Biochemistry 1997, 36, 14975-14984
14975
Mechanism of Formation of Novel Covalent Drug‚DNA Interstrand Cross-Links and Monoadducts by Enediyne Antitumor Antibiotics† Yong-jie Xu,‡,§ Zhen Xi,‡ Yong-su Zhen,§ and Irving H. Goldberg*,‡ Department of Biological Chemistry and Molecular Pharmacology, HarVard Medical School, 250 Longwood AVenue, Boston, Massachusetts 02115, and Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Tiantan, Beijing 100050, China ReceiVed August 25, 1997; ReVised Manuscript ReceiVed September 26, 1997X
ABSTRACT:
The potent enediyne antitumor antibiotic C1027 has been previously reported to induce novel DNA interstrand cross-links and drug monoadducts under anaerobic conditions [Xu et al. (1997) J. Am. Chem. Soc. 119, 1133-1134]. In the present study, we explored the mechanism of formation of these anaerobic DNA lesions. We found that, similar to the aerobic reaction, the diradical species of the activated drug initiates anaerobic DNA damage by abstracting hydrogen atoms from the C4′, C1′, and C5′ positions of the A1, A2, and A3 nucleotides, respectively, in the most preferred 5′GTTA1T/5′ATA2A3C binding sequence. It is proposed that the newly generated deoxyribosyl radicals, which cannot undergo oxidation, likely add back onto the nearby unsaturated ring system of the postactivated enediyne core, inducing the formation of interstrand cross-links, connecting either A1 to A2 or A1 to A3, or drug monoadducts mainly on A2 or A3. Comparative studies with other enediynes, such as neocarzinostatin and calicheamicin γ1I under similar reaction conditions indicate that the anaerobic reaction process is a kinetically competitive one, depending on the proximity of the drug unsaturated ring system or dioxygen to the sugar radicals and their quenching by other hydrogen sources such as solvent or thiols. It was found that C1027 mainly generates interstrand cross-links, whereas most of the anaerobic lesions produced by neocarzinostatin are drug monoadducts. Calicheamicin γ1I was found to be less efficient in producing both lesions. The anaerobic DNA lesions induced by enediyne antitumor antibiotics may have important implications for their potent cytotoxicity in the central regions of large tumors, where relative anaerobic conditions prevail.
The extreme potencies of the enediyne antitumor antibiotics, such as the calicheamicins, esperamicins, C1027, and neocarzinostatin (Figure 1), are believed to be due to their ability to induce single-strand (SS) or double-strand (DS) damage in cellular DNA (1). These agents can be activated by reducing compounds or a spontaneous process to form a diradical species that interacts with DNA in the minor groove and abstracts hydrogen atoms from minor-groove-accessible carbons of the sugar backbone of each complementary DNA strand. So far, three main attack sites (C5′, C4′, and C1′) and the resulting damage products, which include abasic sites and strand breaks, have been characterized (Figure 2). Most of the previously reported DNA damage induced by enediynes was studied, however, in the presence of molecular oxygen (1). In cancer chemotherapy, the relative hypoxic conditions in the centers of solid tumors might influence the DNA damage pathway, causing different lesions and hence modifying the chemotherapeutic efficiency of these antibiotics. Indeed, we have recently shown that when O2 was depleted from drug/DNA reaction systems, enediyne C1027 induces the formation of novel interstrand cross-links and drug monoadducts, instead of strand breaks and abasic lesions † This work was supported by Grant GM 53793 from the National Institutes of Health. * To whom correspondence should be addressed. ‡ Harvard Medical School. § Chinese Academy of Medical Sciences. X Abstract published in AdVance ACS Abstracts, November 15, 1997.
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(2). Some earlier studies with neocarzinostatin have also demonstrated that this agent produces drug monoadducts under anaerobic conditions (3) and that adduction is on the deoxyribose of the attacked nucleotide target (4, 5). In the present study, the mechanism by which C1027 produces interstrand cross-links and drug monoadducts was investigated. We found that by using DNA oligomers with the target 5′GTTA1T/5′ATA2A3C sequence (damaged residues are numbered), where C4′-, C1′-, and C5′-hydrogens at A1, A2, and A3, respectively, are abstracted by the drug diradical, interstrand cross-links are formed by the drug itself connecting either A1 and A2 or A1 and A3. Observation of an isotope selection effect by position-specific deuteriation at the C1′ position of A2 and other experiments strongly indicate that position-specific hydrogen abstraction by the activated drug (Figure 1) from DNA deoxyribose is a key step in initiating the process of cross-linking and adduction. To compare different drug/DNA reaction systems, other enediynes, such as neocarzinostatin and calicheamicin γ1I, were also tried under similar anaerobic reaction conditions. In contrast, neocarzinostatin and calicheamicin γ1I mainly produce drug monoadducts, and their yields depend on the concentrations and the types of thiols and solvents used in the reaction systems. Quantitation of the aerobic and anaerobic damage products in different drug/DNA reaction systems implies that accessibility of O2 and/or hydrogen sources from solvents and proximity of drug to the DNA sugar radicals in the drug/DNA complexes are important factors determining what types of damage products are © 1997 American Chemical Society
14976 Biochemistry, Vol. 36, No. 48, 1997
Xu et al.
FIGURE 1: Diradical forms of enediyne neocarzinostatin chromophore, C1027 chromophore, and calicheamicin γ1I.
FIGURE 2: Proposed mechanisms for enediyne-induced DNA damage by C4′-, C5′-, and C1′-hydrogen abstractions.
produced. Unlike alkylating agents, whose cross-links involve bases, C1027 induces interstrand cross-links by the drug itself most likely connecting the complementary DNA sugar backbones. These findings have significant implications for not only the nature of interactions between the activated enediyne molecules with DNA targets but also for the remarkable therapeutic effects of this class of antibiotics against solid tumors.
MATERIALS AND METHODS Materials. Materials and their sources are as follows: holoantibiotic C1027, purified from the fermentation broth of the producing Streptomyces globisporus C1027 strain as reported (6-8); neocarzinostatin and calicheamicin γ1I, Kayaku Antibiotics and Dr. George Ellestad of Wyeth-Ayerst Research, respectively; [γ-32P]ATP, New England Nuclear
Anaerobic DNA Lesions by Enediyne Antibiotics
FIGURE 3: Sequences of oligodeoxyribonucleotide substrates for enediyne antibiotics. Drug interaction sequences are printed in boldface type. D3 is a hairpin oligomer with a hexaethylene glycol group, X, which holds the positive and the negative strands together via two phosphodiesters.
DuPont; T4 polynucleotide kinase and the Klenow fragment of DNA polymerase I, New England Biolabs; phosphoramidites and chemicals for DNA synthesizer, Glen Research; solutions for making sequencing gels, National Diagnostic; other chemical reagents were from Sigma and Aldrich. Fluorescence. Florescence measurements were performed on a Perkin-Elmer LS 50 B Luminescence Spectrometer. Excitation was at 340 nm, and the emission spectrum was recorded from 370 to 600 nm. Synthesis and Labeling of Oligodeoxyribonucleotides. All oligodeoxyribonucleotides were synthesized on an Applied Biosystems 381A automatic synthesizer by the phosphoramidite method. Synthesis of 5′-(dimethoxytrityl)-1′-deuteriumyl-2′-deoxy-3′-(cyanoethyl)adenosine phosphoramidite was as previously described (9). The specific nucleotide containing the C1′ deuterium label was incorporated manually into the oligomers using standard phosphoramidite methodology. The triethylene glycol chemical linker joining the 5′-end of the positive strand and the 3′-end of the complementary strand via two phosphodiester bonds (D3 in Figure 3) was also incorporated manually. The concentrations of single-strand oligomers were determined by UV absorbance at 260 nm. The 5′-ends of oligomers were labeled with 32P by T4 polynucleotide kinase, and the 3′ends were 32P labeled by the Klenow fragment of DNA polymerase I. Labeled oligomers were purified by electrophoresis on 20% polyacrylmide gels containing 8.3 M urea. DNA/Drug Reaction. The end-labeled oligomers were annealed to their complementary strands at the ratio of 1:1.5 by heating at 90 °C for 5 min and slowly cooling to room temperature. A standard reaction mixture contained 10 mM Tris‚HCl (pH 8.0), 0.5 mM EDTA, and 10 µM oligomers. To initiate the DNA/drug reactions, holo C1027 (50 µM), holo neocarzinostatin (40 µM), and calicheamicin γ1I (15 µM) were separately added to their reaction mixtures. The reactions were usually allowed to proceed in the dark at 37 °C for 3 h or at room temperature overnight and stopped by
Biochemistry, Vol. 36, No. 48, 1997 14977 drying in a Speed-Vac concentrator. Alkaline treatment involved heating one of the duplicate aliquots in 50 µL of 1 M piperidine at 90 °C for 30 min. As for hydrazine and putrescine treatments, either agent was added to a final concentration of 100 mM, and the mixtures were incubated at room temperature or 37 °C, respectively, for 1 h. The anaerobic reactions were performed in a Warburg vessel. The side arm contained drug (or thiols in the reactions with neocarzinostatin and calicheamicin γ1I), and the rest of the components of the reaction mixture were placed in the main chamber. The contents of the flask were frozen in liquid nitrogen and then evacuated at
