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Nitric Oxide-Induced Apoptosis in Tumor Cells Victor Umansky∗ and Volker Schirrmacher Division of Cellular Immunology, Tumor Immunology Program German Cancer Research Center, D-69120 Heidelberg, Germany

I. Introduction II. NO and Antimetastatic Resistance A. NO and Antimetastatic Resistance Mediated by Macrophages B. NO and Antimetastatic Resistance Mediated by Endothelial Cells III. Mechanisms of NO-Mediated Apoptosis A. Death Receptors B. The p53 Response C. Mitochondrial Control D. The Bcl-2 Family E. Caspase Activation IV. Concluding Remarks References

Nitric oxide (NO), an important molecule involved in neurotransmission, vascular homeostasis, immune regulation, and host defense, is generated from a guanido nitrogen of L-arginine by the family of NO synthase enzymes. Large amounts of NO produced for relatively long periods of time (days to weeks) by inducible NO synthase in macrophages and vascular endothelial cells after challenge with lipopolysaccharide or cytokines (such as interferons, tumor necrosis factor-, and interleukin-1), are cytotoxic for various pathogenes and tumor cells. This cytotoxic effect against tumor cells was found to be associated with apoptosis (programmed cell death). The mechanism of NO-mediated apoptosis involves accumulation of the tumor suppressor protein p53, damage of different mitochondrial functions, alterations in the expression of members of the Bcl-2 family, activation of the caspase cascade, and DNA fragmentation. Depending on the amount, duration, and the site of NO production, this molecule may not only mediate apoptosis in target cells but also protect cells from apoptotis induced by other apoptotic stimuli. In this review, we will concentrate on the current knowledge about the role of NO as an effector of apoptosis in tumor cells and discuss the mechanisms of NO-mediated apoptosis.

∗ Address reprint requests to Dr. Victor Umansky, Division of Cellular Immunology, Tumor Immunology Program, German Cancer Research Center, D-69120 Heidelberg, Germany; Tel.: 49-6221-423757; Fax: 49-6221-423702; E-mail: V. Umansky @dkfz-heidelberg.de

107 Advances in CANCER RESEARCH 0065-230X/01 $35.00

C 2001 by Academic Press. Copyright  All rights of reproduction in any form reserved.

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I. INTRODUCTION The small gaseous molecule nitric oxide (NO) is generated from a guanido nitrogen of L-arginine by the family of NO synthase (NOS) enzymes. Despite its short half-life (usually a matter of seconds) and rapid oxidation to the stable, inactive end-products, nitrite and nitrate, NO has been reported as a potent biological modulator for a number of physiological functions, including vasodilation, neurotransmission, and natural defense of the immune system (Bredt et al., 1990; Moncada et al., 1991; Nathan, 1992). Following the identification of NO as endothelial-derived relaxing factor (Ignarro et al., 1987; Palmer et al., 1987), different cell types (such as macrophages, endothelial cells, neurons, fibroblasts, hepatocytes, epithelial and smooth muscle cells) have been shown to produce this free radical (Schmidt and Walter, 1994). At least three distinct isoforms of the NOS encoded by three distinct genes have been isolated (Knowles and Moncada, 1994; Nathan and Xie, 1994; Morris and Billiar, 1994). Two of them, endothelial NOS and neuronal NOS, are expressed constitutively and require intracellular Ca2+ and calmodulin for its activation. The other isoform, inducible NOS (iNOS), is usually induced in the body by bacterial products and/or by some inflammatory cytokines such as interferons, interleukin (IL)-1, and tumor necrosis factor (TNF)-. iNOS activity can also be upregulated by certain viruses (Umansky et al., 1996; Saura et al., 1999). The fourth isoform of NOS has recently been found in the mitochondrial inner membrane (Ghafourifar and Richter, 1997; Giulivi et al., 1998). Mitochondrial NOS is constitutively expressed, is Ca2+-dependent, and exerts control over mitochondrial respiration and mitochondrial transmembrane potential (m). A low level of NO synthesized by constitutive NOS for short periods of time acts as a neurotransmitter and as a regulator of blood pressure and platelet aggregation (Moncada et al., 1991; Schmidt and Walter, 1994; ¨ Forstermann, 2000). In contrast, large amounts of NO produced by iNOS in macrophages and endothelial cells after challenge with lipopolysaccharide or cytokines are effective against various microbes (Nussler and Billiar, 1993; MacMicking et al., 1997), parasites (Green et al., 1991; Diefenbach et al., 1998), and viruses (Karupiah et al., 1993; Saura et al., 1999). Moreover, cytokine-activated macrophages and vascular endothelial cells were reported to kill tumor cells in vitro via NO production (Stuehr and Nathan, 1989; Li et al., 1991a,b). Consistent with these experimental data, iNOS knockout mice were susceptible to infections and showed poor macrophage killer function against microorganisms and tumor cells (MacMicking et al., 1995). The expression of iNOS was also reported in human macrophages from patients with different infectious, autoimmune, and inflammatory dis¨ eases (Nicholson et al., 1996; MacMicking et al., 1997; Kronke et al., 1998).

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It was demonstrated that NO-mediated cytotoxicity involved the inhibition of mitochondrial respiration and DNA synthesis in cell targets, including tumor cells (Stuehr and Nathan, 1989; Moncada et al., 1991; Kwon et al., 1991; Kurose et al., 1993). Moreover, this cytotoxic effect was found to be associated with apoptosis (programmed cell death) in normal (Albina et al., 1993; Sarih et al., 1993; Messmer et al., 1994; Fehsel et al., 1995) and tumor cells (Xie et al., 1993; Cui et al., 1994; Geng et al., 1996; Umansky et al., 1997). Apoptosis results from the action of a genetically encoded suicide program occurring during development and differentiation, in tumor cell deletion, and in response to different stimuli such as TNF, CD95 (Fas/APO-1) ligand, TNF-related apoptosis-inducing ligand (TRAIL) (APO-2 ligand), and shortage of growth factors or certain metabolities (Thompson, 1995). Apoptosis involves an initial commitment phase followed by an execution phase characterized by the activation of a cascade of cytoplasmic cystein proteases (caspases) (Henkart, 1996) and by structural changes including externalization of phosphatidylserine, cell shrinkage, condensation of nuclear chromatin, DNA fragmentation, plasma membrane blebbing, and the breakdown of the cell into small fragments (apoptotic bodies) that are then phagocytosed (Earnshaw, 1995). Aberrant cell survival resulting from inhibition of apoptosis is known to contribute to tumor progression (Williams and Smith, 1993; Krammer et al., 1998), and cancer cells would gain a selective growth advantage by blocking apoptosis (Reed, 1997; Dong et al., 1994). In particular, matrix-independent survival of metastatic carcinoma cells during extravasation may depend upon high resistance to apoptosis, since detachment of epithelial cells from the extracellular matrix induces programmed cell death (Frisch and Francis, 1994). It is important to note that depending on the site, magnitude, and duration of NOS acitivity, NO may not only mediate apoptotic cell death but also protect from apoptosis induced by other agents. This review will concentrate on the current knowledge about the role of NO as an effector of apoptosis in tumor cells.

II. NO AND ANTIMETASTATIC RESISTANCE Tumor cell–host cell interactions play a decisive role for tumor development or regression in various types of cancer. Numerous data from experimental tumor systems and clinical observations have resulted in the conclusion that the majority of tumor cells die rapidly in the circulation, and only a few of them can survive and proliferate to form distant metastases (Fidler, 1990; Hart and Saini, 1992; Nicolson, 1993; Schirrmacher et al., 1996). Both primary tumor lesions and metastases are infiltrated

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by different host cells. Tumor microenvironment includes not only organ parenchymal cells but also T lymphocytes with the potential of mediating specific antitumor immune reactions and antigen nonspecific host cells like natural killers, fibroblasts, granulocytes, endothelial cells, and macrophages (Whitworth et al., 1990; Belloni and Tressler, 1990; Shirrmacher, 1992). The last cell type constitutes a large proportion of stroma cells, and together with endothelial cells can produce in situ cytotoxic amounts of NO after appropriate activation (Thomsen and Miles, 1998; Xie and Fidler, 1998). In addition to cytotoxicity, NO can modulate several steps in the metastatic ¨ process, including suppression of platelet aggregation (Forstermann, 2000), downregulation of expression of adhesion molecules such as VCAM and ICAM (De Caterina et al., 1995), and inhibition of angiogenesis (Sakkoula et al., 1994).

A. NO and Antimetastatic Resistance Mediated by Macrophages 1. NO PRODUCTION DURING TUMOR METASTASIS To study NO production by macrophages infiltrating metastatic lesions, the ESb/ESb-MP mouse lymphoma model was used (Schirrmacher et al., 1982, 1995). ESb cells represent a spontaneous highly metastatic (liver as main site) variant of the chemically induced T cell lymphoma L5178 Y (Eb) of DBA/2 mice. A plastic-adherent variant, ESb-MP, retaining most of its ESb-derived antigenic and biochemical characteristics, has reduced growth capacity in vivo and metastasizes at a lower rate than parental ESb cells. To detect and quantify spontaneous lymphoma metastases at the single cell level, ESbL cells, a subline of ESb lymphoma cells, were transduced with ¨ the bacterial lacZ gene coding for -galactosidase (Kruger et al., 1994a). After intradermal inoculation in immunocompetent syngeneic mice, this lymphoma variant showed a characteristic three-phase kinetics of primary tumor growth and liver metastasis including an initial expansion phase, a plateau ¨ phase, and a final expansion phase leading to the death of the animals (Kruger et al., 1994b). Liver macrophages (Kupffer cells) were isolated during ESbL-lacZ lymphoma metastasis using a new method which provides high cell viability (>93%) and allows the direct ex vivo examination of separated cells without further in vitro culture (Rocha et al., 1996). A correlation between an increased NO production by Kupffer cells and the arrest of tumor growth and metastasis at the plateau phase was demonstrated (Umansky et al., 1995). This increase in NO synthesis may be due to upregulation of iNOS activity by various cytokines, the possible source of which in our model could be

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host T lymphocytes. In support for this assumption, SCID mice, which lack both T and B lymphocytes, and nude (nu/nu) mice, which lack only T lymphocytes, demonstrated no increase in NO production by Kupffer cells and spleen macrophages in response to application of either live or irradiated lymphoma cells. Kupffer cell cytotoxicity mediated by NO has also been reported in a rat hepatoma model in vitro (Kurose et al., 1993; Aono et al., 1994). This cytotoxicity was abrogated by the treatment with the specific iNOS inhibitor N-monomethyl-L-arginine (NMMA). The regression of murine sarcoma liver metastasis in vivo has been reported to correlate with the upregulation of iNOS expression and NO Synthesis within the tumor lesions after treatment with a macrophage activator encapsulated in liposomes (Xie et al., 1995a). application of the iNOS inhibitor prevented NO production and apoptosis in the tumor cells. Interestingly, direct transfection of iNOS into highly metastatic murine melanoma cells caused partial apoptosis of these cells associated with suppression of their tumorigenicity and metastatic potential (Xie et al., 1995b). Tumor cells producing large amounts of NO stimulated not only autocytolysis but also destruction of bystander tumor cells (Xie et al., 1997a). On the other hand, highly metastatic cells may evade NO-mediated apoptosis by development of protective mechanisms. For example, NO production by Kupffer cells was found to be substantially inhibited at the final expansion phase of ESbL-lacZ lymphoma metastasis (Umansky et al., 1995). The downregulation of iNOS activity may be caused by the release of soluble tumor derived factors (Murata et al., 1994). It has been found that overproduction of acidic phosphoprotein osteopontin by metastatic tumor cells suppressed the cytokine-mediated iNOS activation and tumoricidal activity of macrophages (Denhardt and Chambers, 1994; Feng et. al., 1995). In addition, metastatic cells are able to develop different defense mechanisms ¨ to scavenge or to detoxify NO (Kronke et al., 1997; Inai et al., 1996).

2. ROLE OF NF-B IN THE REGULATION OF NO PRODUCTION In has been shown that the transcription of iNOS in stimulated macrophages is predominantly activated by NF-B (Xie et al., 1994; Liang et al., 1999). This transcription factor regulates the expression of various genes encoding cytokines, cell surface proteins, and other genes regulating the immune response and apoptosis (Bauerle and Henkel, 1994). In unstimulated cells, NF-B is presented in the cytoplasm in an inactive form, complexed with its inhibitory subunit IB. Inflammatory cytokines, TNF-, LPS, UVirradiation, or reactive oxygen species (ROS) induce the activity of IB kinases, which allows the degradation of the phosphorylated IB protein and

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the translocation of NF-B into the nucleus, where it binds to DNA enhancer motifs (Stancovski and Baltimore, 1997). Newcastle disease virus (NDV) has also been found to activate NF-B in rat macrophages (Fisher et al., 1994; Bauerle and Henkel, 1994). This virus has received much attention because of its nonspecific immune stimulating potential and its various antitumor activities in tumor mouse models and in cancer patients (Ahlert et al., 1997; Schirrmacher et al., 1998). Using mouse spleen macrophages, we demonstrated that treatment with NDV in vitro and in vivo induced NF-B activation and NO synthesis in these cells (Umansky et al., 1996). This activation of NF-B and NO production was completely inhibited by the antioxidant butylated hydroxyanisole (BHA) and therefore required the participation of ROS. It has been proposed that the stimulation of certain kinases and proteases in the process of NF-B activation is regulated by the intracellular redox state (Westendorp et al., 1995; Cahill et al., 1996). Moreover, NDV-induced NF-B activation and NO synthesis were blocked by an inhibitor of protein tyrosine kinase (genistein) and protein kinase A (H-89) but not by an inhibitor of protein kinase C (staurosporin), which suggests that signaling requirements of both NF-B activation and NO production in NDV-treated macrophages are similar (Umansky et al., 1996). As to the role of the produced NO in the modulation of NF-B activity, there still exists no clear picture. NO was reported to activate the DNAbinding activity of NF-B in T cells (Lander et al., 1995), whereas it inhibited this transcription factor in other cell types (Peng et al., 1995; Matthews et al., 1996). To clarify these apparent contradictions, we incubated endothelial TC10 cells with the NO donor and tested its effect on NF-B activation (Umansky et al., 1998). It was found that at low concentrations, NO increased the DNA-binding activity of NF-B prestimulated with low amounts of TNF-. In contrast, high concentrations of NO inhibited this activation. The following mechanism of interaction between NF-B and iNOS in endothelial cells and macrophages could thus be proposed: The activation of NF-B by cytokines and other stimuli results in the induction of iNOS expression. This leads to an increased NO synthesis, which in turn costimulates NF-B by a self-amplifying mechanism. High amounts of generated NO can not only induce apoptosis in neighboring tumor cells but also prevent the NF-B activation followed by reduced iNOS transcription and NO production in macrophages or endothelial cells. The inhibition of the NF-B activation was proposed to result either from a direct S-nitrosylation of cysteine 62 in the p50 NF-B subunit (Matthews et al., 1996) or from stabilization of the inhibitory IB- protein (Peng et al., 1995). Importantly, NO did not affect the viability of endothelial cells at concentrations causing the inhibition of DNA-binding activity of NF-B, thus allowing the establishment of an autoregulatory loop (Umansky et al., 1998).

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3. NO IN IMMUNOTHERAPY OF METASTASIS In situ activated host macrophages, which are able to produce cytotoxic amounts of NO, were also shown to play an important role in a highly effective adoptive cellular immunotherapy (ADI) of ESb T lymphoma in DBA/2 (H-2d, Mlsa) mice. The therapy protocol consists of a single intravenous injection of anti-tumor-immune spleen lymphocytes from ESb-tumor resistant, MHC identical but superantigen different B10.D2 (H-2d, Mlsb) mice into tumor-bearing DBA/2 mice. This results in rejection of primary tumors (1.5 cm in diameter) from the skin and eradication of liver metastases (Schirrmacher et al., 1991, 1995). This immunotherapy requires only relatively few specific T cells and involves synergistic interactions between CD4 and CD8 tumor-immune donor T cells and host Kupffer cells at the site of ¨ ¨ liver metastases (Schirrmacher et al., 1995; Muerk oster et al., 1999). It was found that Kupffer cells ex vivo isolated at different time points after adoptive transfer of immune spleen cells produced high amounts of NO (Umansky et al., 1995). Furthermore, the depletion of Kupffer cells with liposomes containing chlodronate in tumor-bearing mice during ADI led to ¨ ¨ nearly complete prevention of the immunotherapeutic effect (Muerk oster et al., 1999). This treatment with chlodronate selectively affected Kupffer cells but not other liver cells (e.g., lymphocytes, dendritic, or endothelial cells) and did not result in cytokine release (Rocha et al., 1995; Van Rooijen and Sanders, 1997). After immunohistological staining of livers from ADI-treated mice, iNOSpositive host macrophages were observed at early time points (days 5 and 8 after cell transfer) in close association with apoptotic cells in areas of ¨ ¨ metastases (Muerk oster et al., 2000). Moreover, in vivo experiments, in which iNOS activity was suppressed by constant infusion of the inhibitor N-(3-aminomethylbenzyl)acetamidin (1400W) immediately after ADI treatment, showed a marked reduction of the survival of mice. These findings suggest a cytotoxic role of NO in the regression of liver metastasis (which was completely eradicated at day 12 after ADI). It has been reported that the secretion of NO by macrophages and dendritic cells in vitro is mediated by interaction between CD40 expressed on these cells and CD40 ligand (CD40L) on the surface of T lymphocytes (Tian et al., 1995; Lu et al., 1996). In our therapy model, iNOS-positive macrophages coexpressed the CD40 molecule. Furthermore, the in vivo blockade of CD40L with respective monoclonal antibodies led to a significant reduction of both CD40 and iNOS expression in macrophages and to a considerable inhibition of the immunotherapeutic effect, supporting thereby the involvement of CD40–CD40L interaction in ¨ ¨ the iNOS induction during ADI (Muerk oster et al., 2000). Cytotoxic and regulatory fuctions of NO, including the suppression of T cell proliferation, the inhibition of Thl cytokine secretion, and the

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downregulation of the MHC class II expression (Kolb and Kolb-Bachofen, 1998; Bogdan, 1998, 2000), may also be operative at a later stage of ADI, when local T cell responses should be terminated, and the liver has to be cleared from undesired transferred immune cells. At these time points (in particular, at day 20 after ADI), iNOS expressing cells were detected around periportal veins in close proximity to donor CD8 and CD4 T cells undergoing ¨ ¨ apoptosis (Muerk oster et al., 2000). After day 20, the total numbers of CD8 and CD4 liver-infiltrating T lymphocytes were found to decrease markedly ¨ ¨ (Muerk oster et al., 1998). In contrast, in livers of mice treated with the iNOS-specific inhibitor 1400W at later time points after ADI, the numbers of CD8 and CD4 T cells remained at the high level that correlated with the reduced survival of animals. Similar results were observed when antiCD40L antibodies were injected into ADI-treated mice. Since at these later time points the metastases were already eradicated, the animals died most likely from graft versus host (GvH) disease caused by donor T cells which were not eliminated from the liver via host versus graft (HvG) reactivity and which preserved their proliferative potential. Therefore, CD40–CD40L interactions leading to iNOS induction in Kupffer cells could contribute to the destruction of liver metastases at early time points after ADI and to elimination of the infiltrating allogeneic T cells at a later stage.

B. NO and Antimetastatic Resistance Mediated by Endothelial Cells Interactions between tumor and endothelial cells are not limited only to the promotion of invasion and metastasis through the formation of new capillary vessels or upregulation of adhesion molecule expression which leads to increased tumor cell binding (Belloni and Tressler, 1990; Folkman, 1995). Activated endothelial cells are also able to induce tumor cell death via secretion of cytotoxic factors including NO. It is known that endothelial cells produce NO in vitro and in vivo both by constitutive and inducible mech¨ anisms (Schmidt and Walter, 1994; Moncada, 1997; Forstermann, 2000). The endothelial isoform of constitutive NOS plays an important role in the stimulation of angiogenesis, blood flow, and platelet aggregation (Ziche et al., 1994; Fukumura and Jain, 1998), whereas large amounts of NO produced by iNOS in cytokine-activated vascular endothelial cells are capable of lysing murine and human tumor cells in vitro (Li et al., 1991a, b; Geng et al., 1996). To study whether a similar mechanism may function in vivo, NO production by ex vivo isolated liver endothelial cells was determined during the different phases of ESbL-lacZ lymphoma metastasis (Rocha et al., 1995). A dramatic increase in NO synthesis in liver endothelial cells was found to

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correlate with the arrest of primary tumor growth and a low plateau of liver metastasis. However, when the growth of primary tumor and liver metastasis started again at the final expansion phase, NO production by endothelial cells significantly decreased, reaching the basal level observed in normal mice. Metastatic cells were reported to produce some factors like transforming growth factor-beta (TGF-) or osteopontin, which could downregulate the iNOS activity in both endothelial cells and macrophages (Vodovotz et al., 1993; Denhardt and Chambers, 1994; Feng et al., 1995). In this way, tumor cells appear to be able to defend themselves against being killed by NO produced by cytokine-activated host endothelial cells and/or macrophages. The upregulation of iNOS activity in endothelial cells during tumor growth suppression at the plateau phase may be induced by cytokines such as interferon  (IFN- ) or TNF- produced by activated cells of the tumor microenvironment (macrophages and/or lymphocytes) (Estrada et al., 1992; Bordeling and Murphy, 1995). To investigate this possible regulatory mechanism in the mouse ESbL-lacZ lymphoma model, selective elimination of Kupffer cells was performed by injection of chlodronate entrapped in liposomes (Rocha et al., 1995). Importantly, liver endothelial cells were not functionally affected by this treatment (Boggers et al., 1991; Van Rooijen, and Sanders, 1997). It was found that the depletion of Kupffer cells in lymphoma-bearing DBA/2 mice caused no changes in the high level of NO production by liver endothelial cells during the arrest of tumor growth and metastasis, suggesting that this production is completely independent from Kupffer cells. The role of T lymphocytes in the stimulation of NO synthesis in endothelial cells was studied using different protocols of tumor cell inoculation into immunocompetent or immunocompromised mice. NO production was considerably increased only in liver endothelial cells of tumor cell-injected immunocompetent DBA /2 mice, regardless of the site of lymphoma cell inoculation, whereas injected immunocompromised animals (sublethally  -irradiated, nude or SCID mice) did not show any induction of NO synthesis. The nature and mechanisms of the inductive signals transmitted to the iNOS of liver endothelial cells after tumor cell vaccination at distant sites are still unknown. The systemic effect observed in immunocompetent syngeneic mice might be explained by migration of activated CD4 T lymphocytes from the draining lymph nodes of the vaccination site to the liver where they could produce, among others, IFN- or TNF-, powerful inducers of iNOS activity. A central role of CD4 T cells in the induction of NO synthesis by host cells in the tumor microenvironment associated with a strong antitumor immune responses has been recently demonstrated in mouse tumor models (Hung et al., 1998; Nishimura et al., 1999). The inducible endothelial NO response thus plays an important role in the suppression of lymphoma metastasis in the liver and seems to require mature T lymphocytes and a T cell dependent antitumor immune response.

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In another set of experiments, we demonstrated that endothelial cells isolated ex vivo from livers of ESbL-lacZ lymphoma-bearing mice could induce apoptosis in coincubated ESbL-lacZ target cells. Since it was technically difficult to get ex vivo sufficient amounts of liver endothelial cells to study in detail this effect, we decided to use the well-characterized bovine endothelial cell line (BEC) as a source of effector cells (Umansky et al., 1997). An important advantage of BEC is their fast and strong adherence to plastic, which permits the separation of nonadherent lymphoma target cells after coincubation. It was found that treatment with human TNF- induced BEC iNOS activation and NO production, which could be completely blocked with the iNOS inhibitor NMMA. Coculture of activated BEC with lymphoma cells caused apoptosis in the latter cells, which was considerably reduced after pretreatment of endothelial cells with NMMA. A similar apoptotic effect was also observed after incubation of tumor cells with NO donors such as DETA / NONOate (NOC-18) or glycerol trinitrate (GTN). Interestingly, nonactivated BEC were not able to produce NO and showed a substantially lower level of apoptotic cell death. This might mean that part of the apoptotic effect of endothelial cells against lymphoma cells is mediated not only by NO, but also by other agent(s) as observed in some cases of macrophageinduced tumor cytotoxicity (Mateo et al., 1996; Lavnikova et al., 1997). Besides BEC, TNF- was reported to stimulate iNOS also in human vascular endothelial cells, followed by NO-induced apoptosis in human leukemic cells in vitro (Geng et al., 1996). It might, therefore, be suggested that appropriately activated vascular endothelial cells are able to induce apoptosis in circulating metastasizing cells via NO and thereby contribute to prevention of the extravasation phase of metastasis.

III. MECHANISMS OF NO-MEDIATED APOPTOSIS A. Death Receptors It is known that members of the TNF receptor superfamily play a critical role in the development of apoptosis in various cell types (Nagata, 1997; Krammer et al., 1998). CD95L and TNF- were shown to induce apoptosis by binding to their respective death domain-containing receptors, CD95 and TNFR-1. Binding of CD95L or agonistic antibodies to CD95 leads to a signal transduction cascade initiated by death receptor associated molecules such as FADD/MORT1 (Chinnaiyan et al., 1996) and FADD associated FLICE/MACH (caspase-8), a chimeric molecule containing a death effector domain and a proteolytic ICE-like domain (Boldin et al., 1996). This leads

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to proteolytic processing of caspases into active proteases. Caspase-8 is the first in a cascade of caspases activated by CD95 (Medema et al., 1997). The CD95 system is critical for growth control of T cells. Elimination of peripheral T cells involves the induction of a suicide of fratricide mechanism triggered by CD95 ligand/receptor interaction (Dhein et al., 1995; Ju et al., 1995; Peter and Krammer, 1998). Triggering of CD95 ligand/receptor interaction in lymphoid and nonlymphoid cells may also be caused by cytotoxic drugs or viral proteins (Friesen et al., 1996; Chlichlia et al., 1997). Another member of the TNF superfamily, named TRAIL/APO-2L, has been identified based on the homology of its extracellular domain with CD95L and TNF (Wiley et al., 1995). TRAIL induces rapid apoptosis in a variety of transformed cell lines, but does not appear to be cytotoxic to normal cells in vitro (Walczak et al., 1999). It has been recently reported that human monocytes rapidly expressed TRAIL, but not CD95L or TNF, after activation with IFN- or - and acquire the ability to kill tumor cells through apoptosis (Griffith et al., 1999). The involvement of the CD95/CD95L system in NO-induced apoptosis in human neoplastic lymphoid cells was studied using CD95-sensitive Jurkat cells (APO-S clone) and their CD95-resistant subclone (APO-R), characterized by lack of the CD95 receptor on the cell surface (Peter et al., 1995). APO-S cells were found to be much more susceptible to apoptosis induced by NO donor glycerol trinitrate (GTN) than APO-R cells (Chlichlia et al., 1998). Moreover, NO triggered apoptosis in freshly isolated human leukemic lymphocytes which were also sensitive to anti-CD95 treatment. These findings were confirmed using an in vitro model mimicking a relevant in vivo situation and based on coculture of APO-S target cells with TNF-activated bovine endothelial cells generating NO as effectors. A significant level of lymphoid cell apoptosis was observed which was completely abrogated by the iNOS inhibitor NMMA. Incubation of APO-S cells with the NO donor resulted in a strong increase in the expression of CD95L and TRAIL mRNA. Although CD95L mRNA expression was transient and reduced after prolonged (up to 24 h) incubation of the cells with NO, TRAIL expression was stable for the same time period. Unlike CD95L, whose transcripts are predominantly restricted to stimulated T cells and sites of immune privilege, TRAIL expression is detected in many normal human tissues (Wiley et al., 1995). It was proposed that TRAILmediated apoptosis may play a role in antileukemic growth control in APO-S Jurkat cells, although TRAIL had only 25% of the activity of CD95L in inducing apoptosis (Jeremias et al., 1998). However, the requirement of the CD95/CD95L system in the mechanism of NO-induced apoptosis is not strict. Some neoplastic lymphoid cells resistant to CD95-mediated apoptosis (e.g., BL60 cells) were shown to be sensitive to treatment with NO (Chlichlia et al., 1998). Furthermore, NO was able

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to trigger apoptosis in human breast cancer cell lines derived from primary tumor (BT-20) or from metastases (MCF-7) independently of CD95/CD95L interaction (Umansky et al., 2000a). Thus, in contrast to APO-S Jurkat leukemia cells, NO failed to induce mRNA expression of CD95L in these breast cancer cells. Moreover, BT-20 and MCF-7 cells showed a strong expression of the mRNA of Fas-associated phosphatase-1 (FAP-1). The FAP-1 protein is associated with a negative regulatory domain of CD95 and can thereby inhibit CD95-mediated apoptosis (Sato et al., 1995). Abrogation of this association has been reported to restore CD95-mediated cell death in a colon cancer cell line (Yanagisawa et al., 1997). In addition, FAP-1 expression in some tumor cell lines was found to correlate with their resistance to apoptosis induced by CD95 (Ungefroren et al., 1998).

B. The p53 Response The p53 tumor suppressor gene is a key target for mutation in many types of human cancer (Mowat, 1998; Brown and Wouters, 1999). A main biological function of the p53 protein is positive regulation of apoptosis in response to signals such as genomic damage and aberrant activation of certain oncogenes (Lakin and Jackson, 1999). In many cell types, stressrelated activation of p53 associated with a rapid increase in its levels induces cell-cycle arrrest, which can provide a sufficient time window for DNA repair. When DNA damage is excessive, the cell may undergo apoptosis mediated by p53. The apoptotic activity of p53 has been demonstrated to be crucial for tumor growth suppression in vitro and in vivo (Mowat, 1998; Brown and Wouters, 1999). Evidence for the involvement of accumulation of the p53 gene products in NO-mediated apoptosis was initially provided for normal cells such as RAW 264.7 macrophages (Messmer et al., 1994), pancreatic RINm5F cells (Ankakrona et al., 1994), and murine thymocytes (Fehsel et al., 1995). Upregulation of iNOS activity in macrophages or treatment of these cells with NO donors caused p53 accumulation, which preceded DNA fragmentation at the late apoptotic phase, while inhibition of NO synthesis suppressed p53 ¨ ¨ accumulation (Messmer and Brune, 1996; Brune et al., 1999). Moreover, after incubation with the NO donor S-nitrosoglutathione, macrophages stably transfected with plasmids encoding p53 antisense RNA showed lower p53 levels and significantly reduced DNA fragmentation, suggesting thereby an importance of p53 expression during NO-induced apoptosis. It has recently been reported that NO-mediated accumulation of p53 involves inhibition of the proteasome (Glockzin et al., 1999). The latter molecular complex is responsible for the degradation of many short-lived proteins (including p53) that regulate cell proliferation and cell death.

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The role of wild-type p53 in NO-mediated apoptosis was also demonstrated in tumor systems. In murine melanoma cells, incubation with cytokines such as IFN- and IL-1 stimulated iNOS expression and NO production, which correlated with increased expression of wild-type p53 at the mRNA and protein levels (Xie et al., 1997b). Moreover, this effect on p53 expression could be reversed by inhibiting iNOS activity. A similar induction of p53 expression in melanoma cells was achieved using NO donors and was concentration and time dependent (Xie and Fidler, 1998). In another study, human colon carcinoma cells containing wild-type p53 were more sensitive to NO-mediated apoptosis than cells without p53 or with mutated p53 (Ho et al., 1996). In addition to wild-type p53, NO was found to induce the expression of the p21/ WAF1/CIP1 protein, which may eventually promote apoptosis in these cells. The upregulation of p21/ WAF1/CIP1 expression was also demonstrated in human pancreatic carcinoma cell lines in the process of NO-mediated apoptosis (Gansauge et al., 1998). It is necessary to note that in cancer cells with mutated p53, NO may not induce apoptosis but rather promote tumor growth associated with increased neovascularization, contributing thereby to cancer progression (Ambs et al., 1998). However, p53 negative cells (such as U937 cells) have recently been reported to undergo apoptosis after NO exposure. This observation substantiates the hypothesis for the existence of p53-independent signaling pathways during ¨ ¨ et al., 2000). NO-mediated apoptosis (Brockhaus and Brune, 1998; Brune

C. Mitochondrial Control Recent studies performed in various cellular systems, including cell-free extracts, clearly indicated a crucial role for mitochondrial damage in the effector phase of apoptosis in mammalian cells induced by different agents, including NO (Kroemer et al., 1997; Van der Heiden et al., 1997; Scaffidi et al., 1998; Brown and Borutaite, 1998). This damage includes the early disruption of mitochondrial transmembrane potential  m, the generation of ROS, the opening of permeability transition (PT) pores, and the release of 15-kDa protein cytochrome c release from mitochondria (Marchetti et al., 1996; Susin et al., 1997; Li et al., 1997; Kluck et al., 1997; Kuida et al., 1998).  m results from the asymmetric distribution of ions on both sides of the inner mitochondrial membrane, giving rise to a gradient which is essential for mitochondrial function. Functional experiments show that the mechanism of the early  m loss involves the PT pores, dynamic multiprotein complexes formed at the contact site between the inner and the outer mitochondrial membranes (Kroemer et al., 1997). Cytochrome c, identified as apoptotic protease activating factor 2 (Apaf 2) is required for the formation of the complex between Apaf 1 and caspase-9 (Apaf 3). The latter becomes

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activated under such conditions and in turn activates caspase-3, which leads to DNA fragmentation and apoptosis (Li et al., 1997). Cytochrome c is normally present on the outer surface of the inner mitochondrial membrane and shuttles electrons between complexes III and IV of the respiratory chain (Brown and Borutaite, 1998). The decrease of  m due to the opening of PT pores followed by the depolarization of the inner mitochondrial membrane and massive ROS production was demonstrated in normal thymocytes undergoing apoptosis mediated by NO (Hortelano et al., 1997). To study whether this mechanism is operative also in tumor cells, we treated APO-S Jurkat leukemic cells with the NO donor and observed a time-dependent increase in the number of cells with low  m (Ushmorov et al., 1999). The role of PT in the NO-induced apoptosis in our model was elucidated with the help of bongkrekic acid (BA) which is known to be a specific inhibitor of PT, affecting the molecular conformation of the adenine nucleotide translocator (a protein participating in the formation of PT pores) and was shown to suppress dexamethasoneinduced apoptosis in mouse thymocytes (Marchetti et al., 1996). However, only a limited inhibitory effect on NO-induced apoptosis in APO-S Jurkat cells was observed, and only a moderate decrease in the number of cells with low  m was observed, suggesting that PT is possibly not a major facotor causing NO-induced reduction of  m and apoptosis in Jurkat cells (Ushmorov et al., 1999). Upon NO treatment, an intensive and rapid cytochrome c release into the cytosol of Jurkat cells was found. To clarify the mechanism of this release, we studied the function of respiratory chain complexes and the content of cardiolipin. This major mitochondrial lipid is necessary for the activity of respiratory complexes (Hatch, 1998) and plays a crucial role in cytochrome c attachment to the inner mitochondrial membrane (Choi and Swanson, 1995; Salamon and Tollin, 1996). We found that exposure to NO could significantly inhibit the activity of all complexes of the mitochondrial electron transport chain. These findings are in agreement with publications reporting on NO-mediated inhibiton of cytochrome c oxidase (complex IV) in different cell types via reversible binding to its heme moiety (Takehara et al., 1996; Clementi et al., 1998; Richter et al., 1999). This downregulation of complex IV activity resulted in the upregulation of ROS synthesis, followed by the formation of the strong oxidant peroxynitrite anion (ONOO−) Which can induce irreversible inhibition of complexes I and III but not complex IV (Casina and Radi, 1996). The alternative mechanism of a direct effect of NO on these complexes is also possible in Jurkat leukemic cells, since a long-term exposure to NO in vitro has been recently shown to block complex I activity in murine macrophages, due to S-nitrosylation of this enzyme (Brown and Cooper, 1994; Clementi et al., 1998; Richter et al., 1999).

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After exposure to NO, inhibition of complex IV activity was found to correlate with a significant time-dependent accumulation of Jurkat cells with low cardiolipin concentration (Ushmorov et al., 1999). Moreover, a potent inhibitor of lipid peroxidation, trolox (a vitamin E analog) significantly inhibited NO-induced apoptotic cell death and restored complex IV activity. In addition, trolox considerably reduced the number of NO-treated Jurkat cells with low cardiolipin content. A similar protective effect of trolox has also been reported for NO-exposed rat astrocytes (Heales et al., 1994). To study further the role of mitochondrial lipid cardiolipin in the resistance of Jurkat leukemia cells to NO-mediated apoptosis, we sorted the cells with high cardiolipin concentration (Cardiolipinhigh) which survived after exposure to NO donors for 24 h (Umansky et al., 2000b). These cells were substantially less sensitive to NO-induced apoptosis than unsorted parental Jurkat cells and maintained the same low level of sensitivity during long-term culture. Elevated cardiolipin concentration has recently been reported to be involved in the resistance of hepatocytes to some apoptotic stimuli (Lieser et al., 1998). However, in Cardiolipinhigh Jurkat cells, the increased cardiolipin content quickly returned back to the level observed in parental cells and remained unchanged during the whole period of culture, suggesting that the stimulation of cardiolipin synthesis is associated with Jurkat cell survival after NO treatment but does not play a crucial role in this process. In contrast to cardiolipin, the content of glutathione in Cardiolipinhigh Jurkat cells was significantly and constantly higher than in parental NOsensitive cells (Umansky et al., 2000b). Glutathione as a physiological oxidant plays an important role in maintaining intracellular redox balance ¨ and in the cellular defense against oxidative stress (Droge et al., 1994). It has been reported that glutathione can suppress apoptosis mediated by different agents, including NO, by inhibition of the mitochondrial damage (Clementi et al., 1998; Ghibelli et al., 1998). A depletion of intracellular glutathione has been demonstrated in various apoptotic systems, including exposure of the cells to NO (Van den Dobbelsteen et al., 1996; Clementi et al., 1998; Ghibelli et al., 1998) and has been considered as an early apoptotic event (Macho et al., 1997). In contrast, increased glutathione concentration may downregulate NO-induced apoptosis in smooth muscle cells (Zhao et al., 1997). An antiapoptotic effect is also provided by the inhibition of glutathione efflux via bcl-xL gene overexpression (Bojes et al., 1997) or via pretreatment with methionine (Ghibelli et al., 1998). In our experiments, glutathione depletion in Cardiolipinhigh NO-resistant cells with buthionine-sulfoximine (BSO) resulted in the stimulation of NOmediated apoptosis, whereas the exposure of parental NO-sensitive cells to the glutathione precursor N-acetylcysteine caused a substantial

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suppression of apoptotic cell death. These data suggest an important role of glutathione in the protection against NO-induced apoptosis in human leukemia cells.

D. The Bcl-2 Family Bcl-2 belongs to a growing family of proteins that can either block (Bcl-2, Bcl-xL, etc.) or promote (Bax, Bad, Bak, etc.) apoptosis. Bcl-2-related proteins are integrated in the outer mitochondrial, outer nuclear, and endoplasmic reticular membranes with the help of carboxy-terminal membrane anchor (Hockenbery et al., 1993; Kluck et al., 1997). It was demonstrated that transfection of Bcl-2 specifically prevented normal and tumor cells from apoptosis evoked by NO, which was produced endogenously after exposure to inflammatory cytokines or derived from NO donors (Xie et al., 1996, 1997b; Messmer et al., 1996a; Albine et al., 1996; Melkova et al., 1997). In contrast, expression of the proapoptotic protein Bax increased during NOmediated apoptosis, at least in macrophages and mesangial cells (Messmer et al., 1996a). Mechanisms of Bcl-2 related antiapoptotic effects at the mitochondrial level are linked with (i) inhibition of PT and stabilization of  m (Marchetti et al., 1996); (ii) prevention of caspase activation and cleavage of poly (ADP-ribose) polymerase cleavage (Melkova et al., 1997); (iii) regulation of proton flux (Shimizu et al., 1998); and (iv) blocking of the proapoptotic effect of Bax protein (Antonsson et al., 1997). In addition, Bcl-2 can not only prevent the release of cytochrome c from mitochondria by inhibition of lipid peroxidation (in particular, cardiolipin) (Hockenberry et al., 1993; Tyurina et al., 1997) but can also interfere with cytochrome c already released into the cytosol (Zhivotovski et al., 1998). On the other hand, p53 accumulation remained unchanged in Bcl-2 transfected cells, which led to the conclusion that Bcl-2 acts downstream of p53 but upstream to cytochrome ¨ et al., 1999). c release (Brune In our experiments, we used Jurkat leukemia cells transfected with Bcl-2 and found that Bcl-2 overexpression resulted in a complete resistance to apoptosis in cells treated with NO donors (GTN or DETA / NONOate) even at high concentrations. Furthermore, Bcl-2 was able to block the damage of mitochondrial functions observed after exposure to NO donors. It normalized the content of cardiolipin and ROS production,  m and respiratory complex activities, and prevented cytochrome c release and lipid peroxidation in mitochondria (Ushmorov et al., 1999). From our data on the effect of BA, an inhibitor of mitochondrial PT, and of trolox, an inhibitor of lipid peroxidation, we suggest that Bcl-2-induced suppression of lipid peroxidation plays a more important role in protection against NO-mediated apoptosis in human leukemia cells than the inhibition of PT.

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NO has been reported to induce apoptosis also by direct downregulation of Bcl-2 expression in target cells. Thus, treatment of murine melanoma cells with inflammatory cytokines or with NO donors led to a considerable reduction of Bcl-2 expression followed by apoptosis (Xie et al., 1996). This effect was reversed by inhibiting NO synthesis. However, short-term NO production at low concentrations has been demonstrated to suppress apoptosis in B lymphocytes (Mannick et al., 1994; Genaro et al., 1995) and in endothelial cells (Suschek et al., 1999). This protection was due to the increased expression of Bcl-2 at the mRNA and protein levels. It appears that the influence of NO on Bcl-2 expression is dose and time dependent: prolonged exposure to high-level NO results in Bcl-2 suppression, whereas short incubation with trace-level NO had the reverse effect.

E. Caspase Activation It is assumed that the activation of a cascade of cytoplasmic cysteine proteases (caspases) that specifically cleave a number of cellular substrates is essential for apoptosis, regardless of the initial death signal (Henkart, 1996; Salvesen and Dixit, 1997). Currently, the caspase family consists of 12 members which share several amino acid residues important for substrate binding and catalysis. Caspases are expressed as inactive precursors that are activated by proteolytic processing. According to the model (Cryns and Yuan, 1998), two classes of caspases, initiators and effectors, are involved in apoptosis. Apoptotic agents stimulate initiator caspases such as caspase-2, -8, and -9. This stimulation is autocatalytic and requires the binding of various specific cofactors. Activated initiator caspases process effector caspases (like caspase-3, -6, and -7) which in turn cause cell collapse by cleaving a certain set of substrates. Each initiator caspase seems to be activated only in response to particular apoptotic signals. For example, caspase-8 (FLICE) was shown to be the most upstream in the CD95 signaling pathway (Boldin et al., 1996; Medema et al., 1997). Upon triggering of CD95 receptor, the active subunits of caspase-8 are released into the cytosol, where they may stimulate effector caspases. Caspase-9, another initiator caspase, is known to be activated in the presence of other factors like Apaf 1 and ATP by cytochrome c released from mitochondria (Li et al., 1997; Kuida et al., 1998; Fearnhead et al., 1998). This leads, in turn, to cleavage and activation of caspase-3 followed by DNA fragmentation and apoptosis. It should be noted that caspase activation requires sufficient amount of ATP and could be significantly reduced by ATP depletion (Leist et al., 1997a). In this case, when energy levels are rapidly compromised, cells triggered to undergo apoptosis may instead die by necrosis.

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A series of studies demonstrated that endogenously produced or exogenously supplied NO activates caspases in connection with apoptosis in various normal (Messmer et al., 1996b; Leist et al., 1997b; Melkova et al., ¨ et al., 1999) and tumor cells (Yabuki et al., 1997; Sandau et al., 1998; Brune 1997; Chlichlia et al., 1998; Ushmorov et al., 1999; Umansky et al., 2000a). Importantly, NO-induced caspase-8 activation in human leukemia cells and caspase-3, -6, and -9 activation in human breast cancer cells was completely blocked by zVAD, a broad-spectrum caspase inhibitor. This enzyme inhibition was associated with complete inhibition of NO-mediated apoptosis in these cells (Chlichlia et al., 1998; Umansky et al., 2000a). Cytochrome c accumulation in the cytosol of NO-treated Jurkat leukemia cells was observed before caspase activation (Ushmorov et al., 1999). This suggests that NO-mediated cytochrome c release causes the activation of the caspase cascade followed by apoptosis, as was already shown for CD95 and other apoptotic death signals (Yang et al., 1997; Scaffidi et al., 1998). It has recently been reported by several groups that NO is able not only to increase but also to inhibit caspase activity by S-nitrosylation or oxidation of the catalytically reactive cysteine moiety (Dimmeler et al., 1997; Mohr et al., 1997; Li et al., 1999). However, most of these studies have been performed in cell extracts or with purified proteins. Under cellular conditions (normal hepatocytes) (Li et al., 1999), the concentration of NO suppressing TNF-mediated apoptosis via reduction of caspase activity was much lower than in experiments showing NO-mediated caspase activation. Again, as in the case of Bcl-2, the effect of NO seems to be strictly dependent on its concentration, on the time of NO exposure, and eventually on the type of target cells.

IV. CONCLUDING REMARKS A large body of evidence shows that NO either produced by activated host cells or delivered exogenously by NO donors induces apoptotic cell death in murine and human tumor cells in vitro and in vivo. High concentrations of NO can be produced for a long time in localized “hot spots” by activated iNOS expressed in macrophages within the tumors, endothelial cells in the tumor microvasculature, or in peripheral blood monocytes. The mechanism of NO-mediated apoptosis in tumor cells involves accumulation of the tumor suppressor protein p53, mitochondrial damage (including cytochrome c release, a downregulation of respiratory complex activity, lipid degradation, and glutathione depletion), alterations in the expression of members of Bcl-2 family, followed by activation of the caspase cascade and DNA fragmentation. Thus, stimulation of iNOS in host cells of the tumor

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microenvironment with appropriate inflammatory cytokines could be of importance for effective treatment of cancer patients by induction of apoptosis in tumor lesions and in circulating tumor cells. In addition, NO donors might be promising new agents in therapeutic protocols. However, NO produced in the tumor-bearing host can act as a doubleedged sword against the tumor or the host, depending on the circumstances. For example, NO is able not only to mediate apoptosis but also to protect target cells from apoptosis induced by other agents or to exert regulatory functions. Moreover, NO produced by tumor cells is often conducive to tumor progression and metastasis and is thus detrimental to the host. It appears that the mode of NO effect is dependent on the type of target cells as well as on the magnitude and duration of NO production. Prolonged exposure to high-level NO may result in apoptotic cell death, whereas short incubation with low-level NO may cause a cell protective effect.

ACKNOWLEDGMENTS The authors thank the members of our research group, collaborators, and colleagues who have contributed to the research described in this review. This work was supported in part by Grant No. 10-0980-Schi2 from the Dr. Mildred Scheel Stiftung and by the D. Hopp Stiftung.

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