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-ouly 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(ADP-ribose) 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.