Adozelesin is a member of a family of extraordinarily cytotoxic DNA damaging agents that bind to the DNA minor groove in a sequence-specific manner and form covalent adducts with adenines. Previous studies employing purified enzymes and adozelesin-modified template DNAs suggested that adozelesin-DNA adducts inhibit DNA replication at the level of nascent DNA chain elongation. In this study, neutral/neutral two-dimensional agarose gel electrophoresis was employed to analyze simian virus 40 (SV40) DNA replication intermediates recovered from adozelesin-treated SV40 virusinfected cells. SV40 replication intermediates rapidly disappeared from infected cells when they were treated with adozelesin, but not when the cells were also treated with aphidicolin to block maturation of replicating SV40 DNA. We conclude that the disappearance of SV40 replication intermediates induced by adozelesin treatment was a consequence of maturation of these intermediates in the absence of new initiation events. Adozelesin inhibition of nascent chain elongation is first observed at concentrations above those needed to block initiation. Adozelesin treatment inhibits SV40 DNA replication at concentrations that produce adducts on just a small fraction of the intracellular population of SV40 DNA molecules.Adozelesin (U-73,975) is a synthetic analog of the antitumor antibiotic CC-1065 (1). The cytotoxic activity of adozelesin is orders of magnitude more potent than many common antineoplastic agents such as doxorubicin, cisplatin, 5-fluorouracil, or cytosine arabinoside (2-6). It is highly effective against a broad spectrum of murine tumors and human tumor xenografts without the lethal hepatotoxicity caused by CC-1065 (7). Adozelesin was chosen for clinical development based upon this in vivo potency and efficacy, and is currently undergoing phase II clinical evaluation (8).A primary intracellular target of adozelesin is DNA (3, 9). Like CC-1065, adozelesin elicits its potent cytotoxic and antitumor effects by alkylating DNA (10). As shown in Fig. 1, adozelesin is composed of three heterocyclic subunits linked serially by amide bonds. Similar to the parent compound, CC-1065, this structure conforms to the curved shape of DNA by mimicking the twist of the helix and allowing the drug to bind within the minor groove in a sequence-specific manner (11-16). The indole and benzofuran substituents of adozelesin (Fig. 1, subunits B and C, respectively) form non-covalent interactions with DNA that may contribute to the sequence preference of the drug (11,(17)(18)(19)(20)(21)(22). These non-covalent interactions cause bending and stiffening of the DNA helix (11-13). After binding to the minor groove, the cyclopropyl ring of the left-hand cyclopropylpyrroloindole substituent (Fig. 1) forms a covalent bond with N-3 of adenine at the 3Ј end of the binding site (23, 24). Two consensus sequences have been identified for this alkylation event: 5Ј-(A/T)(A/T)A* and 5Ј-(A/T)(G/C)(A/T)A*, where A* is the alkylated 3Ј adenine (9,22).Inhibition of DNA synthesis as ...

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We examined DNA damage induced by the enediyne-containing antitumor antibiotic C-1027 in intracellular nuclear and mitochondrial DNA targets using the episome-containing cell line 935.1. Strand-scission activity of the C-1027 holoantibiotic was measured by the topological forms conversion assay in episomal and mitochondrial DNA, as well as in cell-free plasmid DNA. Genomic DNA damage was quantitated by filter elution analysis. Comparisons were made to the well-characterized enediyne neocarzinostatin. From these studies, mixed single- and double-strand breaks were observed not only in cell-free, plasmid DNA but also in intracellular episomal, mitochondrial, and genomic DNA at low nanomolar concentrations. C-1027 cleaved DNA 285-fold more efficiently in cells than in a cell-free environment, and displayed preference for intracellular DNA species in the following rank order: episome > mitochondrial DNA >> genomic. NCS also damaged the non-histone-associated mitochondrial DNA, but not the episome. Cleavage of the 935.1 cell episome by C-1027 occurred at specific sites including the BPV origin of replication and E6/E7 open reading frame regions, as well as the MMTV LTR promoter region.

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