BIOLOGY CONTRIBUTION DIFFERENTIAL RADIOSENSITIZING POTENTIAL OF TEMOZOLOMIDE IN MGMT PROMOTER METHYLATED GLIOBLASTOMA MULTIFORME CELL LINES KRISTA A. VAN NIFTERIK, M.SC.,* y JAAP VAN DEN BERG, B.S.,* LUKAS J. A. STALPERS, M.D., PH.D., z M. VINCENT M. LAFLEUR,PH.D.,* SIEGER LEENSTRA, M.D., PH.D., x BEN J. SLOTMAN, M.D., PH.D.,* THEO J. M. HULSEBOS,PH.D., y AND PETER SMINIA,PH.D.* * Department of Radiation Oncology, VU University Medical Center, and Departments of y Neurogenetics, z Radiotherapy, and x Neurosurgery, Academic Medical Center, Amsterdam, The Netherlands Purpose: To investigate the radiosensitizing potential of temozolomide (TMZ) for human glioblastoma multiforme (GBM) cell lines using single-dose and fractionated g-irradiation. Methods and Materials: Three genetically characterized human GBM cell lines (AMC-3046, VU-109, and VU-122) were exposed to various single (0–6 Gy) and daily fractionated doses (2 Gy per fraction) of g-irradiation. Repeated TMZ doses were given before and concurrent with irradiation treatment. Immediately plated clonogenic cell- survival curves were determined for both the single-dose and the fractionated irradiation experiments. To estab- lish the net effect of clonogenic cell survival and cell proliferation, growth curves were determined, expressed as the number of surviving cells. Results: All three cell lines showed MGMT promoter methylation, lacked MGMT protein expression, and were sensitive to TMZ. The isotoxic TMZ concentrations used were in a clinically feasible range of 10 mmol/L (AMC-3046), 3 mmol/L (VU-109), and 2.5 mmol/L (VU-122). Temozolomide was able to radiosensitize two cell lines (AMC 3046 and VU-122) using single-dose irradiation. A reduction in the number of surviving cells after treatment with the combination of TMZ and fractionated irradiation was seen in all three cell lines, but only AMC 3046 showed a radiosensitizing effect. Conclusions: This study on TMZ-sensitive GBM cell lines shows that TMZ can act as a radiosensitizer and is at least additive to g-irradiation. Enhancement of the radiation response by TMZ seems to be independent of the epigenetically silenced MGMT gene. Ó 2007 Elsevier Inc. Human GBM cell lines, Temozolomide, MGMT, Radiosensitization, Fractionated irradiation. INTRODUCTION Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor in adults. Despite standard treatment consisting of surgery and postoperative radiother- apy, patient survival remains poor, owing to radio- and che- moresistance of the tumor. Recently it was demonstrated that the combination of temozolomide (TMZ) with standard daily fractionated irradiation therapy followed by adjuvant TMZ improves the prognosis (1). Since then, TMZ has become part of the standard therapy for patients with newly diagnosed GBM. The benefit of TMZ is most prominent for tumors with a methylated O 6 -methylguanine-DNA methyltransferase (MGMT) promoter. Methylation of the MGMT promoter has been associated with longer overall survival of GBM patients treated with radiotherapy and TMZ compared with radiotherapy alone (2, 3). However, this study also showed relatively long-term survivors—both in the methylated and unmethylated GBM groups—treated with radiotherapy plus TMZ (3). This might indicate interaction between TMZ and irradiation. Temozolomide is a chemotherapeutic prodrug that trans- forms under physiologic conditions into its active unstable methylating metabolite, 5-(3-methyl-1-triazeno)imidazole- 4-carboxamide (MTIC). Methylation of the DNA by MTIC results in O 6 -methylguanine adducts, which are considered to be responsible for the cytotoxic effect of TMZ (4–6). O 6 -methylguanine adducts can result in futile attempts of the mismatch repair system, leading to DNA double-strand breakage and eventually cell death (7, 8). Reprint requests to: Peter Sminia, Ph.D., Division of Radiobiology, Department of Radiation Oncology, VU University Medical Center, Bldg: Faculty of Medicine, Room J-392, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Tel: (+31) 20-4448355; Fax: (+31) 20-4448285; E-mail: p.sminia@vumc.nl Supported by Dutch Cancer Society Grant No. VU 2000-2149. Conflict of interest: none. Acknowledgments—The authors thank J.C.M. Vos, R.J. van Andel, R.H. Wessel, L.E. Wedekind, and N. Ameziane for their technical assistance; and G.P. Bieger-Smith for correction of the English text. Temozolomide was generously provided by Schering-Plough RS. Received April 25, 2007, and in revised form June 28, 2007. Accepted for publication July 30, 2007. 1246 Int. J. Radiation Oncology Biol. Phys., Vol. 69, No. 4, pp. 1246–1253, 2007 Copyright Ó 2007 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/07/$–see front matter doi:10.1016/j.ijrobp.2007.07.2366