Apoptosis in UV-C light irradiated p53 wild-type, apaf-1 and p53 knockout mouse embryonic fibroblasts: interplay of receptor and mitochondrial pathway.
Mouse embryonic fibroblasts (MEFs) deficient for the transcription factor p53 are hypersensitive to UV-C light. They also show a reduced recovery from UV-C induced replication blockage and are unable to repair UV-C photoproducts. In this study, we utilized wild-type (wt), Apaf-1 deficient (apaf-1(-/-)) and p53 deficient (p53(-/-)) MEFs in order to elucidate the role of non-repaired UV-C lesions in apoptotic signalling. Corresponding with the cellular sensitivity determined by the WST assay, p53(-/-) cells displayed the highest level of apoptosis, whereas wt cells showed moderate apoptosis after UV-C irradiation. Apaf1(-/-) cells were most resistant. In wt cells apoptosis was executed both via the mitochondrial and the receptor-mediated pathway, as shown by Bcl-2 decline, induction of fasR and activation of caspases-3,8,9. In apaf-1(-/-) (p53(+/+)) cells, the mitochondrial pathway was blocked downstream of Bcl-2, indicating that in this case apoptosis was mediated via the induction of fasR and caspase-3,8 activation. In p53 deficient cells, non-repaired UV-C induced DNA lesions triggered sustained up-regulation of fas ligand (fasL) mRNA, which was not seen in wt and apaf-1(-/-) cells. Therefore, in p53(-/-) MEFs, the receptor/ligand triggered pathway appeared to be dominant. This was confirmed by significant reduction of apoptosis after DN-FADD transfection. As opposed to wt and apaf-1(-/-) cells, p53 deficient MEFs showed no induction of Fas receptor and no Bcl-2 decline. Nevertheless, the resulting caspase-8 and -3 activation was stronger compared to wt and apaf-1(-/-) cells. The data indicate that UV-C light activates in MEFs both the Fas (CD95, Apo-1) receptor and the mitochondrial damage pathways. In p53(-/-) cells, however, the high level of non-repaired DNA damage forces signalling by fasL upregulation, leading to enhanced UV-C-induced apoptosis.
Tomicic
M T
MT
Department of Toxicology, University of Mainz, Germany.
Christmann
M
M
Kaina
B
B
D000818:Animals; D017209:Apoptosis / Q000528:radiation effects*; D053477:Apoptotic Protease-Activating Factor 1 / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D004622:Embryo, Mammalian / Q000166:cytology / Q000201:enzymology / Q000528:radiation effects; D004789:Enzyme Activation / Q000528:radiation effects; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000201:enzymology / Q000528:radiation effects; D005799:Genes, Dominant; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism* / Q000528:radiation effects; D009336:Necrosis; D011740:Pyrimidine Dimers / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013816:Thermodynamics; D015533:Transcriptional Activation / Q000528:radiation effects; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000378:metabolism*; D014466:Ultraviolet Rays
C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D053200:Fas-Associated Death Domain Protein; D011740:Pyrimidine Dimers; D011956:Receptors, Cell Surface; D016159:Tumor Suppressor Protein p53; D020169:Caspases