Apoptotic Signaling Pathways Induced by Nitric Oxide in Human Lymphoblastoid Cells Expressing Wild-Type or Mutant p53

Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, USA.
Cancer Research (Impact Factor: 9.33). 06/2004; 64(9):3022-9. DOI: 10.1158/0008-5472.CAN-03-1880
Source: PubMed


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.

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    • "NO-mediated apoptotic processes are associated with p53 gene activation, which is essential for regulation of the cell cycle and/or apoptotic signaling occurring through p21Waf1/Cip1 or Bax [61, 62]. Here, we show upregulation of both p53 and p21 mRNA expression following treatment with MG/HG, which was blocked upon pretreatment with PTIO and NAC (Figure 6). "
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    ABSTRACT: Hyperglycemia and elevation of methylglyoxal (MG) are symptoms of diabetes mellitus (DM). In this report, we show that co-treatment of human mononuclear cells (HMNCs) with MG (5 microM) and high glucose (HG; 15 - 30 mM) induces apoptosis or necrosis. HG/MG co-treatment directly enhanced the reactive oxygen species (ROS) content in HMNCs, leading to decreased intracellular ATP levels, which control cell death via apoptosis or necrosis. Concentrations of 5 microM MG and 15 mM glucose significantly increased cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. In contrast, no apoptotic biochemical changes were detected in HMNCs treated with 5 microM MG and 25 mM glucose, which appeared to undergo necrosis. Pretreatment with nitric oxide (NO) scavengers inhibited apoptotic biochemical changes induced by 5 microM MG/15 mM glucose, and increased the gene expression levels of p53 and p21 involved in apoptotic signaling. The results collectively suggest that the treatment dosage of MG and glucose determines the mode of cell death (apoptosis vs. necrosis) of HMNCs, and that both ROS and NO play important roles in MG/HG-induced apoptosis.
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    • "Experiments in p53 null mice as well as in p53wt versus p53mut lymphoblastoid cells have demonstrated that NO induces apoptosis, and that this effect is dependent upon the presence of wild type p53 [68]. We detected elevated NO and apoptosis in MCF-7 cells treated with SXR activators, and increases in iNOS mRNA as well as mRNAs encoding p53 and p53-target genes in MCF-7 and ZR75-1 cell lines. "
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    • "The human PPM1D and mouse Ppm1d proteins are 83% homologous and are similarly induced in response to stress (2). PPM1D transcription is induced following exposure to many DNA damage-inducing agents, including NO (3), H2O2 (4) and MNNG (5). PPM1D was identified as a component of a 16-gene signature characterizing the p53-dependent DNA-damage response (6). "
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