Abstract
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(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.
Highlights
NO1⁄7 has been shown to initiate apoptotic cell death in many in vitro and in vivo experimental models [1, 2]; the pathways involved are still not completely understood
We previously found that NO1⁄7 treatment resulted in mitochondrial membrane depolarization and cytochrome c release in a pair of closely related human lymphoblastoid cells harboring either wild-type (TK6 cells) or mutant p53 (WTK1 cells), but the apoptotic response was substantially different in the two cell types [24]
Our mRNA and protein analyses of lymphoblastoid cells undergoing apoptosis indicate that both mitochondria- and receptormediated pathways of apoptosis are activated by exposure to NO1⁄7 and modulated by p53
Summary
NO1⁄7 has been shown to initiate apoptotic cell death in many in vitro and in vivo experimental models [1, 2]; the pathways involved are still not completely understood. Wild-type p53 is a critical cellular gatekeeper for growth and division that mediates DNA damage-induced cell-cycle arrest and apoptosis [5] through, at least in part, the transcriptional activation of p21WAF1, an inhibitor of cyclin-dependent kinases [6]. Biochemical sion of these and other proteins that control mitochondrial membrane permeability and, the release of mitochondrial proteins during apoptosis [23]. We explore the involvement of the Bcl-2 family of proteins in NO1⁄7-induced cell death as well as release of the mitochondrial proapoptotic factors Smac, AIF, and endonuclease G. AIF and endonuclease G translocate from mitochondria to nuclei during apoptosis, where they induce caspase-independent chromatin condensation and large-scale (50 kb) DNA cleavage (26 –28)
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