Apoptosis mediated by p53 in rat neural AF5 cells following treatment with hydrogen peroxide and staurosporine

Abstract

AF5 neural cells derived from fetal rat mesencephalic tissue were immortalized with a truncated SV40 LT vector lacking the p53-inactivating domain to maintain long-term cultures with a p53-responsive phenotype. This study examined p53 function in producing programmed cell death in propagating AF5 neural cells after exposure to hydrogen peroxide (H 2O 2) and the kinase inhibitor staurosporine (STSP). Concentration-dependent exposure of AF5 cells to 0–800 mM H 2O 2 and STSP at 0–1000 nM revealed increasing cytotoxicity from MTS cell viability assays. Apoptosis occurred at 400 mM H 2O 2 as evidenced by subG 1 DNA and Annexin V flow cytometry analyses and cellular immunofluorescence staining with propidium iodide, anti-Annexin V and DAPI. DNA fragmentation, caspase-3/7 activity and cytochrome c release into cytosol also confirmed H 2O 2-mediated apoptotic events. p53 protein levels were increased over 24 h by H 2O 2 in a coordinated fashion with mdm2 expression. p53 activation by H 2O 2 was evidenced by elevated Ser15 phosphorylation, increased luciferase p53 reporter activity and upregulation of the downstream p53 targets p21 waf1 and apoptotic proteins, bax, Noxa and PUMA. STSP exposure produced apoptosis demonstrated by DNA fragmentation, caspase-3/7 activity, cytochrome c release and over 24 h was accompanied by sustained increase in p53 and Ser15 phosphorylation, rise in p21 waf1 and bax and a transient increase in p53 reporter activity but without Annexin V binding. These findings demonstrate that AF5 cells undergo apoptosis in response to H 2O 2-mediated oxidative stress and signal pathway disruption by STSP that therefore would be useful in studies related to p53-dependent neuronal cell death and neurodegeneration.