Caspase-activation and induction of inducible nitric oxide-synthase during TNF alpha-triggered apoptosis.
Activation of the intracellular "death domain" (DD) of the 55kD-TNF alpha-receptor by TNF alpha initiates signal and effector cascades with pro- and anti-apoptotic function. Co-activation of the adjacent "NO-domain" is followed by induction of inducible nitric oxide-synthase (iNOS) and generation of nitric oxide radicals (NO.). Recently, we have shown NO.-generation to be essential for TNF alpha-induced apoptosis of various tumor cell lines. However, the impact of iNOS activation in relation to other promoters of apoptosis, such as the caspases, is still unclear. Caspase activation, iNOS induction and death rate were therefore investigated in TNF alpha-treated MCF-7 cells. Incubation with TNF alpha (+/- cycloheximide) led to activation of the caspase cascade and was followed by apoptosis. Simultaneously, TNF alpha stimulated induction of iNOS and generation of NO.. Caspase inhibitors DEVD-CHO, YVAD-cmk and YVAD-CHO effectively inhibited caspase activation and prevented apoptosis. Apoptotic cell death was decreased to a similar degree following inhibition of iNOS by L-nitro-arginine-methyl-ester (L-NAME). Cell death suppression by caspase inhibition did not result in reduced iNOS activity, as well as L-NAME-dependent prevention of apoptosis was not associated with caspase inactivation. Taken together, TNF alpha induces apoptosis in MCF-7 cells by initiating a two-sided effector pathway including iNOS-induction and activation of caspase 1- and 3-like proteases. Both mechanisms seem to be equally essential for the execution of the death program. The exact nature of their cooperation needs further clarification.
Binder
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C
Department of Haematology/Oncology, Georg-August-University, Göttingen, Germany.
Schulz
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M
Hiddemann
W
W
Oellerich
M
M
D000230:Adenocarcinoma / Q000473:pathology; D015703:Antigens, CD / Q000187:drug effects; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000473:pathology; D020169:Caspases / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D018124:Receptors, Tumor Necrosis Factor / Q000187:drug effects; D047888:Receptors, Tumor Necrosis Factor, Type I; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D015703:Antigens, CD; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; C110881:S-nitro-N-acetylpenicillamine; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D003513:Cycloheximide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D020169:Caspases; D010396:Penicillamine; D019331:NG-Nitroarginine Methyl Ester