Cell Death and Differentiation (1999) 6, 937 ± 942 ã 1999 Stockton Press All rights reserved 13509047/99 $15.00 http://www.stockton-press.co.uk/cdd Review NO: an inhibitor of cell death Limin Liu1 and Jonathan S Stamler*,1,2 process in particular, are among NO's major biological functions. 1 Howard Hughes Medical Institute, Duke University Medical Center, Durham Inhibition of apoptosis by NO was first demonstrated in B NC, 27710, USA lymphocytes5,6 (although a protective effect of NO had 2 Department of Medicine and Cell Biology, Duke University Medical Center, been reported in rat liver7 and rat neurons8). Anti-apoptotic Durham NC, 27710, USA NO effects were subsequently shown in a variety of cells, * corresponding author: JS Stamler, Duke University Medical Center, MSRB Room 321 (Box 2612), Durham N.C. 27710, USA. including T cells, hepatocytes, endothelial cells, neurons, tel: 919-684-6933; fax: 919-684-6998; ovarian follicle cells, eosinophils, thymocytes and embryo- e-mail: [email protected] nic kidney cells (see Table 1). A remarkable feature of these studies is that NO inhibition of cell death is largely independent of the apoptotic stimulus: protection is seen Received 28.6.99; accepted 15.7.99 against anti-Fas antibody, tumor necrosis factor a (TNFa) Edited by G Melino and ceramide on the one hand, and to ultraviolet radiation, growth factor withdrawal and NO synthase blockade on the other hand. Moreover, these salutary effects are broadly Abstract conferred by multiple sources of NO-related activity, Nitric oxide (NO) or related molecules of both endogenous and including constitutive and cytokine-inducible NO exogenous origin inhibit programmed cell death in a variety of synthases, overexpression of NOS and several classes of cells and tissues. This general protective function is largely NO donors. This review will concentrate on the antiapopto- independent of the apoptotic stimulus. S-nitrosylation of the tic effects of endogenous NOS activity. catalytic-site cysteine of caspases is a well-established and possibly widespread mechanism of enzyme inhibition that Anti-apoptotic effects of NO protects from cell death. However, NO may inhibit apoptosis by additional mechanisms. The physiological and pathologi- Lymphocytes cal significance of NO's anti-apoptotic activity remains to be Epstein-Barr virus (EBV)-infected as well as EBV negative determined in most cases. Burkitt's lymphoma cell lines express iNOS (`inducible' NOS) constitutively.6 Inhibition of such iNOS activity with NG- Keywords: nitric oxide; apoptosis; caspase S-nitrosylation monomethyl-L-arginine (L-NMMA) or by substrate depletion resulted in more than threefold increases in the rate of Abbreviations: ActD, actinomycin D; Casp, caspase; Cyt, spontaneous apoptosis, which in these cells was *3% over 6 cytochrome; DETA/NO, (Z)-1-[N-(2-aminoethyl)-N-(2-ami- several days. This increase in apoptosis was partially noethyl)diazen-1-ium-1,2-diolate; FADD, Fas-associating protein blocked by excess L- but not D-arginine and was abrogated with death domain; GSNO, S-nitrosoglutathione; L-NMMA, NG- by concomitant application of the NO donor S-nitroso- monomethyl-L-arginine; NGF, nerve growth factor; NOR3, (+)-(E)- penicillamine. Thus, constitutive low level NOS activity 4-ethyl-2-[(E)-hydroxyimino]-5-nitroso-3-hexenamide; NTR, neu- protects human lymphocyte cell lines from spontaneous rotrophin receptor; SIN-1, 3-morpholinosydnonimine; SNAP, S- apoptosis. Constitutive expression of iNOS also blocked nitroso-N-acetyl-penicillamine; SNP, sodium nitroprusside apoptosis in primary B cell cultures from patients with B-cell chronic lymphocytic leukemia (B-CLL),9 a disorder which results principally from decreased cell death rather than increased cell proliferation.10 In particular, application of the Introduction NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME), Apoptosis is a highly organized process that is characterized either alone or in combination with anti-Fas antibody, by caspase activation, chromatin condensation and DNA substantially increased apoptotic DNA fragmentation in B- fragmentation.1 It functions to remove excess, autoreactive, CLL cells. Thus, endogenous NO is probably anti-apoptotic in damaged or infected cells in development, immune reactions vivo, and might contribute to the extended survival of B-CLL and many other biological processes.2 Apoptosis is tightly cells that is characteristic of the disease. It is not regulated; too little may cause cancer and autoimmune inconceivable that the anti-apoptotic activity of NOS may diseases while too much can manifest in fulminant hepatitis, contribute to the abnormal survival or pathologic proliferation heart failure or neurodegenerative disease.3,4 The inhibition of of cells and, more generally, to accumulation of mutations and such programmed cell death by nitric oxide (NO) or related development of tumors. Indeed, NOS is often highly molecules is a common and widespread phenomenon that expressed in malignant cells.11,12 has attracted considerable interest in the last few years. NOS not only protects B cells against spontaneous Regulation of apoptosis in general, and attenuation of the apoptosis but also from apoptosis induced by Fas NO: an inhibitor of cell death LLiuandJSStamler 938 activation.9,13 The same apparently holds true for T cells antibody, overexpression of FADD or caspase-8 and by and monocytic cell lines.13,14 However, the NO synthases various alternative stimuli (see Table 1).6,16,17 The involved and the mechanism of NO action may be quite biological relevance of this antiapoptotic effect, however, different in each case (Table 1). For instance, constitutive is not known. iNOS expression was detected in the B cells, monocytic cells and two T cell lines,9,13 whereas nNOS was co- expressed with iNOS in Jurkat T cells15 and only eNOS Hepatocytes expression was detected in allogeneically activated gdT The protective effects of NO in the liver were discovered 9 cells.14 years ago.7 Mice were injected with Corynebacterium parvum NO production was not detected in splenic B lympho- and then lipopolysaccharide (LPS)7 in a model of acute cytes from wild-type mice,5 and addition of a NOS inhibitor hepatic necrosis. The LPS resulted in a substantial increase in (nitromethylarginine) to the culture medium did not change NOx production, which clearly protected from liver damage. the apoptotic response. Nevertheless, exogenous NO or Specifically, suppression of NO production with L-NMMA NO donors significantly delayed and inhibited both markedly exacerbated hepatic necrosis and other markers of spontaneous and antigen-induced apoptosis in these injury. It has been more recently reported that L-NMMA or L- cells.5 Moreover, exogenous NO substantially repressed NAME but not the iNOS-specific inhibitor L-N6-(1-imi- apoptosis in various T cells or B cells activated by anti-Fas noethyl)lysine (L-NIL), potentiate LPS-induced necrosis in Table 1 Inhibition of apoptosis by NO Cell type NO source Apoptosis induced by Target or pathway* References Lymphocytes B lymphoma cell lines iNOS; SNAP L-NMMA; A23187; Fas+ (7) Casp S-nitrosylation; 6, 13, 15 L-NMMA cGMP-independent B cell chronic lymphocytic iNOS L-NMMA+Fas Unknown 9 leukemia cells Mouse splenic B cells SIN-1; NO; isosorbide dinitrate spontaneous; soluble MHC-I (+) cGMP; (7) decline in 5 Bcl-2 T cell lines iNOS; SNAP; SNP; GSNO Fas+L-NMMA; FADD (7) Casp S-nitrosylation; 13, 15, 16, 17 or Casp8 over expression cGMP-independent Human gdT cells Allogenetically activated eNOS Fas (+) cGMP; (+) ceramide 14 Rodent hepatocytes Cytokine-induced or Spontaneous; TNFa (Fas)+ (7) Casp S-nitrosylation; 20, 23 and liver overexpressed iNOS; SNAP; ActD; TNFa+galactosamine; (+) cGMP; (7) Bcl-2 7, 18, 58 V-PYRRO/NO LPS; hyperthermia cleavage; (7) Cyt c 54, 55 release; (+) HSP70 Endothelial cells Human umbilical vein Shear stress-stimulated NOS; TNFa; OxLDL; oxidative (7) Casp S-nitrosylation; 29, 33, 34 SNP; SNAP; PAPA NONOate stress; angiotensin II; cGMP-independent 27, 28, 35 caspase-3 overexpression Rat aorta Cytokine-induced iNOS; UVA (+) Bcl-2; (7) Bax 38 DETA/NO Sheep pulmonary artery Overexpressed iNOS LPS (7) Casp 30, 31 Porcine aorta DETA/NO; SNAP LPS then arsenite cGMP-independent 32 Neurons Rat embryonic motor eNOS L-NMMA (+) cGMP 40 neuron Differentiated PC12 cell NOS; NO donor NOS inhibitor; NGF withdrawal Unknown 41 Neuroblastoma NB-39-nu SNAP; NOR3 vincristine (7) Casp; cGMP- 44, 59 independent Neuroblastoma SK-N-BE SNAP P75NTR overexpression; cGMP-independent 43 ceramide Human eosinophils Activated U937 cells; SNAP; Fas; spontaneous (+) cGMP 50, 51 ceramide; azide; hydroxylamine U937 monocytic cell line iNOS; GSNO; SIN-1 Fas+L-NMMA (7) Casp; cGMP- 13, 60 independent Rat thymocyte GSNO (2 mM) Dexamethasone Unknown 64 Rat ovarian follicle NOS induced by IL-1b; SNP Spontaneous; L-NMMA Probably cGMP-related 49 Human kidney KEK-293 cell nNOS overexpression Caspase-1 overexpression (7) Casp 57 MCF-7 breast cancer cell SNAP TNFa+ActD (7) Casp; (7) Bcl-2 56 cleavage; (7) Cyt c Abbreviations: ActD, actinomycin D; Casp; caspase; Cyt, cytochrome; DETA/NO, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)diazen-1-ium-1,2-diolate; GSNO, S- nitrosoglutathione; L-NMMA, NG-monomethyl-L-arginine; NGF, nerve growth factor; NOR3, (+)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitroso-3-hexenamide; NTR, neurotrophin receptor; SIN-1, 3-morpholinosydnonimine; SNAP, S-nitroso-N-acteyl-penicillamine; SNP, sodium nitroprusside, UVA, ultraviolet light A. *(+), increase in level; (7), inhibition involving/via. NO: an inhibitor of cell death LLiuandJSStamler 939 rat liver.18 This finding has been interpreted in terms of a endothelial survival. It was also shown that cutaneous protective role of eNOS but not iNOS in inflammation-induced iNOS expression and NO production were activated by UV necrosis. In contrast, both L-NAME and L-NIL increased irradiation.39 Whether this UV-induced NO production levels of apoptosis in LPS-treated livers. LPS, L-NAME and L- protects against the apoptotic effects of UV irradiation in NIL do not induce apoptosis when given alone.