Nitric oxide-induced genotoxicity, mitochondrial damage, and apoptosis in human lymphoblastoid cells expressing wild-type and mutant p53.
Nitric oxide (NO(*)) is mutagenic and, under appropriate conditions of exposure, also induces apoptosis in many in vitro and in vivo experimental models. Biochemical and cellular mechanisms through which NO(*) induces apoptosis are incompletely understood, but involve p53/mitochondria-dependent signaling pathways. In this study, we exposed human lymphoblastoid cells harboring either wild-type (TK6 cells) or mutant p53 (WTK-1 cells) to NO(*), delivered by diffusion through Silastic tubing. Cells were exposed for 2 h at constant rates of 100-533 nM/s, similar to levels estimated to occur in vivo in inflamed tissues. DNA double-strand breaks and fragmentation detected 8-48 h after NO(*) treatment were more extensive in TK6 cells than in WTK-1 cells, whereas NO(*)-induced mutant fractions in both HPRT and TK1 genes were significantly lower in TK6 cells than in WTK-1 cells (P < 0.01-0.05). Treatment of TK6 cells with NO(*) caused extensive apoptosis, but this response was delayed and greatly reduced in magnitude in WTK-1 cells. Mitochondrial membrane depolarization and cytochrome c release were induced in both cell types. However, elevation of apoptotic protease-activating factor-1 (Apaf-1) protein and reduction of X-chromosome-linked inhibitor of apoptosis (XIAP) protein were observed only in TK6 cells. These results indicate that p53 status is an important modulator of NO(*)-induced mutagenesis and apoptosis, and suggest that levels of the Apaf-1 and XIAP proteins, but not mitochondrial depolarization and cytochrome c release, are regulated by p53 in these human lymphoblastoid cells. Thus, Apaf-1 and XIAP may play important roles in the regulation of p53-mediated apoptotic responses.
Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Chun-Qi
CQ
Li
Laura J
LJ
Trudel
Gerald N
GN
Wogan
D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group / Q000378:metabolism; D004249:DNA Damage; D053938:DNA Fragmentation; D006412:Hematopoietic Stem Cells; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D019169:Jurkat Cells; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology*; D009569:Nitric Oxide / Q000494:pharmacology*; D014176:Protein Biosynthesis; D011506:Proteins; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D051636:X-Linked Inhibitor of Apoptosis Protein
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D009153:Mutagens; D011506:Proteins; D016159:Tumor Suppressor Protein p53; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D009569:Nitric Oxide