The cell death response to the ROS inducer, cobalt chloride, in neuroblastoma cell lines according to p53 status

Abstract

Cobalt chloride (CoCl 2), a hypoxia-mimetic agent, induces reactive oxygen species (ROS) generation, leading to cell death. Divergent data have been reported concerning p53 implication in this apoptotic mechanism. In this study, we studied cobalt-induced cell death in neuroblastoma cell lines carrying wild-type (WT) p53 (SHSY5Y) and a mutated DNA-binding domain p53 [SKNBE(2c)]. CoCl 2 induced an upregulation of p53, p21 and PUMA expression in WT cells but not in SKNBE(2c). In SHSY5Y cells, p53 serine-15 phosphorylation appeared early (6 h) in the mitochondria, and also in the nucleus after 12 h. In contrast, in SKNBE(2c) cells, the slight nuclear signal disappeared with CoCl 2 treatment. In SHSY5Y cells, a mitochondrial pathway dependent on caspases [collapse of mitochondrial transmembrane potential (ΔΨ mt), caspase 3 and 9 activation], was activated in a time-dependent manner. SKNBE(2c) cells exhibited a delay in the cell death executive phase linked to a caspase-independent pathway, involving apoptosis inducing factor nuclear translocation, but also an autophagic process attested by LC3-II expression and cathepsin-B activation. The downregulation of p53 in SHSY5Y cells by siRNA induced a cell death pathway related to the one observed in SKNBE(2c) cells. Finally, CoCl 2 induced time-dependent canonical p53 mitochondrial apoptosis in the WT p53 cell line, and caspase-independent cell death in cells with a mutated or KO p53.