphocytes.     

Chromatin-associated DNA endonucleases from xeroderma pigmentosum cells are defective in interaction with damaged nucleosomal DNA

The influence of nucleosome structure on the activity of 2 chromatin-associated DNA endonucleases, pIs 4.6 and 7.6, from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells was examined on DNA containing either psoralen monoadducts or cross-links. As substrate a reconstituted nucleosomal system was utilized consisting of a plasmid DNA and either core (H2A, H2B, H3, H4), or total (core plus H1) histones from normal or XPA cells. Both non-nucleosomal and nucleosomal DNA were treated with 8-methoxypsoralen (8-MOP) plus long-wavelength ultraviolet radiation (UVA), which produces monoadducts and DNA interstrand cross-links, and angelicin plus UVA, which produces monoadducts. Both normal endonucleases were over 2-fold more active on both types of psoralen-plus-UVA-damaged core nucleosomal DNA than on damaged non-nucleosomal DNA. Addition of histone H1 to the system reduced but did not abolish this increase. By contrast, neither XPA endonuclease showed any increase on psoralen-treated nucleosomal DNA, with or without histone H1. Mixing the normal with the XPA endonucleases led to complementation of the XPA defect. These results indicate that interaction of these endonucleases with chromatin is of critical importance and that it is at this level that a defect exists in XPA endonucleases.