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Cell Cycle, Cell Death, and Senescence

Ribotoxic Stress Sensitizes Glioblastoma Cells to Death Receptor–Induced Apoptosis: Requirements for c-Jun NH₂-Terminal Kinase and Bim

¹ The Kennedy-Krieger Institute and Departments of ² Neurology, ³ Oncology, and ⁴ Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, Maryland; and ⁵ Department of Oncology, Lombardi Cancer Center, Washington, District of Columbia

Requests for reprints: John Laterra, Room 400, Kennedy Krieger Research Institute, 707 N. Broadway, Baltimore, MD 21205. Phone: 443-923-2683; Fax: 443-923-2695. E-mail: laterra{at}kennedykrieger.org

A prominent feature of glioblastoma is its resistance to death receptor–mediated apoptosis. In this study, we explored the possibility of modulating death receptor–induced cell death with the c-Jun-NH₂-terminal kinase (JNK) activator anisomycin. Anisomycin activates JNK by inactivating the ribosome and inducing "ribotoxic stress." We found that anisomycin and death receptor ligand anti-Fas antibody CH-11 or tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) synergistically induce apoptosis in multiple human glioblastoma cell lines. For example, in U87 cells, anisomycin reduced the IC₅₀ of CH-11 by more than 20-fold (from 500 to 25 ng/mL). Cell viability in response to anisomycin, CH-11, and their combination was 79%, 91%, and 28% (P < 0.001), respectively. Anisomycin and TRAIL were found to be similarly synergistic in glioblastoma cells maintained as tumor xenografts. The potentiation of death receptor–dependent cell death by anisomycin was specific because emetine, another ribosome inhibitor that does not induce ribotoxic stress or activate JNK, did not have a similar effect. Synergistic cell death was predominantly apoptotic involving both extrinsic and intrinsic pathways. Expression of Fas, FasL, FLIP, and Fas-associated death domain (FADD) was not changed following treatment with anisomycin + CH-11. JNK was activated 10- to 22-fold by anisomycin + CH-11 in U87 cells. Inhibiting JNK activation with pharmacologic inhibitors of JNKK and JNK or with dominant negative mitogen-activated protein kinase (MAPK) kinase kinase 2 (MEKK2) significantly prevented cell death induced by the combination of anisomycin + CH-11. We further found that anisomycin + CH-11 up-regulated the proapoptotic protein Bim by 14-fold. Simultaneously inhibiting Bim expression and JNK activation additively desensitized U87 cells to anisomycin + CH-11. These findings show that anisomycin-induced ribotoxic stress sensitizes glioblastoma cells to death receptor–induced apoptosis via a specific mechanism requiring both JNK activation and Bim induction. (Mol Cancer Res 2007;5(8):783–92)