Topoisomerase IIβ Deficiency Enhances Camptothecin-induced Apoptosis

Ren-Kuo Lin,Chia-Wen Ho,Leroy F Liu,Yi Lisa Lyu

doi:10.1074/jbc.m112.415471

Abstract

Camptothecin (CPT), a topoisomerase (Top) I-targeting drug that stabilizes Top1-DNA covalent adducts, can induce S-phase-specific cytotoxicity due to the arrest of progressing replication forks. However, CPT-induced non-S-phase cytotoxicity is less well characterized. In this study, we have identified topoisomerase IIβ (Top2β) as a specific determinant for CPT sensitivity, but not for many other cytotoxic agents, in non-S-phase cells. First, quiescent mouse embryonic fibroblasts (MEFs) lacking Top2β were shown to be hypersensitive to CPT with prominent induction of apoptosis. Second, ICRF-187, a Top2 catalytic inhibitor known to deplete Top2β, specifically sensitized MEFs to CPT. To explore the molecular basis for CPT hypersensitivity in Top2β-deficient cells, we found that upon CPT exposure, the RNA polymerase II large subunit (RNAP LS) became progressively depleted, followed by recovery to nearly the original level in wild-type MEFs, whereas RNAP LS remained depleted without recovery in Top2β-deficient cells. Concomitant with the reduction of the RNAP LS level, the p53 protein level was greatly induced. Interestingly, RNAP LS depletion has been well documented to lead to p53-dependent apoptosis. Altogether, our findings support a model in which Top2β deficiency promotes CPT-induced apoptosis in quiescent non-S-phase cells, possibly due to RNAP LS depletion and p53 accumulation.

Highlights

The sensitivity of non-S-phase cells to Top1-targeting drugs has been under-explored
Top2␤ Deficiency Sensitizes Quiescent mouse embryonic fibroblasts (MEFs) to Camptothecin-induced Cell Death—Top1-targeting drugs (e.g. CPT) are known to induce S-phase-specific cytotoxicity; this is largely attributed to the arrest of the advancing replication forks by the drug-induced Top1 cleavage complexes [22]
Using serum-starved primary MEFs as a model, we have previously shown that Top2␤ knock-out MEFs (Top2␤Ϫ/Ϫ) were highly resistant to Top2-targeting drugs (e.g. VP-16) as compared with wild-type MEFs (Top2␤ϩ/ϩ) [36, 37]

Summary

Background

The sensitivity of non-S-phase cells to Top1-targeting drugs has been under-explored. Our findings support a model in which Top2␤ deficiency promotes CPT-induced apoptosis in quiescent nonS-phase cells, possibly due to RNAP LS depletion and p53 accumulation. Camptothecin (CPT), a Top1-targeting drug, can interfere with the catalytic cycle of Top by inhibiting the religation reaction and increases the half-life of the transient Top1DNA covalent adducts ( known as Top cleavage complexes) [1, 20]. It has been demonstrated that the collision of Top cleavage complexes with DNA replication machineries and the subsequently generated DNA double strand breaks are responsible for the S-phase-specific cytotoxicity of CPT [22]. CPT treatment, possibly due to the reduced replication-mediated Top cleavage complex processing [25] This S-phase-selective cytotoxicity enables Top1-based chemotherapy to be efficacious in treating several types of cancers, including ovarian, lung, and colon cancers [20, 24, 26]. The increased sensitivity is associated with the depletion of RNA polymerase II large subunit (RNAP LS), p53 accumulation, general transcription inhibition, and apoptosis induction

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION