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The Journal of Immunology, 2006, 176: 3923-3930.
Copyright © 2006 by The American Association of Immunologists

Nitric Oxide Promotes Resistance to Tumor Suppression by CTLs1

Zhanhai Su*, Jürgen Kuball{dagger}, Ana-Paula Barreiros*, Daniela Gottfried*, Edite Antunes Ferreira{dagger}, Matthias Theobald{dagger}, Peter R. Galle*, Dennis Strand* and Susanne Strand2,*

* Department of Internal Medicine, Johannes Gutenberg-University, Mainz, Germany; and {dagger} Department of Hematology and Oncology, Johannes Gutenberg-University, Mainz, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Many human tumors express inducible NO synthetase (NOS2), but the roles of NO in tumor development are not fully elucidated. An important step during tumor development is the acquisition of apoptosis resistance. We investigated the dose-dependent effects of endogenously produced NO on apoptosis using ecdysone-inducible NOS2 cell lines. Our results show that short-term NOS2 expression enhances CD95-mediated apoptosis and T cell cytotoxicity dose dependently. Furthermore, we could show that during chronic exposure to NO, besides the primary cytotoxic NO effect, there is selection of cell clones resistant to NO that show cross-resistance to CD95-induced apoptosis and the killing by CTLs. We propose that NO production could initially act as an autocrine suicide or paracrine killing mechanism in cells undergoing malignant transformation. However, once failed, the outcome is fatal. NO promotes tumor formation by enhancing the selection of cells that can evade immune attack by acquiring apoptosis resistance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Nitric oxide plays key roles in physiological as well as pathological processes (1, 2, 3). NO functions as an intra- and intercellular signaling molecule shaping the immune response (4). During inflammation, NO produced from the inducible NO synthetase (NOS2) in response to proinflammatory cytokines is critical for short-term defense against infection and tumor cells (5). But during longer episodes of inflammation, NO is capable of causing tissue damage and can be genotoxic and mutagenic (6, 7, 8). Extensive evidence indicates that tissues undergoing chronic inflammation have a significantly higher risk for cancer (9). Furthermore, aberrant production of NO by induction of NOS2 can be acquired by tumors. Human tumors such as breast cancer, melanoma, bladder cancer, hepatocellular carcinoma, and colorectal cancer often express high levels of NOS2 (10, 11, 12, 13). The underlying roles that NO plays in tumor-host cell interactions are complex and may be dependent on the context and source of NO. For example, recent data using a genetic strategy in mice has provided evidence that NO can suppress tumorigenesis as p53–/–NOS2–/– mice developed tumors more rapidly than the corresponding wild-type mice (14). Furthermore, antitumor activity through NO production has been attributed to host macrophages (15). In contrast, significant experimental and clinical evidence suggests that tumor-derived NO is conducive to tumor progression and is detrimental to the host (16, 17, 18, 19).

One of the major apoptosis signaling pathways is the CD95 receptor/ligand system. Alterations in the control of apoptosis mediated through the CD95 system contribute to the pathogenesis of disorders such as cancer, autoimmunity, AIDS, and liver diseases (20, 21, 22, 23, 24, 25, 26). Acquiring reduced sensitivity to CD95-mediated apoptosis is common in cancer cells and may provide them with critical survival advantages ultimately promoting malignancy (27, 28, 29, 30). Furthermore, the selection for resistance to apoptosis-inducing signals may hinder elimination of tumors through immune surveillance. Tumor escape from immune surveillance has been hypothesized to result from the inability of the immune system to react to the tumor. CTLs have been consistently implicated in tumor surveillance and regression. One system which plays an effector role in immune surveillance is CD95-mediated apoptosis of tumor cells by infiltrating T lymphocytes. Thus, resistance to CD95-mediated apoptosis signals can conceivably lead to escape of tumor cells from immune surveillance (31, 32, 33, 34, 35, 36).

In many studies, cells are exposed to NO through the use of donor drugs. The methods and substances used to deliver NO can significantly affect responses to NO, resulting in conflicting reports with respect to cytotoxicity and apoptosis. The total doses can be very high and rates of exposure can be well above physiological levels. Furthermore, the donor drugs themselves and their reaction by-products may affect cellular responses (37). Cell culture experiments with stable cell lines constitutively expressing NO have the disadvantage that selection occurs under constant NOS2 production. The possibility cannot be excluded that selection pressure is in part due to coping with constant NO exposure.

The molecular mechanisms underlying NO cytotoxicity are not completely unraveled. NO toxicity is generally thought to be mediated by mitochondria dysfunction, the DNA damage-p53 pathway (6, 38), and, more recently, endoplasmic reticulum (ER)3 dysfunction (39, 40, 41, 42). Recent work has identified signals derived from the ER during states of stress which can induce apoptosis. In mice, high levels of ER stress can activate caspase-12, an ER-associated death effector caspase, which cleaves the downstream caspases such as caspase-9-inducing apoptosis (43). In humans, a functional caspase-12 protein is not produced, because the gene is interrupted by a frame shift mutation resulting in a premature stop codon (44). The gene in humans with the highest homology to rodent caspase-12 is caspase-4. Human caspase-4 is localized to the ER membrane and is cleaved when cells are treated with ER stress-inducing reagents (45). Thus, caspase-4 can function as an ER stress-specific caspase in humans.

In this study, we examined the effects of endogenously produced NO on the CD95 system, on the action of CTLs, and of long-term NO exposure in cells with ecdysone-regulatable NOS2 expression. Our results demonstrate that the primary effect of endogenously produced NO is to enhance the sensitivity for CD95-induced apoptosis and the killing by CTLs. We could show that persistent exposure to NO induces apoptosis without additional stimuli and selects for cells with reduced sensitivity to NO and apoptosis. We report that NO-induced apoptosis is mediated through an ER stress mechanism, involving the induction of C/EBP-homologous protein (CHOP)/growth arrest and DNA damage-inducible gene 153 (GADD153), the ER chaperone Ig-binding protein (BiP), and caspase-4. Collectively, our findings suggest that NO production in tumor cells is initially detrimental for their survival but promotes selection of variant apoptosis-resistant cells favoring tumorigenic growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Chemicals

The following materials were purchased from commercial sources: anti-NOS2 Ab, anti-caspase-3, -8, and -9 Ab (Apotech); anti-BiP Ab (Affinity Bioreagents), anti-CHOP Ab (Santa Cruz Biotechnology); anti-{alpha}-actin Ab (Biocarta); anti-rabbit, anti-mouse, and anti-goat secondary Abs, alkaline phosphatase-conjugated (Sigma-Aldrich); propidium iodide (Sigma-Aldrich); JC-1 (Molecular Probes); Griess assay reagent (Alexis Biochemicals); protein concentration determination kit (Pierce); ponasterone A (Invitrogen Life Technologies); Cell Titer Glo cell viability assay kit (Promega); Lipofectamine 2000 reagent (Invitrogen Life Technologies), CDP-star Western blot detection system (Applied Biosystems).

Cell culture and cloning

EcR293 cells (human embryonic kidney (HEK) 293, stably transfected with pVgRXR/zeocin regulator vector), were grown in DMEM supplemented with 10% FCS, 5% penicillin and streptomycin, 5% L-glutamine, 5% HEPES buffer at 37°C in a humidified incubator with 5% CO2. The cDNA of NOS2 (inducible NO synthase) containing the entire open reading frame, and partial 5' and 3' untranslated sequences of NOS2 were cloned into an ecdysone-inducible mammalian expression vector, pIND(SP1)/neomycin (Invitrogen Life Technologies). EcR293 cells were seeded in 6-well plates at 50% confluence, and transfected with pIND-NOS2 plasmid by Lipofectamine 2000 reagent according to the manual. The stable transfected cells were selected in medium containing both G418 (400 µg/ml) and zeocin (400 µg/ml). The clones growing up after ~4 wk of selection were picked up and further analyzed.

Western blot analysis

EcR293-NOS2 clones were seeded in 6-well plates, induced by ponasterone A. After a 48-h induction, the cells were washed two times with PBS and lysed in 1% Nonidet P-40 solution. For Western blot, 40 µg of proteins were loaded on a 8% SDS-PAGE gel, separated, and transferred to a polyvinylidene difluoride membrane. The proteins were detected by indirect immunofluorescence and visualized by the CDP-star detection system.

Apoptosis analysis

EcR293-NOS2 cells were seeded in 12-well plates at 60% confluency, and the cells were induced for 24 h with ponasterone A. For apoptosis induction, the cells were treated with 300 ng/ml anti-APO-1 Ab for 24 h. DNA fluorescence was measured by FACS analysis after fixation with cold 100% ethanol and staining by propidium iodide according to a published method (46). Apoptotic sub-G1 cell nuclei were quantified using a FACSCalibur cytometer.

Griess assay

EcR293-NOS2 clones were seeded in 6-well plates, induced by ponasterone A at 0, 0.5, 1, 3, 10 µM concentrations for 48 h. Medium was collected for nitrite determination and the cells were harvested for normalizing each sample to total protein concentration. Griess assay was performed according to manufacturer’s protocol. Nitrite concentrations were determined at an OD570 by comparison with standard solutions of sodium nitrite prepared in the same culture media and presented in correlation to protein content.

Caspase activity analysis

The EcR293-NOS2 cells were seeded in 6-well plates, induced by 5 µM ponasterone A for 24 h and treated with 300 ng/ml anti-APO-1. After 0, 3, 6, 12, 24, 36 h treatment (or until 120 h for caspase-4 activation assay during ER stress), the cells were harvested in 1x PBS/1% Nonidet P-40 solution, and centrifuged at 13,000 x g for 5 min at 4°C. Protein concentration of the resulting supernatant was determined by using the Micro BCA protein assay kit (Pierce) protein assay kit. Supernatant containing, respectively, 10, 40, 50, and 50 µg of proteins was diluted with assay buffer and incubated for 2 h at 37°C with 500 µM caspase-3, -4, -8, or -9 substrate (Ac-DEVD-AFC, Ac-LEVD-AFC, Ac-IETD-AFC, Ac-LEHD-AFC), respectively. Cleavage of the substrate was monitored at 405 nm and expressed in relative proteolytic activity.

Mitochondrial membrane potential analysis

Cells were seeded in 6-well plates, induced by ponasterone A for 24 h and treated with anti-APO-1 (300 ng/ml) for another 24 h. Then the cells were harvested and stained with JC-1 for 1 h at 37°C. After washing with PBS, the cells were resuspended and stained with JC-1. Red fluorescence of mitochondrial JC-1 aggregates was measured immediately by FACS analysis.

Cytotoxic T cell killing

An alloreactive A2.1-specific polyclonal CTL line (CD8 Allo-A2) was established as reported (47). An A2.1-restricted CTL clone specific for the Flu M1 58–66 peptide (CD8 x A2KbFlu) has been described previously (48). CTLs were cocultured with 51Cr-labeled EcR293-NOS2 cells at the indicated E:T ratios. 51Cr release was measured after 4.5 h. The peptide specificity of the CD8 x A2KbFluM1 CTLs was controlled by their failure to lyse target cells loaded with an irrelevant peptide.

Viability measurement

Cell viability was determined by the CellTiter-Glo viability assay kit (Promega). For this purpose, the cells were seeded in 96-well plates and treated as described. After culturing for the indicated times, CellTiter-Glo reagent was added as recommended by the manufacturer. Luminescence was determined in a luminescence ELISA reader (Tecan).

Immunofluorescence

The cells were seeded in an 8-chamber slide with coverslip bottoms (BD Biosciences) and induced with 5 mM ponasterone A for 48 h. After washing with PBS, the cells were fixed in 4% paraformaldehyde and stained for NOS2 or CHOP. Cy3-labeled anti-rabbit IgG (Dianova) was used as secondary Ab. Hoechst 33342 (Molecular Probes) was used to stain nuclei. The cells were imaged directly in the chambers using a Zeiss LSM 510UV laser scanning microscope.

Selection of NO-resistant clones

EcR293-NOS2 no. 33 cells were seeded in 6-well plates at ~5–10% confluency and cultured in selection medium (containing 300 µg/ml G418, 300 µg/ml zeocin, and 10 µM ponasterone A). The medium was changed every 2 days. After ~3 mo of continuous selection, the clones were analyzed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Construction and regulatable expression of NOS2 in HEK-293 cells

The full-length human NOS2 gene was cloned into pIND/neomycin, which contains the ecdysone-responsive element sequences and the neomycin-resistance gene. The resulting vector, pIND-NOS2, was transfected in EcR293 cells which have stably integrated pVgRxR vector carrying both the retinoid X receptor and the ecdysone receptor and a zeocin gene. Stable transfectants were selected which showed NOS2 expression in response to ponasterone A, an ecdysone analog, in a dosage-dependent manner (Fig. 1A). Immunofluorescent staining of EcR293-NOS2 clone no. 33 for NOS2 was consistent with Western blot results. NOS2 expression was detected in induced cultures localized in punctate structures (Fig. 1B). We monitored ecdysone-inducible NO production by Griess assay (Fig. 1C). These data demonstrate that EcR293-NOS2 cells produce NO in a regulatable and dose-dependent manner in response to ponasterone A treatment. NO levels correlate with NOS2 protein levels as characterized by Western blot and immunofluorescence analysis. A time course for induction of NOS2 after treatment with ponasterone A revealed maximal expression levels between 12 and 48 h, however, continuous expression of NOS2 could be detected throughout the time course of the experiment (Fig. 1D).


Figure 1
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  		FIGURE 1. Establishing ecdysone regulatable EcR293-NOS2 clones A, Western blot for NOS2 expression. EcR293-NOS2 clone nos. 45, 53, 33, 35, 15 were induced by ponasterone at concentrations of 0, 0.5, 1, 3, 10 µM for 2 days before Western blot analysis. B, Immunofluorescence staining of NOS2 (red) in EcR293-NOS2 cells. Cells were uninduced (–Pon) or induced by 5 µM ponasterone (+Pon), immunostained for NOS2, and imaged by a Zeiss LSM 510 UV laser scanning microscopy. Nuclei are stained in blue. Bar in lower panel depicts 10 µm. C, NO production was analyzed by Griess assay after induction by ponasterone A. Nitrite levels in medium of EcR293-NOS2 nos. 15, 33, 35, 45, 53 in response to ponasterone A are dose dependent. D, Time kinetic of NOS2 expression. Clone no. 33 was induced by 5 µM ponasterone A for 0, 8, 12, 24, 36, 48, 60 h. Western blot analysis indicates that NOS2 expression peaks between 12 and 48 h.

 
Effect of NO on CD95-mediated apoptosis

The effect of endogenously produced NO on CD95-mediated apoptosis was examined in EcR293-NOS2 cell clones. Fig. 2A shows CD95 apoptosis induction monitored by the amount of cells with a sub-G1 DNA content in the absence or presence of ponasterone A. As the doses of ponasterone A increased, apoptosis induced by treatment with a constant concentration of the agonistic CD95 Ab, anti-APO-1, proportionally increased compared with controls (Fig. 2B). This effect was observed in three independently derived cell clones. These results indicate that induced NOS2 expression is responsible for an increased CD95 apoptosis sensitivity.


Figure 2
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  		FIGURE 2. NO increases apoptosis sensitivity. A, CD95-induced apoptosis in EcR293 and EcR293-NOS2 cells. EcR293 and EcR293-NOS2 no. 33 cells were untreated or treated with 10 µM ponasterone A for 24 h. Apoptosis was induced by addition of 300 ng/ml anti-APO-1 for 24 h to the cultures. Apoptotic sub-G1-peak indicates increasing apoptosis sensitivity in NOS2-expressing EcR293-NOS2 no. 33 cells. B, Apoptosis after CD95 activation in NOS2-expressing clones (EcR293-NOS2 nos. 45, 53, 33). The cells were induced with ponasterone A at concentrations of 0, 3, or 10 µM for 24 h and treated with 300 ng/ml anti-APO-1 for an additional 24 h. Apoptotic sub-G1 cells were quantified by FACS analysis. C, CTL-mediated apoptosis is increased in NOS2-expressing cells. Coculture experiments were performed with allogenic or peptide-specific CTL (CD8 Allo A2 and CD8 x A2kbFluM1) and EcR293-NOS2 no. 33 target cells, in which NOS2 was induced with 5 mM ponasterone A (+Pon) or uninduced (–Pon) at the indicated E:T ratios. Target cells were pulsed with the relevant FluM1 peptide ({diamondsuit}, {blacksquare}) or an irrelevant peptide ({blacktriangleup}, x). Dose-dependent induction of NOS2 in EcR293-NOS2 no. 33 cells correlates with apoptosis induced by CTLs (CD8 allo A2) at an E:T ratio of 3:1.

 
Sensitivity of EcR293-NOS2 cells to CTL-induced apoptosis

The CD95/CD95L system (APO-1/Fas) mediates perforin-independent cytotoxic T cell killing of tumor cells. To test whether cytolytic effector functions of CTLs are influenced by NO, we performed coculture experiments with allogenic or peptide-specific CTL (CD8 Allo A2 and CD8 x A2kbFluM1) and EcR293-NOS2 cells as targets. We found a substantial increase in the cytotoxic action of T cells in EcR293-NOS2 targets when NOS2 was expressed by induction with ponasterone A (Fig. 2C). This effect was dependent on the doses of ponasterone A (Fig. 2C) indicating that increasing NO levels in targets correspondingly increased their susceptibility for the killing by CTLs. The NO-producing target cells pulsed with an irrelevant peptide were not lysed during the time course of the coculture experiments excluding the possibility of autocrine suicide (Fig. 2C). Taken together, our results demonstrate that NO increases the sensitivity of EcR293-NOS2 cells to apoptosis induced by CD95 stimulation and the cytolytic action of CTLs.

Apoptosis signaling

To investigate whether the increased sensitivity to CD95-induced apoptosis is possibly due to increased CD95R expression, we measured CD95 surface expression by immunofluorescence staining and FACS analysis. No changes in CD95 levels were detected between the induced and uninduced cells (data not shown). We further tested the effect of NOS2 induction on caspases activated during CD95 apoptosis in cells uninduced and induced for production of NO. As shown in Fig. 3A, activation of caspases-3, -8, and -9 after CD95 stimulation was enhanced in cells when NOS2 was induced. These results indicate that NO can increase the CD95 apoptosis sensitivity in EcR293-NOS2 cells. Mitochondria act as a central integrator of the apoptotic response. Using JC-1, we analyzed the loss of mitochondrial membrane potential after expression of NOS2 and activation of the CD95 apoptotic pathway. Qualitative changes in the mitochondria membrane potential induced by exposure to NO and treatment with agonistic CD95 Abs (anti-APO-1) were observed using laser scan microscopy (data not shown). Aggregates of JC-1 in normal cells appeared red in the mitochondria, whereas damaged cells with mitochondrial membrane potential loss appeared with decreasing red due to loss of JC-1 aggregates. Quantitative analysis by flow cytometry revealed that short-term NO induction sensitized cells to mitochondria membrane potential loss during CD95-induced apoptosis in a dose-dependent manner (Fig. 3B).


Figure 3
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  		FIGURE 3. Apoptosis signaling and mitochondrial membrane depolarization. A, Caspase activity in EcR293 and EcR293-NOS2 no. 33 cells. The cells were induced with ponasterone A at concentrations of 0, 0.5, 1, 3, 10 µM for 24 h and treated with 300 ng/ml anti-APO-1 for an additional 24 h. Caspase-3, -8, and -9 activity was analyzed with fluorescent caspase substrates. B, Loss of mitochondrial membrane potential in EcR293-NOS2 no. 33 cells after CD95 stimulation. NOS2 expression was induced by treatment with 0, 0.5, 1, 3, 10 µM ponasterone A for 24 h, then 300 ng/ml anti-APO-1 was added to induce apoptosis for an additional 24 h. JC-1 staining shows increased loss of mitochondrial membrane potential dependent on NOS2 expression. Quantification is shown in the right panel.

 
NO induces apoptosis

We examined the effects of long-term exposure to NO in EcR293-NOS2 cells. Fig. 4A shows growth curves of EcR293 cells in the presence or absence of 5 µM ponasterone A. There was no apparent differences in the cell numbers of the control EcR293 cells treated with or without ponasterone A. However, induction of NOS2 in EcR293-NOS2 cells led to drastic reduction in cell number compared with cultures left untreated (Fig. 4B). To ensure that the growth retardation is dependent on the expression level of NOS2, we treated EcR293 and EcR293-NOS2 with various doses of ponasterone A for 6 days, and then measured the cell viability. Compared with untreated controls, ponasterone A treatment did not influence the viability of the EcR293 cells, whereas in EcR293-NOS2 cells a dose-dependent reduction of the cell viability reaching 50% reduction with 10 µM ponasterone A was observed (Fig. 4C). Thus, the striking growth reduction was due to the effect of NO production.


Figure 4
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  		FIGURE 4. The influence of endogenous NO on cell growth. A, Growth properties of EcR293 cells. The cells treated with (+) or without (–) ponasterone A (5 µM) throughout the time of the experiment. At the indicated time points, cells were measured with Cell-Titer Glo viability assay. B, Growth properties of EcR293-NOS2 no. 33 cells. The cells were treated with or without ponasterone A (5 µM) throughout the time of the experiment and the cells were measured with luminescence viability assays at the indicated time points. C, Growth properties of EcR293 and EcR293-NOS2 no. 33 cells treated with different concentrations of ponasterone A for 8 days and the viability measured. Experiments were performed in quadruplicates and mean values were calculated and plotted. Error bars indicate SDs. D, Apoptosis in EcR293-NOS2 no. 33 after prolonged NOS2 induction. Apoptotic sub-G1 cells were quantified by FACS analysis.

 
To investigate whether long-term NO exposure induced apoptosis or caused growth arrest, we measured the cell cycle status and apoptosis in uninduced and induced EcR293-NOS2 cells. No significant alternations in the cell cycle of cultures were observed as measured by FACS analysis (data not shown). As shown in Fig. 4D, NOS2 expression increased apoptosis in EcR293-NOS2 cells substantially over uninduced controls after 8 days of culture. These results indicate that endogenous NO reduces the cell number by inducing apoptosis in the cultures.

ER and mitochondrial stress after NO induction

To explore the possible mechanisms underlying the induction of apoptosis in cells exposed to longer periods of NO, NOS2-expressing EcR293 cells were treated with thapsigargin, an ER-stress inducer. The cytotoxic effects of thapsigargin increased with higher levels of NOS2 suggesting that NO enhanced the sensitivity of cells to toxic stimuli including ER stress (Fig. 5A). Expression of BiP, CHOP/GADD153, both induced during ER stress, and caspase-4, mediating ER-derived apoptosis signals, was analyzed. Procaspase-9, the mitochondrial caspase, that is recruited to the apoptosome where it is activated and released, was also investigated. EcR293-NOS2 cells were treated with increasing concentrations of ponasterone A to induce NOS2 expression and then Western blot analysis was performed for the expression level of the ER chaperone BiP (glucose-regulated protein 78). In untreated cells, low levels of BiP were detected. As the time of NO exposure increased, the amount of BiP increased (Fig. 5B) indicating that NO stress induced BiP expression. The expression of the transcription factor CHOP/GADD153, which is induced during ER stress, was analyzed by immunostaining and Western blot. Compared with uninduced cells, the expression of CHOP/GADD153 was increased and was localized primarily in the nucleus under induction of NOS2 by ponasterone A (Fig. 5C). Caspase-9 expression was monitored by immunoblotting during 72 h of continuous NO exposure. Caspase-4 activity, measured by caspase assay was observed after 48–72 h of NO exposure suggesting that NO induced ER stress leading to activation of caspase-4 (Fig. 5D). Similarly, the activated form of caspase-9 increased during these cultivation conditions (Fig. 5D). Long-term exposure to NO resulted in extensive mitochondrial membrane potential loss without additional apoptosis stimulation (Fig. 5E). Together, these results show that endogenously produced NO activates ER and mitochondrial stress pathways which may play a direct or indirect role in triggering apoptosis.


Figure 5
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  		FIGURE 5. NO activates ER stress. A, NOS2 expression enhances ER-stress induced apoptosis in EcR293-NOS2 no. 33 cells. Cells were induced with the indicated ponasterone A concentrations for 2 days and treated with the ER-stress inducer thapsigargin at 0, 0.5, 1, 2.5 µM for an additional 2 days. Viability of the cells was measured by a luminescence assay. B, Time kinetic of BiP expression in EcR293-NOS2 no. 33 cells after NOS2 induction. Cells were induced with ponasterone A with 5 µM ponasterone A for the designated times before Western blot analysis. C, Confocal laser scanning image of EcR293-NOS2 no. 33 cells with NOS2 induction (+Pon) and without induction (–Pon) stained for CHOP (green). CHOP localizes primarily to cell nuclei (red) after induction. Overlay shows colocalization of CHOP and stained nuclei as yellow in the lower right panel. Western blot analysis shows increased expression level of CHOP after NOS2 induction. D, Caspase-4 activity and procaspase-9 cleavage after NOS2 induction. The cells were induced with 5 µM ponasterone A for the designated times and caspase-4 activity was measured with a fluorescent caspase substrate. E, JC-1 staining shows a time-dependent loss of the mitochondrial membrane potential in cells where NOS2 is induced with ponasterone A for 48 and 72 h.

 
Continuous NO stress selects for NO- and apoptosis-resistant cells

If long-term NO exposure induces apoptosis, then all NOS2-expressing cells should be eliminated or counteract this death stimuli by acquiring apoptosis resistance. To test this hypothesis, we cultured EcR293-NOS2 cells in the presence of 10 µM ponasterone A to continuously produce NO. We observed significant cell death in these cultures but cell colonies grew which were selected for further cultivation. After ~3 mo of culturing in the presence of ponasterone A, the cell clones were analyzed for NO production by Griess assay. All the cell clones which survived cultivation in the presence of 10 µM ponasterone A also produced NO (Fig. 6A). These results demonstrate that the survival of the colonies was due to NO resistance and not merely to loss of ponasterone A-inducible NO production. To assess the effects of the NO-resistant clones on apoptosis, cells were treated with agonistic CD95 Abs and apoptosis was analyzed by quantification of sub-G1 cells. Interestingly, we found that the NO-resistant cell lines were also cross-resistant to CD95-induced apoptosis (Fig. 6B). Coculture experiments with different peptide-specific CTLs demonstrate an acquired resistance to CTL killing (Fig. 6, C and D). These results support the idea that chronic exposure to NO selects cells with reduced sensitivity to apoptosis.


Figure 6
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  		FIGURE 6. NO-resistant cells are cross-resistant to apoptosis. A, NO-resistant clones produce NO. The media from cells, resistant for continuous NO stress were analyzed for nitrite concentrations by Griess assay. B, CD95-mediated apoptosis is attenuated in NO-resistant EcR293-NOS2 cell clones. Induced cells were treated for 24 h with 400 ng/ml anti-APO-1 and apoptotic sub-G1 cells were quantified by FACS analysis. C and D, CTL-mediated apoptosis is decreased in NO-resistant clones. Coculture experiments were performed with allogenic (C) or peptide-specific (D) CTLs (CD8 allo A2 and CD8 x A2kbFluM1) and EcR293-NOS2 no. 33 or NO-resistant clones R7 or R8.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
NO is associated with tumor development and progression (2, 6, 7, 17, 18, 19). We have analyzed the cellular mechanisms by which endogenous NO exposure in tumor cells can modulate apoptosis sensitivity, giving particular attention to apoptosis mediated by the CD95 system. To this end, we constructed an NOS2 expression vector driven by an ecdysone-inducible promoter; the vector was then introduced into EcR293 cells. Ecdysone-dependent NOS2 expression and NO production was detectable in a number of cell clones. Using this cell culture model, we tested short- and long-term effects of endogenous NO exposure on CD95-mediated apoptosis. Our results demonstrate a dramatic enhancement of CD95-mediated apoptosis during acute episodes, with increased caspase activity and mitochondrial membrane depolarization. Furthermore, in coculture experiments, we show that the cytolytic action of T cells against NO-producing tumor cells is enhanced. In summary, short-term NO exposure should reduce tumor formation. In line with this observation, continuous intermediate-term exposure to NO initially reduced cell numbers and induced apoptosis. However, finally, we observed a selection for cells that were resistant to both NO and apoptosis after chronic NOS2 expression.

Diseases associated with chronic inflammation predispose individuals to cancer in the affected tissues. NO has attracted considerable attention as a possible link between inflammation and tumorigenesis for several reasons. First, due to its free radical nature, NO is capable of reacting at different points in the cell by modifying proteins and DNA. Second, NO is produced during inflammation by induction of NOS2. Third, NOS2 expression is observed in many tumors and is continued to be expressed in advanced tumors suggesting that it has a positive role for the tumor (49). Moreover, there is extensive experimental evidence to support a role for NO in accelerating tumor development but the cellular and molecular mechanisms by which NO promotes tumor formation and growth are not fully understood. One of the proposed mechanisms linking NO to carcinogenesis is the ability of NO to act as an endogenous mutagen and to modify intracellular signaling. For example, recent reports have demonstrated an important role for p53 as modulator of genotoxicity and mutagenesis during NO-induced oxidative DNA damage (6, 7, 50, 51). Furthermore, the accumulation of DNA lesions during NO stress may be in part attributed to the inhibition of DNA repair enzymes by NO (52). Thus, continuous genomic insults due to NO production may have negative effects on genome stability promoting tumor progression.

However, short-term cytotoxic effects of NO have been clearly documented; the possible targets being the ER and mitochondria. Our studies demonstrate that one of the short-term effects of endogenous NO production is enhanced sensitivity for the CD95 apoptosis pathway. Prolonged exposure to NO induced ER and mitochondrial stress causing activation of caspase-4 and -9 leading to apoptosis. Furthermore, cytokines, secreted by lymphokine-activated killer cells, can induce endogenous NO synthesis and apoptosis in colon cancer cells (53). This non-contact-dependent cell toxicity mechanism supports a role for NO in T cell-mediated tumor defense.

Our results highlight a paradoxical role of NO during tumorigenesis. On one hand, NO induces apoptosis and sensitizes cells to apoptosis mediated by CD95, and on the other hand, NO promotes survival of tumor cells. On the basis of our results, we propose that the apoptosis-enhancing effect of NO initially reduces tumor formation but finally results in the elimination of cells with a fully functional apoptotic response and the retention of a subpopulation of cells with an aberrant or attenuated response to death-inducing signals. Chronic exposure to NO thus facilitates the clonal evolution of a population of cells that can circumvent normal death-inducing signals, including those derived from CTLs during immune surveillance. One of the hallmarks of cancer cells is apoptosis resistance. We postulate selection for apoptosis-resistant tumor cells may be the product of repeated genomic and cellular insults due to continuous NO exposure. Analysis of the cell clones we have obtained during these experiments will allow us to further characterize the molecular mechanisms involved in developing NO and apoptosis resistance in tumor cells.


   Acknowledgments
 
We thank Christina Groessgen and Mirjam Weisser for excellent technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 553 C15) and the Mainzer Forschungsförderung program (to S.S.), the Deutsche Forschungsgemeinschaft (SFB 432 B6), and the Deutsche Krebshilfe (to S.S. and P.R.G.), and in part by grants from the Deutsche Forschungsgemeinschaft (SFB 432 A3) (to M.T.), the MAIFOR program (to J.K. and M.T.), the Sixth Framework Programme of the European Commission (Contract No. 502983) (to M.T.), and the Laupitz Foundation (to M.T.). M.T. is a José Carreras Leukemia Foundation Professor. Back

2 Address correspondence and reprint requests to Dr. Susanne Strand, I. Dept. of Internal Medicine, Obere Zahlbacher Strasse 63, Johannes Gutenberg-University, 55101 Mainz, Germany. E-mail address: sstrand{at}uni-mainz.de Back

3 Abbreviation used in this paper: ER, endoplasmic reticulum; CHOP, C/EBP-homologous protein; GADD153, growth arrest and DNA damage-inducible gene 153; BiP, Ig-binding protein. Back

Received for publication July 8, 2005. Accepted for publication January 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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