Induction of inducible nitric oxide synthase is an essential part of tumor necrosis factor-alpha-induced apoptosis in MCF-7 and other epithelial tumor cells.

TNF-alpha-induced cytotoxicity is mediated by the intracellular "death domain" of the 55-kDa TNF-alpha receptor and has been demonstrated to be coupled with induction of inducible nitric oxide synthase (iNOS), leading to generation of nitric oxide radicals (NO*). Because it is still widely unknown, to what extent NO* participates in the execution of TNF-alpha-induced apoptosis, NO* production, iNOS expression, and enzyme activity in relation to TNF-alpha-induced apoptotic cell death were investigated in the human breast cancer cell line MCF-7 and various other malignant cell lines. Incubation with TNF-alpha led to induction of iNOS mRNA and protein as well as enhancement of NOS activity. Augmented synthesis of NO2, the stable end product of NO* generation, was significantly correlated with augmented rates of cell death. Measurement of TNF-alpha-triggered production of reactive oxygen species (ROS) suggested a major role for NO* within the generated oxygen radicals. Dying cells showed characteristic features of apoptosis. Addition of cycloheximide (CX) enhanced apoptotic cell death by increasing iNOS activity. L-Nitro-arginine-methylester (L-NAME), a competitive NOS-inhibitor, and iNOS antisense oligonucleotides effectively prevented NO2 generation and apoptosis. Evaluation of iNOS expression during TNF-alpha-induced cell death in various malignant cell lines demonstrated iNOS positivity for all TNF-alpha-sensitive cells. The only primarily resistant line was iNOS negative. In a resistant variant of MCF-7, iNOS mRNA was still detectable; however, treatment with TNF-alpha did not enhance NOS activity. TNF-alpha sensitivity and NO2 production were completely restored by the addition of CX. Taken together, iNOS induction plays an essential role in TNF-alpha-induced apoptosis of the investigated cell lines. Further studies are necessary to define the impact of NO* in relation to other specific effectors of apoptosis such as the caspases.

Binder C ，Schulz M ，Hiddemann W ，Oellerich M ... -  《LABORATORY INVESTIGATION》

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 C ，Schulz M ，Hiddemann W ，Oellerich M ... -  《ANTICANCER RESEARCH》

Bryostatin-1 and IFN-gamma synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages.

Bryostatin-1 (Bryo) is a nontumor-promoting protein kinase C modulator that has been shown to have both in vitro and in vivo activity against several murine and human tumors. In this study, we investigated the effects of Bryo on nitric oxide production, measured as accumulated nitrite (NO2-) in culture supernatant, and inducible nitric oxide synthase (iNOS) gene expression in the murine macrophage cell line ANA-1. ANA-1 macrophages did not produce NO2- or iNOS mRNA constitutively, and very little or no NO2- or iNOS mRNA were detectable upon exposure to IFN-gamma. Bryo, although ineffective alone, and IFN-gamma synergized to produce high levels of NO2- and iNOS mRNA. The activity of Bryo was evident at a concentration of 0.1 ng/ml and reached its maximum at 1 ng/ml. The effects of Bryo were time dependent because expression of iNOS mRNA was detectable as early as 6 h and increased through 24 h. Analyses of the molecular mechanisms involved indicate that Bryo and IFN-gamma mainly regulate iNOS gene expression posttranscriptionally through stabilization of iNOS mRNA. Experiments designed to investigate the role of tumor necrosis factor alpha (TNF-alpha) in NO2- production by Bryo- and IFN-gamma-activated macrophages revealed that ANA-1 macrophages expressed low levels of TNF-alpha mRNA constitutively that were not augmented in the presence of IFN-gamma. However, Bryo alone augmented the TNF-alpha mRNA expression, which was only slightly increased with the addition of IFN-gamma. A polyclonal antibody to TNF-alpha was able to completely neutralize TNF-alpha secreted in either medium or Bryo plus IFN-gamma-treated cultures. Neutralizing concentrations of anti-TNF-alpha antibody suppressed the Bryo plus IFN-gamma-induced NO2- production approximately by 50%, suggesting that NO2- produced by Bryo plus IFN-gamma-treated ANA-1 macrophages may involve both TNF-alpha-dependent and TNF-alpha-independent mechanisms. Overall, these findings provide the first evidence that Bryo and IFN-gamma can synergize for the induction of NO2- production as well as iNOS gene expression and show the involvement of posttranscriptional mechanisms in the induction of iNOS mRNA.

Taylor LS ，Cox GW ，Melillo G ，Bosco MC ，Espinoza-Delgado I ... -  《CANCER RESEARCH》

Involvement of iNOS-dependent NO production in the stimulation of osteoclast survival by TNF-alpha.

Osteoclasts, cells primarily responsible for bone resorption, differentiate from hematopoietic progenitor cells under the influence of various hormones, cytokines, and differentiation factors. Once fully differentiated, osteoclasts rapidly die in the absence of any survival factor. We have previously shown that tumor necrosis factor alpha (TNF-alpha) promotes the survival of differentiated osteoclasts. The expression of inducible nitric oxide synthase (iNOS) and consequent NO production is often stimulated under inflammatory conditions. In this study, we found that TNF-alpha, but not receptor activator of nuclear factor kappa B ligand and interleukin 1, increased the expression of iNOS both at the mRNA and protein levels. Subsequently, an enhanced NO level was detected both inside the cells and the culture medium of TNF-alpha-stimulated osteoclasts. Blocking NOS activity with L-NAME prevented TNF-alpha-induced NO generation by osteoclasts and the osteoclast survival stimulated by TNF-alpha. The iNOS selective inhibitor L-NIL also suppressed TNF-alpha-induced osteoclast survival, whereas low concentrations of NO releaser NOC-18 were sufficient to promote osteoclast survival. Furthermore, antiapoptotic and caspase suppressive effects of TNF-alpha on osteoclasts were abolished by L-NAME. Our findings indicate that iNOS-dependent NO generation contributes to the survival-promoting function of TNF-alpha in osteoclasts.

Lee SK ，Huang H ，Lee SW ，Kim KH ，Kim KK ，Kim HM ，Lee ZH ，Kim HH ... -  《EXPERIMENTAL CELL RESEARCH》

Human NK cells express endothelial nitric oxide synthase, and nitric oxide protects them from activation-induced cell death by regulating expression of TNF-alpha.

Although NO appears important in rodent immune responses, its involvement in the human immune system is unclear. We report that human NK cells express constitutive endothelial NO synthase mRNA and protein, but not detectable levels of inducible NO synthase. They produce NO following activation by coculture with target cells or cross-linking with anti-CD16 mAb, and production is increased in the presence of IL-2. N-monomethyl-L-arginine (L-NMA), a NOS inhibitor, partially inhibited NK cell lysis of four different target cells (<40% inhibition at 500 microM L-NMA), but not granule release following coculture with target cells, or Fas ligand induction following cross-linking with anti-CD16 mAb. However, L-NMA augmented apoptosis of NK cells induced by activation through CD16 ligation or coculture with K562. An NO donor, S-nitroso-N-acetylpenicillamine (SNAP), suppressed apoptosis of NK cells induced by CD16 cross-linking or coculture with target cells, suggesting that endogenous NO production is involved in protection of NK cells from activation-induced apoptosis, thereby maintaining NK activity. SNAP also suppressed, and L-NMA enhanced, expression of TNF-alpha, reported to be involved in activation-induced NK cell death, in response to CD16 cross-linking. Suppression of anti-CD16-induced apoptosis by SNAP was reversed by the addition of rTNF-alpha. DNA-binding activity of the transcription factor, NF-AT, which is involved in TNF-alpha induction upon ligation of CD16, was inhibited by SNAP and enhanced by L-NMA. Our results suggest that down-regulation of TNF-alpha expression, possibly due to suppression of NF-AT activation, is a mechanism by which endogenous NO protects NK cells from activation-induced apoptosis, and maintains lytic capacity.

Furuke K ，Burd PR ，Horvath-Arcidiacono JA ，Hori K ，Mostowski H ，Bloom ET ... -  《JOURNAL OF IMMUNOLOGY》