S-Nitrosation regulates the activation of endogenous procaspase-9 in HT-29 human colon carcinoma cells.
Nitric oxide-mediated signals have been suggested to regulate the activity of caspases negatively, yet literature has provided little direct evidence. We show in this paper that cytokines and nitric-oxide synthase (NOS) inhibitors regulate S-nitrosation of an initiator caspase, procaspase-9, in a human colon adenocarcinoma cell line, HT-29. A NOS inhibitor, N(G)-methyl-l-arginine, enhanced the tumor necrosis factor-alpha (TNF-alpha)-induced cleavage of procaspase-9, procaspase-3, and poly-(ADP-ribose) polymerase, as well as the level of apoptosis. N(G)-Methyl-l-arginine, however, did not affect the cleavage of procaspase-8. These results suggest that nitric oxide regulates the cleavage of procaspase-9 and its downstream proteins and, subsequently, apoptosis in HT-29 cells. Labeling S-nitrosated cysteines with a biotin tag enabled us to reveal S-nitrosation of endogenous procaspase-9 that was immunoprecipitated from the HT-29 cell extracts. Furthermore, the treatment with TNF-alpha, as well as NOS inhibitors, decreased interferon-gamma-induced S-nitrosation in procaspase-9. Our results show that S-nitrosation of endogenous procaspase-9 occurs in the HT-29 cells under normal conditions and that denitrosation of procaspase-9 enhances its cleavage and consequent apoptosis. We, therefore, suggest that S-nitrosation regulates activation of endogenous procaspase-9 in HT-29 cells.
Kim
Ji-Eun
JE
Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Tannenbaum
Steven R
SR
D017209:Apoptosis; D001205:Ascorbic Acid / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death; D003110:Colonic Neoplasms / Q000201:enzymology*; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019073:HT29 Cells; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D008954:Models, Biological; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D015538:Nitrosation; D011233:Precipitin Tests; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004791:Enzyme Inhibitors; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D001710:Biotin; D007371:Interferon-gamma; D004247:DNA; D019001:Nitric Oxide Synthase; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003545:Cysteine; D001205:Ascorbic Acid