S-Nitrosation regulates the activation of endogenous procaspase-9 in HT-29 human colon carcinoma cells.

Abstract

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.

• Authors: Ji-Eun Kim, Steven R Tannenbaum
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 279 Issue 11 Pg. 9758-64 (Mar 12 2004) ISSN: 0021-9258 [Print] United States
• PMID: 14701803 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Enzyme Inhibitors
• Tumor Necrosis Factor-alpha
• Nitric Oxide
• Biotin
• Interferon-gamma
• DNA
• Nitric Oxide Synthase
• CASP3 protein, human
• CASP8 protein, human
• CASP9 protein, human
• Caspase 3
• Caspase 8
• Caspase 9
• Caspases
• Cysteine
• Ascorbic Acid

Topics

• Apoptosis
• Ascorbic Acid (metabolism)
• Biotin (metabolism)
• Blotting, Western
• Caspase 3
• Caspase 8
• Caspase 9
• Caspases (metabolism)
• Cell Death
• Colonic Neoplasms (enzymology)
• Cysteine (metabolism)
• DNA (metabolism)
• Enzyme Activation
• Enzyme Inhibitors (pharmacology)
• Enzyme-Linked Immunosorbent Assay
• HT29 Cells
• Humans
• Interferon-gamma (metabolism)
• Models, Biological
• Nitric Oxide (metabolism)
• Nitric Oxide Synthase (antagonists & inhibitors, metabolism)
• Nitrosation
• Precipitin Tests
• Time Factors
• Tumor Necrosis Factor-alpha (metabolism)