Abstract
Camptothecin derivatives are powerful anticancer drugs because of their ability to trap topoisomerase I (Top1)-DNA cleavage complexes. However, they exhibit clinical limitations due to the instability of their α-hydroxylactone six-membered E-ring structure. In addition, they exhibit bone marrow and intestinal toxicity, especially in adults, and are drug efflux substrates. Here, we report a novel Top1 inhibitor, Genz-644282. We show that Genz-644282 and its metabolites induce Top1 cleavage at similar, as well as unique genomic positions, compared with camptothecin. The compound also induces protein-linked DNA breaks and Top1-DNA cleavage complexes that persist longer after compound removal than camptothecin. Concentration-dependent and persistent γH2AX formation was readily observed in cells treated with Genz-644282, and was present in greater than 50% of the cell population following 24 hours compound exposure. The compound shows partial cross-resistance in cell lines resistant to camptothecin. These cell lines include the human prostate DU145RC0.1 and the leukemic CEM/C2 cells. Limited cross-resistance to Genz-644282 was also found in the Top1 knockdown colon cancer (HCT116) and breast cancer (MCF7) cell lines and in human adenocarcinoma cells (KB31/KBV1) that overexpress (P-glycoprotein, ABCB1), a member of the ATP-binding cassette family of cell surface transport proteins known to confer MDR. Together, our results provide the first molecular and cellular characterization of Genz-644282 and its clinically relevant metabolites.
Highlights
The main activity of DNA topoisomerase I (Top1) is the relaxation of DNA supercoils generated during DNA replication, transcription, chromatin folding, and possibly DNA repair
Top1 inhibitors effectively trap the enzyme as it cleaves DNA by creating a Top1–DNA cleavage complex (Top1cc), which leads to cell killing after Top1ccs are converted into DNA damage by DNA replication and/or transcription [1]
Aliquots were stored at À20C, and additional dilutions were made in dimethyl sulfoxide (DMSO) immediately before use
Summary
The main activity of DNA topoisomerase I (Top1) is the relaxation of DNA supercoils generated during DNA replication, transcription, chromatin folding, and possibly DNA repair. The enzyme nicks DNA by forming a reversible covalent tyrosyl–DNA bond, which allows the broken DNA strand to rotate around the complementary intact strand, followed by the religation of the breaks through reversal of the Top1–DNA covalent bond [1,2,3]. Top inhibitors effectively trap the enzyme as it cleaves DNA by creating a Top1–DNA cleavage complex (Top1cc), which leads to cell killing after Top1ccs are converted into DNA damage by DNA replication and/or transcription [1]. This DNA damage can be detected by the phosphorylation of the histone H2A variant H2AX [7, 8]. Within minutes following the formation of a Top1-
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