title: Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine.
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 O(6)-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O(6)-benzylguanine showed that, in gliomas, the apoptotic signal originates from O(6)-methylguanine (O(6)MeG) and that repair of O(6)MeG by MGMT prevents apoptosis. We further demonstrate that O(6)MeG-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. O(6)MeG-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. O(6)MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by gammaH2AX 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.
authors.affiliation: Department of Toxicology, University of Mainz, Mainz, Germany.
authors.forename: W P
authors.initials: WP
authors.lastname: Roos
authors.forename: L F Z
authors.initials: LF
authors.lastname: Batista
authors.forename: S C
authors.initials: SC
authors.lastname: Naumann
authors.forename: W
authors.initials: W
authors.lastname: Wick
authors.forename: M
authors.initials: M
authors.lastname: Weller
authors.forename: C F M
authors.initials: CF
authors.lastname: Menck
authors.forename: B
authors.initials: B
authors.lastname: Kaina
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology*; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage / Q000187:drug effects*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D053222:Fas Ligand Protein / Q000378:metabolism; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D005434:Flow Cytometry; D005910:Glioma / Q000378:metabolism / Q000473:pathology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D000077204:Temozolomide; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D018906:Antineoplastic Agents, Alkylating; C505533:FADD protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D008769:Methylnitronitrosoguanidine; D006147:Guanine; D003606:Dacarbazine; C008449:O-(6)-methylguanine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D020169:Caspases; D000077204:Temozolomide