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 0 -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 MeGtriggered 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 MeGinduced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by cH2AX formation. Glioma cells mutated in DNA-PK cs , which is involved in non-homologous endjoining, 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.

p53 promotes cellular survival in a context-dependent manner by directly inducing the expression of haeme-oxygenase-1

“…Similarly, p53 was shown to function as a survival factor in the gastrointestinal tract under conditions of severe irradiation, which was attributed to increased sensitivity of vascular endothelial cells (Komarova et al, 2004;Burdelya et al, 2006). Furthermore, other reports have identified conditions in which absence of p53 led to elevated apoptosis, but a direct causal role for p53 in providing survival signals has not been described (Lassus et al, 1996;Lackinger and Kaina, 2000;Hermisson et al, 2006;Batista et al, 2007;Roepke et al, 2007;Roos et al, 2007). Notwithstanding these occasional reports, the major observations that p53 activation often leads to apoptosis or DNA repair have been overwhelming, and hence, have overshadowed the exploration of the survival-promoting properties of p53.…”

“…For example, absence of p53 has been shown to promote taxol-mediated death owing to failure of cell arrest in the G 1 phase of the cell cycle, and hence, allowing cells to accumulate in the G 2 /M phase where they undergo mitotic cell death (Vikhanskaya et al, 1998). In addition, cells with wild-type p53 have been shown to be more resistant to cytotoxic drug treatment in some cases (Hermisson et al, 2006;Batista et al, 2007;Roos et al, 2007). Other reports have also shown that absence of p53 leads to sensitization to UV irradiation and alkalyting agents in mouse embryonic fibroblasts (MEFs), and to thymoquinone-induced apoptosis in human cells (Lackinger and Kaina, 2000;Roepke et al, 2007).…”

Mouse embryonic stem cells are hypersensitive to apoptosis triggered by the DNA damage O6-methylguanine due to high E2F1 regulated mismatch repair

“…the mechanism observed in other cell systems in response to alkylating agents, 24,25 the following marker proteins were assayed: phosphorylated H2AX, p53, p21, Fas/CD95/Apo-1, Bcl-2, Bcl-2-associated X protein (Bax), cytochrome c, active fragments of caspase-7 and caspase-3. As shown in Figure 6, in the nucleus of ES (R1) cells histone H2AX becomes phosphorylated after MNNG treatment, which was was performed using an E2F1-specific antibody.…”

“…Western blot analysis was performed as described. 25 Antibodies used were anti-P53 and anti-cytochrome c (Oncogene), anti-Fas, antiBcl-2, anti-Bax and ERK2 (Santa Cruz Biotechnology Inc.), anti-caspase-3, anticaspase-7 and anti-phospho-Rb (ser807/811) (Cell Signaling technology), anti-Rb (Pharmingen), MSH2 (Calbiochem) and MSH6 (Transduction Laboratories).…”

Glioblastoma multiforme: the role of DSB repair between genotype and phenotype

“…Notably, cells with mutated DNA-PKcs were more sensitive to TMZ-induced apoptosis. Since both p53 wild-type and p53 mutant glioma cells undergo apoptosis in response to TMZ, TP53 is not necessarily required for TMZ-induced apoptosis (Roos et al, 2007). Inhibition of DNA-PKcs may offer a way to improve both the efficiency of TMZ therapy and radiotherapy.…”