O 6 -methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells Mirjam Hermisson,* Andrea Klumpp,* Wolfgang Wick,* Jo ¨rg Wischhusen,* Georg Nagel,  Wynand Roos, Bernd Kaina and Michael Weller* *Laboratory of Molecular Neuro-Oncology, Department of General Neurology, Hertie Institute for Clinical Brain Research, University of Tu ¨bingen, School of Medicine, Tu ¨ bingen, Germany  Institute of Toxicology, University of Mainz, Mainz, Germany Abstract Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O 6 -methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O 6 -alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X L . We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clono- genic survival after TMZ exposure are highly correlated (p< 0.0001, r 2 ¼ 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O 6 -benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X L protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ion- izing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ. Keywords: BCL-X L , glioblastoma, O 6 -methylguanine DNA methyltransferase, p53, temozolomide. J. Neurochem. (2006) 96, 766–776. Temozolomide (TMZ) is a cytotoxic alkylating agent which has shown activity in recurrent anaplastic glioma and glioblastoma (Yung et al. 1999, 2000). Moreover, the recent European Organisation for Research and Treatment of Cancer/National Cancer Institute of Canada trial on con- comitant and adjuvant TMZ in addition to radiotherapy as the first-line treatment of glioblastoma produced an increase in median survival from 12.1 to 14.6 months and an increase in the 2-year survival rate from 10 to 26% compared with radiotherapy alone (Stupp et al. 2005). Temozolomide is rapidly and completely absorbed after oral administration and undergoes spontaneous hydrolysis at physiological pH to its active metabolite 5-(3-methyl- triazeno)-imidazole-4-carboxamide. 5-(3-methyltriazeno)-im- idazole-4-carboxamide causes DNA damage by methylation of the O 6 position of guanine. However, DNA adducts different from O 6 -methylguanine might also be involved in the cytotoxicity induced by methylating agents and poly (ADP-ribose) polymerase (PARP) inhibitors may also sensi- tize tumour cells to TMZ-induced cell death (Tentori et al. 1998, 2001). Further, DNA lesions induced by alkylating Received August 22, 2005; revised manuscript received September 29, 2005; accepted October 13, 2005. Address correspondence and reprint requests to Dr Mirjam Hermisson, Department of General Neurology, University of Tu ¨bingen, School of Medicine, Hoppe-Seyler-Strasse 3, 72076 Tu ¨bingen, Germany. E-mail: mirjam.hermisson@uni-tuebingen.de Abbreviations used: MGMT: O 6 -methylguanine DNA methyltransf- erase; MSH, mutS homologue; O 6 -BG, O 6 -benzylguanine; PARP, poly(ADP-ribose) polymerase; PMS2, post-meiotic segregation increased 2; TMZ, temozolomide. Journal of Neurochemistry , 2006, 96, 766–776 doi:10.1111/j.1471-4159.2005.03583.x 766 Journal Compilation Ó 2006 International Society for Neurochemistry, J. Neurochem. (2006) 96, 766–776 Ó 2006 The authors