Topoisomerase I-DNA Complexes Contribute to Arsenic Trioxide-induced Apoptosis

Olivier Sordet,Zhiyong Liao,Hong Liu,Smitha Antony,Ellen V Stevens,Glenda Kohlhagen,Haiqing Fu,Yves Pommier

doi:10.1074/jbc.m404620200

Abstract

Topoisomerase I is an essential enzyme that relaxes DNA supercoiling by forming covalent DNA cleavage complexes, which are normally transient. Topoisomerase I-DNA complexes can be trapped by anticancer drugs (camptothecins) as well as by endogenous and exogenous DNA lesions. We show here that arsenic trioxide (a potent inducer of apoptosis that induces the intracellular accumulation of reactive oxygen species and targets mitochondria) induces cellular topoisomerase I cleavage complexes. Bcl-2 overexpression and quenching of reactive oxygen species, which prevent arsenic trioxide-induced apoptosis, also prevent the formation of topoisomerase I-DNA complexes, whereas enhancement of reactive oxygen species accumulation promotes these complexes. The caspase inhibitor, benzyloxycarbonyl-VAD partially prevents arsenic trioxide-induced topoisomerase I-DNA complexes and apoptosis, suggesting that activated caspases further maintain intracellular levels of reactive oxygen species that induce the formation of topoisomerase I-DNA complexes. Down-regulation of topoisomerase I expression decreases arsenic trioxide-induced apoptotic DNA fragmentation. Thus, we propose that arsenic trioxide induces topoisomerase I-DNA complexes that participate in chromatin fragmentation and programmed cell death during apoptosis.

Highlights

A key underlying mechanism for As2O3-induced apoptosis is the dissipation of the mitochondrial transmembrane potential (⌬␺m), an event inhibited by Bcl-2 overexpression (3, 10 –12), which is followed by the release of soluble intermembrane proteins into the cytosol, including apoptosis inducing factor, caspase activation, DNA fragmentation, and the classic morphologic changes of apoptosis (10 –15)
Because Top1 cleavage complexes can be produced by oxidative DNA lesions, we investigated whether modulation of reactive oxygen species (ROS) levels by changing the cellular GSH content would affect the generation of Top1-DNA
This study demonstrates the formation of Top1 cleavage complexes in cells undergoing apoptosis after As2O3 treatment

Summary

The abbreviations used are

As2O3, arsenic trioxide; APL, acute promyelocytic leukemia; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Z, benzyloxycarbonyl; AFC, 7-amino-4trifluoromethylcoumarin; ROS, reactive oxygen species; BSO, DL-buthionine-(SR)-sulfoximine; CPT, camptothecin; fmk, fluoromethyl ketone; siRNA, small interfering RNA; PARP, poly(ADP-ribose)-polymerase; NAC, N-acetyl-L-cysteine; LA, lipoic acid; Top, topoisomerase I. (H2O2), hydroxyl Inhibition of GSH radical (HO1⁄7), and superperoxidase [16] and activation of flavoprotein-dependent superoxide-producing enzymes (such as NADPH oxidase) [13] by As2O3 have been implicated in ROS accumulation These As2O3-induced ROS generate oxidative DNA lesions, mainly 8-oxoguanine and. Top cleavage complexes have been detected after formation of endogenous and exogenous DNA lesions [32] These include oxidative lesions (8-oxoguanine, abasic sites, and strand breaks), UV-induced base modifications, guanine alkylation, polycyclic aromatic carcinogenic adducts, base mis-. The Top poisoning effect of As2O3 is indirect because As2O3 does not induce the formation of Top cleavage complexes when incubated with recombinant Top1 These Top1-DNA complexes occur in association with As2O3-induced apoptosis, and we propose that they are due to oxidative DNA lesions generated by As2O3-induced ROS and caspase activation. Top1-mediated DNA cleavage fragments are indicated by the arrowheads

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION