Radiation-Induced Caspase-8 Mediates p53-Independent Apoptosis in Glioma Cells Golnar Afshar, 1,2 Nannette Jelluma, 1,2 Xiaodong Yang, 1,2 Daniel Basila, 1,2 Nils D. Arvold, 1,2 Amelia Karlsson, 1,2 Garret L. Yount, 3 Tobias B. Dansen, 2 Erich Koller, 4 and Daphne A. Haas-Kogan 1,2 1 Departments of Radiation Oncology and 2 Comprehensive Cancer Center, University of California at San Francisco; 3 California Pacific Medical Center Research Institute, San Francisco, California; and 4 Functional Genomics, Isis Pharmaceuticals, Inc., Carlsbad, California Abstract Malignant gliomas are almost uniformly fatal and display exquisite radiation resistance. Glioma cells lacking wild-type (WT) p53 function are more susceptible to radiation-induced apoptosis than their isogenic counterparts expressing WT p53. We explored the mechanisms of such apoptosis and found that, in the absence of WT p53, radiation increases caspase- 8 expression and activity. Inhibition of caspase-8 expression using caspase-8 antisense or small interfering RNA (siRNA) oligonucleotides partially blocks radiation-induced apoptosis. In contrast, inhibition of the mitochondrial death pathway by expression of Bcl-2 has no effect on radiation-induced caspase-8 activity or apoptosis. Our data indicate that, in contrast to commonly accepted models of p53-dependent radiation-induced apoptosis, in our cell system, radiation relies on caspase-8 activity to help mediate p53-independent cell death. In a system of inducible E2F1 activity, E2F1 activated caspase-8 and, accordingly, decreased cellular viability, effects that were abolished by caspase-8 siRNA. In this model, in the absence of WT p53, p21 Cip1 is not induced, and E2F1 activity is sustained and allows transcription and activation of caspase-8. This model may explain why p53 mutations in adult gliomas paradoxically correlate with improved survival and enhanced response to radiation. (Cancer Res 2006; 66(8): 4223-32) Introduction Glioblastoma multiforme is the most common and malignant central nervous system tumor. Patients with glioblastoma multi- forme have a median survival time of <1 year (1). Glioblastoma multiformesaretreatedbysurgicalresectionfollowedbyradiation therapy;however,inevitably,tumorsrecurusuallyintheproximity oftheoriginalmass(2,3).Althoughradiationisthemosteffective adjuvant treatment, glioblastoma multiformes exhibit radiation resistance that ultimately precludes their cure. In response to radiation, mammalian cells undergo apoptosis and/or cell cycle arrest. Although p53 mediates both cellular responses, the dominant response in each tumor is influenced by cellular lineage, biological context, and determinants that hitherto remainpoorlyunderstood(4).Solidtumors,inwhichp53mediates growth arrest after radiation, pose compelling clinical quandaries becausemoreoftenthannot,followingagrowtharrest,clonogens regrow and lead to tumor recurrence. Although in many solid tumors the dominant p53-mediated radiation response is a growth arrest, more than half of human malignancies harbor mutations in p53 and are thus deficient in their p53-dependent responses to radiation (5). Specifically, abrogation of wild-type (WT) p53 function in isogenic glioma cell lines renders them more susceptible to radiation-induced apopto- sis(6,7).Therefore,theclinicaluseofradiationforthetreatmentof humanmalignanciesmustcapitalizeonp53-independentformsof radiation-inducedcelldeath.p53-independentapoptosisisinduced byradiationinseveralmalignancies(8),andpreviousstudieshave revealed the importance of p53-independent apoptosis in gliomas (8). We sought to explore the molecular mechanism of p53- independent apoptosis induced in gliomas by ionizing radiation. Cysteine proteases, known as caspases, constitute key compo- nents of the apoptotic pathway (9). Two distinct pathways of apoptosishavebeenidentifiedasmitochondriainitiatedanddeath receptor initiated (9, 10). DNA-damaging agents, such as ionizing radiation, trigger release of cytochrome c from mitochondrial intermembrane spaces. Cytochrome c forms a complex with an adaptormolecule,Apaf1,whichbindsandactivatescaspase-9(11). Activated caspase-9, in turn, cleaves and activates downstream caspases, effector proteases that execute the cell death program. Release of cytochrome c is regulated by the balance between antiapoptotic and proapoptotic members of the Bcl-2 family of proteins. Whereas mitochondria-initiated apoptosis occurs through caspase-9, the death receptor–mediated pathway requires caspase-8 (12–14). In the death receptor–mediated pathway, binding of a ligand, such as Fas ligand or tumor necrosis factor- a (TNF-a), induces conformational changes of oligomerized death receptors (15, 16). The death domain–containing cytoplasmic region of the receptor recruits an adaptor molecule known as Fas-associated death domain (FADD) either by direct binding to thedeathdomainofFADDorthroughyetanotherdeathdomain– containing protein named TRADD. The death effector domain of FADD binds with procaspase-8. Binding to FADD triggers proteolyticprocessingofprocaspase-8totheactivecaspase-8form, which in turn activates downstream caspases (9, 10). Ionizing radiation increases protein levels of the tumor suppressor p53, which in turn regulates many cellular responses to DNA damage. Caspase-9 has been shown to execute p53- dependent radiation-induced apoptosis (11, 17). In contrast, radiation-induced apoptotic pathways executed in the absence of Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). G. Afshar and N. Jelluma contributed equally to this work. Requests for reprints: DaphneA.Haas-Kogan,DepartmentofRadiationOncology, University of California at San Francisco, 1600 Divisadero Street, Suite H1031, San Francisco, CA 94143-1708. Phone: 415-353-7175; Fax: 415-353-9883; E-mail: hkogan@Radonc17.ucsf.edu. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-1283 www.aacrjournals.org 4223 Cancer Res 2006; 66: (8). April 15, 2006 Research Article Research. on February 12, 2016. © 2006 American Association for Cancer cancerres.aacrjournals.org Downloaded from