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Research Articles: Therapeutics, Targets, and Development

S-phase checkpoints regulate Apo2 ligand/TRAIL and CPT-11–induced apoptosis of prostate cancer cells

¹ Department of Cancer Biology, Lerner Research Institute; ² Department of Radiation Oncology, Cleveland Clinic, Cleveland, Ohio; and ³ Department Biological Sciences, Kent State University, Kent, Ohio

Requests for reprints: Alex Almasan, Departments of Cancer Biology and Radiation Oncology, NB40, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: 216-444-9970; Fax: 216-445-6269. E-mail: almasaa{at}ccf.org

Abstract

As S-phase checkpoints play critical roles in maintaining genomic integrity and replicating the human genome correctly, understanding the molecular mechanism by which they regulate the therapeutic response is of great interest. Previously, we reported that the cytotoxic effect of a zinc-bound form of Apo2 ligand/tumor necrosis factor–related apoptosis-inducing ligand (Apo2L/TRAIL), which is currently evaluated in clinical trials, in combination with low-dose CPT-11, induces apoptosis of C4-2 human prostate cancer cells and tissues. Here, we show that apoptosis, induced synergistically by this combination treatment, was associated with accumulation of cells in early S phase, indicated by cell cycle analyses, increased proliferating cell nuclear antigen, and Chk2-Thr⁶⁸ phosphorylation in tumors xenografted in mice. The combination treatment induced an S-phase checkpoint response through activation of Chk2 and Chk1 by the ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3 related kinases, leading to phosphorylation and decreased Cdc25A levels. Cdc25A-dependent regulation of cyclin-dependent kinase 2 (Cdk2) and changes in association of p21^WAF1/CIP1 and hSpy1 with Cdk2 resulted in inhibition of Cdk2-associated kinase activity. Knockdown of ataxia telangiectasia mutated/Chk2 and ataxia telangiectasia mutated and Rad3 related/Chk1 by small inhibitory RNAs abrogated the S-phase checkpoint and accelerated apoptosis, resulting in caspase-3 activation and poly(ADP-ribose) polymerase 1 cleavage following combination treatment. Thus, Apo2L/TRAIL + CPT-11 treatment–induced apoptosis is regulated through an S-phase checkpoint controlled by the Chk2-Cdc25A and Chk1-Cdc25A pathways and inhibition of Cdk2-associated kinase activity. Low-dose CPT-11 and aphidicolin increased the proportion of S-phase cells and sensitized cells to Apo2L/TRAIL, by inducing phosphatidylserine externalization, caspase activation, and poly(ADP-ribose) polymerase 1 cleavage. Combinations with S-phase arrest–inducing chemotherapeutic drugs may represent promising avenues for clinical development of Apo2L/TRAIL. [Mol Cancer Ther 2007;6(4):1368–78]

Grant support: NIH grant CA81504.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/10/05; revised 12/13/06; accepted 2/ 8/07.

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