Topoisomerase I Requirement for Death Receptor-induced Apoptotic Nuclear Fission

Abstract

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...

Genome-wide Analysis of Novel Splice Variants Induced by Topoisomerase I Poisoning Shows Preferential Occurrence in Genes Encoding Splicing Factors

“…Under these conditions, DRB abrogated CPT-induced Pol II hyperphosphorylation (Fig. 6D) (37). These results suggest a relationship between CPT-induced Pol II hyperphosphorylation and splicing alterations.…”

“…β2 microglobulin (β2) mRNA was used as control. The percentage of cells in sub-G1 (37) is indicated at the bottom of the panel. B , Downregulation of Top1 does not induce the alternative splicing of RBM8A or SF1.…”

8-Oxo-7,8-dihydroguanine: Links to gene expression, aging, and defense against oxidative stress

“…Topoisomerase I (Top I) is ubiquitously expressed and essential in eukaryotic cells, and among its diverse functions are several that contribute to the recognition and elimination of apoptotic cells [198, 199]. Specifically, Top I plays a critical role in tumor necrosis factor-related, apoptosis-inducing ligand (TRAIL)-induced apoptosis during the formation and release of apoptotic nuclear bodies [199].…”

“…Topoisomerase I (Top I) is ubiquitously expressed and essential in eukaryotic cells, and among its diverse functions are several that contribute to the recognition and elimination of apoptotic cells [198, 199]. Specifically, Top I plays a critical role in tumor necrosis factor-related, apoptosis-inducing ligand (TRAIL)-induced apoptosis during the formation and release of apoptotic nuclear bodies [199]. Sordet and colleagues [199] demonstrated that an apoptotic Top I cleavage complex (Top Icc, an 80-kDa C-terminal fragment of Top I, generated by caspase-3) is trapped at the proximity of oxidative 8-oxoG DNA lesion generated by TRAIL-induced ROS.…”

“…Specifically, Top I plays a critical role in tumor necrosis factor-related, apoptosis-inducing ligand (TRAIL)-induced apoptosis during the formation and release of apoptotic nuclear bodies [199]. Sordet and colleagues [199] demonstrated that an apoptotic Top I cleavage complex (Top Icc, an 80-kDa C-terminal fragment of Top I, generated by caspase-3) is trapped at the proximity of oxidative 8-oxoG DNA lesion generated by TRAIL-induced ROS. Importantly, this phenomenon may be general, as the ROS-dependent formation of 8-oxoG appears to represent a common mechanism for the trapping of Top Icc during apoptosis induced by arsenic trioxide and chemotherapeutic agents, including staurosporine and etoposide [200–202].…”