Transcription-dependent Activation of Ataxia Telangiectasia Mutated Prevents DNA-dependent Protein Kinase-mediated Cell Death in Response to Topoisomerase I Poison

Hirobumi Teraoka,Ryo Sakasai,Masatoshi Takagi,Randal S. Tibbetts

doi:10.1074/jbc.m110.101808

Abstract

Camptothecin (CPT) is a topoisomerase I inhibitor, derivatives of which are being used for cancer chemotherapy. CPT-induced DNA double-strand breaks (DSBs) are considered a major cause of its tumoricidal activity, and it has been shown that CPT induces DNA damage signaling through the phosphatidylinositol 3-kinase-related kinases, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase). In addition, CPT causes DNA strand breaks mediated by transcription, although the downstream signaling events are less well characterized. In this study, we show that CPT-induced activation of ATM requires transcription. Mechanistically, transcription inhibition suppressed CPT-dependent activation of ATM and blocked recruitment of the DNA damage mediator p53-binding protein 1 (53BP1) to DNA damage sites, whereas ATM inhibition abrogated CPT-induced G(1)/S and S phase checkpoints. Functional inactivation of ATM resulted in DNA replication-dependent hyperactivation of DNA-PK in CPT-treated cells and dramatic CPT hypersensitivity. On the other hand, simultaneous inhibition of ATM and DNA-PK partially restored CPT resistance, suggesting that activation of DNA-PK is proapoptotic in the absence of ATM. Correspondingly, comet assay and cell cycle synchronization experiments suggested that transcription collapse occurring as the result of CPT treatment are converted to frank double-strand breaks when ATM-deficient cells bypass the G(1)/S checkpoint. Thus, ATM suppresses DNA-PK-dependent cell death in response to topoisomerase poisons, a finding with potential clinical implications.

Highlights

Have been intensively studies for their tumoricidal properties
The most salient findings in the study include: (i) CPT induces transcription-dependent and -independent activation of ATM leading to 53BP1 foci formation and checkpoint activation; (ii) the absence of ATM-dependent G1/S checkpoint leads to severe DNA damage and DNA-PK hyperactivation in the S phase; (iii) hyperactivation of DNA-PK by CPT in the absence of ATM causes cell death
The mechanism of DNA strand breakage induced by CPT and clinically useful derivatives, irinotecan and topotecan, has been the subject of intensive study, and it is well established that topoisomerase I (TopI)-DNA cleavage complexes (TopI-cc) are converted to frank doublestrand breaks (DSBs) during S phase [1]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Irradiation—HeLa, U2OS, and HCT116 cells were obtained from American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Cell preparation for staining of 53BP1, ␥H2AX and incorporated BrdUrd was performed as described [9]. The fixed cells were incubated with primary antibodies specific for 53BP1, BrdUrd, ␥H2AX, and Rad. After incubation with secondary antibodies, cell nuclei were stained with 4Ј,6-diamidino-2-phenylindole (2 ␮g/ml). Cell Cycle Synchronization and Flow Cytometry—To synchronize cells in the S phase, cells were treated with thymidine at 2.5 mM for 18 h and incubated with a fresh medium for 5 h. Propidium iodide-stained cells were analyzed using a FACSCalibur (BD Biosciences). Comet tails were stained with SYBR Green I (BMA) and analyzed by fluorescent microscope

RESULTS

Substantiation of the above model required us to test whether

DISCUSSION