Topoisomerase I (Top1) is a ubiquitous enzyme that removes DNA supercoiling generated during transcription and replication. Top1 can be trapped on DNA as cleavage complexes by the anticancer drugs referred to as Top1 inhibitors as well as by alterations of the DNA structure. We reported recently that Top1 cleavage complexes (Top1cc) are trapped during apoptosis induced by arsenic trioxide and staurosporine. In the present study, we generalize the occurrence of apoptotic Top1cc in response to anticancer drugs, which by themselves do not directly interact with Top1: the topoisomerase II inhibitors etoposide, doxorubicin, and amsacrine, and the tubulin inhibitors vinblastine and Taxol. In all cases, the Top1cc form in the early phase of apoptosis and persist throughout the apoptotic process. Their formation is prevented by the caspase inhibitor benzyloxycarbonyl-Val-Ala-DLAsp(OMe)-fluoromethylketone and the antioxidant N-acetyl-L-cysteine. We propose that the trapping of Top1cc is a general process of programmed cell death, which is caused by alterations of the DNA structure (oxidized bases and strand breaks) induced by caspases and reactive oxygen species. [Mol Cancer Ther 2006; 5(12):3139 -44]

Topoisomerase I (Top1) is known to relax DNA supercoiling generated by transcription, replication, and chromatin remodeling. However, it can be trapped on DNA as cleavage complexes (Top1cc) by oxidative and carcinogenic DNA lesions, base damage, and camptothecin treatment. We show here that Top1 is also functionally involved in death receptor-induced programmed cell death. In cells exposed to TRAIL or Fas ligand, Top1cc form at the onset of apoptosis. Those apoptotic Top1cc are prevented by caspase inhibition and Bax inactivation, indicating that both caspases and the mitochondrial death pathway are required for their formation. Accordingly, direct activation of the mitochondrial pathway by BH3 mimetic molecules induces apoptotic Top1cc. We also show that TRAIL-induced apoptotic Top1cc are preferentially formed by caspase-3-cleaved Top1 at sites of oxidative DNA lesions with an average of one apoptotic Top1cc/100 kbp. Examination of Top1 knockdown cells treated with TRAIL revealed similar DNA fragmentation but a marked decrease in apoptotic nuclear fission with reduced formation of nuclear bodies. Thus, we propose that Top1 contributes to the full apoptotic responses induced by TRAIL.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) 2 is a promising therapeutic agent because it induces apoptosis in a wide variety of cancer cells without affecting normal tissues (1, 2). TRAIL belongs to the TNF family of cytokines, including TNF␣ and Fas ligand, which induce apoptosis by binding to their cognate plasma membrane receptors (3). The binding of TRAIL to the DR4 or DR5 receptors and the binding of Fas ligand to Fas receptor cause the intracellular death domains of those receptors to trimerize, which leads to the recruitment of FADD and the activation of caspase-8. Caspase-8 then cleaves and thereby activates caspase-3 either directly (type I cells) or/and indirectly (type II cells) (4) by activating the mitochondrial death pathway through the cleavage of Bid (5). Cleaved Bid binds to and activates the pro-apoptotic Bcl-2 relatives Bax and Bak proteins, causing the release of mitochondrial cytochrome c and the activation of caspase-9 and caspase-3 (6). Activated caspase-3 (and other downstream caspases) cleaves a broad array of intracellular targets including DNA topoisomerase I (Top1) (7). It is also required to induce the controlled rearrangement and degradation of nuclear structures with chromatin condensation, DNA fragmentation, nuclear fission, and release of apoptotic nuclear bodies in the extracellular space (8). TRAIL-induced apoptosis also involves an accumulation of intracellular reactive oxygen species (ROS) (9).Top1 removes DNA superhelical tensions generated during transcription, replication, and chromatin remodeling (10) and is essential in higher eukaryotes (11). It relaxes DNA by forming transient DNA single-strand breaks that are produced as Top1 forms a covalent bond between its active site tyrosine (Tyr 723 ) and a 3Ј-DNA phosphate. These Top1 cleavage complexes (Top1cc) allow con...

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