Apaf-1 deficient mouse fibroblasts are resistant to MNNG and MMS-induced apoptotic death without attenuation of Bcl-2 decline.
Simple alkylating agents induce cell death by activating the apoptotic pathway. In rodent fibroblasts, apoptosis triggered by DNA methylation lesions is executed via the mitochondrial damage pathway. Here, we studied cell death induced by the methylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in mouse fibroblasts wild-type (wt) and deficient for Apaf-1 (apaf-1 knockout cells). Apaf-1 is an essential component of the apoptosome complex that becomes activated upon cytochrome c release from mitochondria. We show that apaf-1 knockout cells are more resistant to the cytotoxic effect (as measured by WST assay) of methylating agents. This is due to a reduced frequency of apoptosis and necrosis, which appeared at late times (72 h) after exposure. Caspase-3 and -9 became activated in wt but not in apaf-1 knockout cells, whereas caspase-8 was not activated in either one of the lines. The data indicate that MMS and MNNG-induced cell death in mouse fibroblasts is triggered by the activation of the mitochondrial damage pathway and that apaf-1 is required for eliciting this response. A hallmark of mitochondria-mediated apoptosis induced upon alkylation is decline of Bcl-2 protein level. Bcl-2 decline occurred to similar extent in wt and apaf-1 knockout cells suggesting that it is an upstream event in MMS and MNNG-induced apoptosis triggered by non-repaired DNA damage.
Tomicic
Maja T
MT
Institute of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany.
Christmann
Markus
M
Fabian
Kerstin
K
Kaina
Bernd
B
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics / Q000502:physiology*; D002461:Cell Line, Transformed; D008741:Methyl Methanesulfonate / Q000633:toxicity*; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D051379:Mice; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*
C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D019253:Proto-Oncogene Proteins c-bcl-2; D008769:Methylnitronitrosoguanidine; D008741:Methyl Methanesulfonate