Survivin enhances radiation resistance in primary human glioblastoma cellsvia caspase-independent mechanismsArnab Chakravarti*,1, Gary G Zhai1, Min Zhang1, Rajeev Malhotra1, Douglas E Latham1,Meaghan A Delaney1, Pierre Robe2, Ulf Nestler3, Qinhui Song1and Jay Loeffler11Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA;2Department ofNeurosurgery, Brigham and Women’s Hospital/Harvard Medical School, Boston, MA, USA;3Department of Neurosurgery, JustusLiebig University, Giessen, GermanyThe observed radioresistance of human glioblastomamultiforme (GBM) poses a major challenge, which, ifovercome, may lead to significant advances in themanagement of this patient population. There is accumu-lating evidence from correlative studies that Survivinexpression is associated with increased malignant poten-tial of human gliomas. The purpose of this study was toinvestigate whether Survivin plays a direct role inmediating radiation resistance in primary human gliomacell lines, and, if so, investigating the underlying mechan-isms. Our panel of GBM cell lines included two that wererelatively radiation resistant (GM20 and GM21) and twothat were more radiation sensitive (GM22 and GM23),which demonstrated differential levels of Survivin expres-sion between the two groups. Through the use ofadenoviral vectors containing either dominant-negative(pAd-S(T34A)) or wild-type Suvrivin (pAd-S(WT)), wewere able to inactivate or overexpress Survivin, respec-tively. Our findings suggest that Survivin plays a criticalrole in mediating radiation resistance in primary GBMcells, in part through suppression of apoptotic cell deathvia a caspase-independent manner. We have identifiednovel mechanisms by which Survivin may enhance tumorcell survival upon radiation exposure such as regulation ofdouble-strand DNA break repair and tumor cell metabo-lism, which were most evident in the radiation-resistantcell lines. These differences in Survivin function both inradiation-resistant vs radiation-sensitive cell lines and inthe presence vs absence of radiation exposure warrantfurther investigation and highlight potentially importantmechanisms of radiation resistance in these tumors.Oncogene (2004) 23, 7494–7506. doi:10.1038/sj.onc.1208049Published online 23 August 2004Keywords: Survivin; radiation resistance; primary hu-man glioblastomaIntroductionHuman gliomas are among the most aggressive tumors,with no curative therapies currently available. Molecu-lar and genetic mechanisms that serve to enhance themalignant potential of these tumors are becoming betterunderstood (Maher et al., 2001). Patients with glioblas-toma multiforme (GBM), a WHO Grade IV/IV glioma,are routinely managed by surgery followed by adjuvantradiation and chemotherapy. Despite such aggressivetreatment approaches, median survival times remainunder 1 year in most series (Walker et al., 1978, 1980;Davis et al., 1999). Identifying the molecular and geneticmechanisms underlying treatment resistance representsan important first step for the development of moreeffective therapies against GBMs.Survivin expression has been associated with adverseclinical outcome and enhanced malignant potential ofgliomas (Chakravarti et al., 2002; Sasaki et al., 2002;Kajiwara et al., 2003; Katoh et al., 2003). Survivin is a16.5 kDa member of the inhibitor of apoptosis (IAP)family of proteins, which has been found to be expressedin the G2/M phases of the cell cycle almost exclusively intumor cells, but absent in most normal adult differ-entiated tissues (Ambrosini et al., 1997, 1998; Kobayashiet al., 1999; Li and Altieri, 1999; Skoufias et al., 2000;Wright et al., 2000; Grossman et al., 2001; Jiang et al.,2001; Fortugno et al., 2002a). It is thought that Survivinenhances survival of tumor cells primarily throughsuppression of apoptotis-related cell death perhaps viadirect inhibition of caspase-related proteins (Conwayet al., 2000; Wright et al., 2000; Shin et al., 2001).Whether Survivin plays a direct role in promotingradiation resistance of GBMs and, if so, the precisemechanisms involved are outstanding issues. We reporthere that Survivin does, indeed, appear to play amechanistic role in enhancing cell survival upon radia-tion exposure in a manner that appears to beindependent of its known caspase-mediated functions.ResultsSurvivin expression levels in radiation-resistant vsradiation-sensitive primary GBM cell linesPrimary cell lines were established from four GBMtumor specimens as described above. As Figure 1aReceived 18 May 2004; revised 9 July 2004; accepted 9 July 2004;published online 23 August 2004*Correspondence: A Chakravarti, Department of Radiation Oncol-ogy, Massachusetts General Hospital/Harvard Medical School, 100Blossom Street, Founders House, Room 536, Boston, MA 02114,USA; E-mail: [email protected] (2004) 23, 7494–7506&2004 Nature Publishing GroupAll rights reserved 0950-9232/04 $30.00www.nature.com/oncdemonstrates, GM20 and GM21 were found to berelatively radiation resistant compared to GM22 andGM23, as determined by clonogenic survival assays.Survivin expression levels were found to be higher inGM20 and GM21 compared with GM22 andGM23. Further, after radiation exposure, levels ofphospho-Survivin were found to increase in all fourprimary GBM cell lines (Figure 1b), indicatingthat radiation can lead to more stable (phosphorylated)isoforms of Survivin. Although Survivin expressionwas most evident in the G2/M phases of the cellcycle under physiologic conditions (Figure 1c),after radiation exposure, Survivin expression wasevident throughout all phases of the cell cycle in themore radioresistant GM 20 and GM21 cell lines(Figure 1d).Antagonism of Survivin enhances radiation responseThe four primary GBM cell lines were treated withadenoviral vectors containing either dominant-negative(Ad-S(T34A)) or with wild-type (Ad-S(WT)) Survivin toeither inactivate or further activate Survivin function,respectively, in these cells. It is apparent that in all fourcell lines, cells treated by Ad-S(T34A) demonstratedincreased sensitivity to radiation compared to untreatedcells (Po0.0001, Figures 2a–d). In contrast, cells over-expressing wild-type Survivin through treatment withAd-S(WT) demonstrated increased resistance to radia-tion compared to untreated cells (Po0.0001, Figures2a–d). Interestingly, when normal fetal astrocytes wereinfected with either Ad-S(T34A) or Ad-S(WT), therewere no appreciable