Apoptotic signaling pathways induced by nitric oxide in human lymphoblastoid cells expressing wild-type or mutant p53

Loss of p53 function by inactivating mutations results in abrogation of NO.-induced apoptosis in human lymphoblastoid cells. Here we report characterization of apoptotic signaling pathways activated by NO. in these cells by cDNA microarray expression and immunoblotting. A p53-mediated transcriptional response to NO. was observed in p53-wild-type TK6, but not in closely related p53-mutant WTK1, cells. Several previously characterized p53 target genes were up-regulated transcriptionally in TK6 cells, including phosphatase PPM1D (WIP1), oxidoreductase homolog PIG3, death receptor TNFRSF6 (Fas/CD95). and BH3-only proteins BBC3 (PUMA) and PMAIP1 (NOXA). NO. also modulated levels of several gene products in the mitochondria-dependent and death-receptor-mediated apoptotic pathways. Inhibitors of apoptosis proteins X-chromosome-linked inhibitor of apoptosis, cellular inhibitor of apoptosis protein-1, and survivin were significantly down-regulated in TK6 cells, but not in WTK1 cells. Smac release from mitochondria was induced in both cell types, but release of apoptosis-inducing factor and endonuclease G was detected only in TK6 cells. Fas/CD95 was increased, and levels of the antiapoptotic proteins Bcl-2 and Bcl-x/L were reduced in TK6 cells. Activation of procaspases 3, 8, 9, and 10, as well as Bid and poly(ADPribose) polymerase cleavage, were observed only in TK6 cells. NO. treatment did not alter levels of death receptors 4 and 5, Fas-associated death domain or proapoptotic Bax and Bak proteins in either cell line. Collectively, these data show that NO. exposure activated a complex network of responses leading to p53-dependent apoptosis via both mitochondrial and Fas receptor pathways, which were abrogated in the presence of mutant p53.