1. ¹Department of Toxicology, University of Mainz, Mainz, Germany
2. ²Department of Microbiology, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil
3. ³Department of General Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, School of Medicine, Tübingen, Germany

Correspondence: Professor B Kaina, Department of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. E-mail: kaina@uni-mainz.de

Received 7 March 2006; Revised 22 May 2006; Accepted 23 May 2006; Published online 3 July 2006.

Abstract

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O⁶-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O⁶-benzylguanine showed that, in gliomas, the apoptotic signal originates from O⁶-methylguanine (O⁶MeG) and that repair of O⁶MeG by MGMT prevents apoptosis. We further demonstrate that O⁶MeG-triggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O⁶MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O⁶MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by H2AX formation. Glioma cells mutated in DNA-PK_cs, which is involved in non-homologous end-joining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.