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(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.
Li
Chun-Qi
CQ
Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, USA.
Robles
Ana I
AI
Hanigan
Christin L
CL
Hofseth
Lorne J
LJ
Trudel
Laura J
LJ
Harris
Curtis C
CC
Wogan
Gerald N
GN
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D005609:Free Radicals / Q000737:chemistry / Q000494:pharmacology; D020869:Gene Expression Profiling; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D009154:Mutation; D009569:Nitric Oxide / Q000737:chemistry / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*
D005609:Free Radicals; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D009569:Nitric Oxide