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[Cancer Research 65, 8461-8470, September 15, 2005]
© 2005 American Association for Cancer Research

Immunology

Nitric Oxide Sensitizes Tumor Cells to Dendritic Cell–Mediated Apoptosis, Uptake, and Cross-Presentation

Jian Huang1, Tomohide Tatsumi6, Eva Pizzoferrato2,5, Nikola Vujanovic1,3,5 and Walter J. Storkus1,4,5

Departments of 1 Immunology, 2 Medicine, 3 Pathology, and 4 Dermatology, University of Pittsburgh School of Medicine; 5 University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania; and 6 Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan

Requests for reprints: Walter J. Storkus, Department of Dermatology and Immunology, University of Pittsburgh School of Medicine, 1.32e Hillman Cancer Center, University of Pittsburgh Cancer Institute Research Pavilion, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-3240; Fax: 412-623-7704; E-mail: storkuswj{at}msx.upmc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells are professional antigen-presenting cells associated with efficient antigen processing and presentation to T cells. However, recent evidence also suggests that dendritic cells may mediate direct tumoricidal functions. In this study, we investigated the mechanism by which murine dendritic cells mediate the apoptotic death of murine lymphoma cell lines, and whether dendritic cell effector function could be enhanced by preconditioning tumor cells with the protein phosphatase inhibitor nitric oxide (NO) by altering the balance of proapoptotic/antiapoptotic proteins in the treated cells. We observed that NO donor compound sensitized lymphomas to dendritic cell–mediated cytotoxicity in vitro. Both immature and spontaneously matured bone marrow–derived dendritic cells (SM-DC) were capable of inducing tumor cell apoptosis, with SM-DCs serving as comparatively better killers. Fas ligand (FasL)-Fas engagement proved important in this activity because elevated expression of membrane-bound FasL was detected on SM-DCs, and dendritic cells derived from FasL-deficient mice were less capable of killing NO-sensitized tumor cells than wild-type dendritic cells. As FasL-deficient dendritic cells were still capable of mediating a residual degree of tumor killing, this suggests that FasL-independent mechanisms of apoptosis are also involved in dendritic cell–mediated tumor killing. Because NO-treated tumor cells displayed a preferential loss of survivin protein expression via a proteasome-dependent pathway, enhanced tumor sensitivity to dendritic cell–mediated killing may be associated with the accelerated turnover of this critical antiapoptotic gene product. Importantly, NO-treated tumor cells were also engulfed more readily than control tumor cells and this resulted in enhanced cross-presentation of tumor-associated antigens to specific T cells in vitro.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells are the most efficient antigen-presenting cells (APC) and seem largely responsible for the induction of primary immune responses (1). In a tumor-bearing host, dendritic cells are believed to cross-present tumor peptide epitopes in the context of MHC class I and II molecules to stimulate specific (CD8+ and CD4+) T-cell responses, respectively. Interestingly, apoptotic tumor cells have been reported to provide dendritic cells with a comprehensive source of tumor antigens used to cross-prime effector T cells (2, 3). The origin of apoptotic tumor bodies in situ has been generally presumed to be the result of spontaneous tumor cell death or to be mediated by cytotoxic effector natural killer (NK) or T cells. However, tumor infiltration by these two killer cell populations may not be a common event early in tumor progression and cross-priming would occur more efficiently if dendritic cells themselves were able to directly induce tumor cell apoptosis as an innate function thereby generating an antigenic substrate for consequent T-cell cross-priming in situ.

It has been previously shown that human dendritic cells, particularly after treatment with proinflammatory cytokines, are capable of mediating the in vitro apoptosis of tumor cells via a mechanism involving membrane-bound Fas ligand (FasL) or tumor necrosis factor (TNF)–related apoptosis inducing ligand (TRAIL; refs. 4–7). Recently, we reported that immature human dendritic cells are preferentially able to directly induce the apoptotic death of cancer cell lines and fresh tumor cells in vitro. This cytotoxicity was antagonized by inclusion of blocking antibodies or receptor-Fc constructs specific to TNF family ligands, showing that human immature dendritic cells mediate tumoricidal activity by simultaneous engagement of multiple transmembrane TNF family ligands, TNF, FasL, LT{alpha}1/ß2, and TRAIL (8, 9). In the current report, we have determined that spontaneously matured murine bone marrow–derived dendritic cells (SM-DC) are superior to immature dendritic cells in mediating the apoptotic death of B and T lymphoma cells in vitro.

Because our current observations and previous reports (5–7) suggest that dendritic cell–mediated killing of tumor cells in vitro was most evident at high dendritic cell-to-tumor cell ratios, which may not accurately reflect the physiologic situation where low frequencies of tumor-infiltrating dendritic cells are observed (10), we sought means by which tumor cells could be further sensitized to this lytic pathway to define a potentially translatable therapy for established cancers. In particular, we chose to sensitize tumor cells to dendritic cell–mediated apoptosis by pharmacologically altering the balance of the antiapoptotic/proapoptotic protein expression in tumor cells using a nitric oxide (NO) donor compound PAPA-NO. This agent is capable of oxidizing and ablating the enzymatic activity of protein phosphatases that regulate the proteasome-dependent turnover of many cellular proteins (11–14), including the proapoptotic/antiapoptotic proteins in the Bcl-2 and the inhibitor of apoptosis (IAP) families, as well as the inhibitor {kappa}B (I{kappa}B; refs. 15–18). In the present study, we report that NO induces the enhanced proteasome-dependent degradation of survivin in tumor cells, in association with the increased sensitivity of treated tumor cells to dendritic cell–mediated apoptosis and uptake, leading to enhanced cross-presentation of tumor cell–expressed antigens to specific T cells in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and mice. The A20 cell line is an H-2d B lymphoma cell line (American Tissue Culture Collection, Manassas, VA). The EL4 cell line is an H-2b T lymphoma cell line and EG7 is a derivative of EL4 cells that contains the chicken ovalbumin (OVA) transgene (ref. 19; both cell lines were the kind gifts of Dr. Louis Falo III, University of Pittsburgh). All cell lines were maintained in complete media (CM: RPMI 1640 supplemented with 10% FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, 10 mmol/L L-glutamine; all reagents from Invitrogen, Carlsbad, CA) and 50 µmol/L 2-mercaptoethanol (Sigma Chemical Co., St. Louis, MO) at 5% CO2 and 37°C in a humidified incubator. The mock- and mouse FasL-transfected L5178Y T lymphoma cell lines were obtained from Dr. Hideho Okada (Department of Surgery, University of Pittsburgh, PA). The cells were cultured in CM supplemented with 1 mmol/L sodium pyruvate and 0.1 mmol/L nonessential amino acid (both reagents from Invitrogen). G418 sulfate (Geneticin, from Invitrogen) was used at 1 mg/mL for maintenance of the transfected line. The DO11.10 cell line is a T-cell hybridoma that recognizes I-Ad MHC class II/OVA peptide323-339 complex (a generous gift from Dr. Louis Falo III). This CD4+ T cell line was maintained in DMEM supplemented with 10% FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, and 10 mmol/L L-glutamine. Female 6- to 8-week-old BALB/c and FasL-deficient (gld) mice (both from Jackson Laboratory, Bar Harbor, ME) were maintained in microisolator cages and handled under aseptic conditions per an Institutional Animal Care and Use Committee–approved protocol.

Peptide. The OVA323-339 and control C. Falciparum (MCS326-345) I-Ad binding peptides were synthesized using 9-fluorenylmethoxycarboxyl chemistry by the University of Pittsburgh Cancer Institute's (UPCI) Peptide Synthesis Faculty. The peptides were >95% pure based on high-performance liquid chromatography, with identities validated by tandem mass spectrometric (MS/MS) analyses done by the UPCI Protein Sequencing Facility.

Generation of dendritic cells in vitro from bone marrow. Dendritic cells were generated as previously described (20). Briefly, bone marrow cells were cultured in CM supplemented with 1,000 units/mL recombinant murine granulocyte/macrophage colony-stimulating factor (mGM-CSF; Schering-Plough, Kenilworth, NJ) and 500 units/mL recombinant mIL-4 (Peprotech, Inc., Rocky Hill, NJ) at 37°C in a humidified, 5% CO2 incubator for 7 days (immature) or 9 days (mature). Dendritic cells were then purified using CD11c magnetic beads (MACS; Miltenyi Biotec., Auburn, CA) and subjected to the phenotypic and functional analyses described below. In some experiments, an additional metrizamide gradient centrifugation (20) was done before CD11c MACS isolation, resulting in purities exceeding 99%.

Experimental design of nitric oxide sensitization of tumor cells. Tumor cells were pretreated with the NO donor compound PAPA-NO (a generous gift from Dr. Lawrence Keefer, National Cancer Institute, Bethesda, MD). After a 1-hour incubation, the media were removed and cells maintained in fresh media for additional 18 hours at 37°C. The cells were then harvested and used as targets in cytotoxicity assays or lysed for Western blot analyses.

Flow cytometry. All the flow cytometric analyses were done using a Coulter Epics XL (Beckman Coulter, Fullerton, CA) flow cytometer. The phenotypic analyses of dendritic cells and cell surface expression of Fas or FasL were done as described previously (8, 21).

To evaluate the cytotoxicity of dendritic cells against tumor cells, dendritic cells were cocultured with tumor cells for 4 hours at an effector/target (E/T) ratio of 5:1 at 37°C. The cells were then harvested and stained on ice for 30 minutes with PE-conjugated anti-mouse B220 monoclonal antibody (mAb, BD PharMingen, San Jose, CA, for A20) or anti-mouse H-2Kd mAb (BD PharMingen, for EG7) to distinguish tumor cell populations from dendritic cells, and FITC-conjugated pan-caspase inhibitor z-VAD-FMK (VAD-FMK; Promega, Madison, WI) as an early apoptosis marker. Results are reported based on the percentage of cytotoxicity calculated as the number of apoptotic tumor cells divided by total number of tumor cells gated.

To evaluate antigen uptake, untreated or PAPA-NO-treated tumor cells were stained with Hoechst 33342 (Molecular Probes, Carlsbad, CA) for 30 minutes before coculture with dendritic cells for 18 hours at a dendritic cell/tumor ratio of 5:1. Uptake of Hoechst-positive tumor cells by dendritic cells was then analyzed using a MoFlo cytometer (Cytomation, Fort Collins, CO).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. To assess the apoptotic sensitivity of A20 cells to agonist anti-mouse Fas mAb (Jo2; BD PharMingen), A20 cells were cultured in the presence or absence of the antibody at the indicated concentrations for 24 hours. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were then done, as described previously (8).

In vitro antigen cross-presentation assays. EG7 and EL4 cells were pretreated with PAPA-NO before coculture with dendritic cells for 24, 36, or 48 hours at a dendritic cell/tumor ratio of 5:1. DO11.10 T hybridoma cells were then added to wells at a T/dendritic cell ratio of 1:1. After 18 hours, the supernatants were harvested and interleukin 2 (IL-2) production was quantitated by specific ELISA (Endogen, Woburn, MA). IL-2 secretion by hybridoma cells stimulated with dendritic cells pulsed with 400 ng/mL of the OVA323-339 synthetic peptide served as a positive control in these experiments.

Western blot analyses. A20 cells were treated with PAPA-NO before lysed and subjected to Western blot analysis as described previously (22). Mouse anti-ß-actin antibody (Abcam, Cambridge, MA) was used as a loading control. Densitometric quantitations were done using a White/UV Transilluminator (UVP Products, Upland, CA) and analyses done using Labworks software (UVP Products).

Statistical analyses. Statistical differences between groups were evaluated using a two-tailed Student's t test with statistical significance defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Spontaneously matured bone marrow–derived murine dendritic cells mediate superior killing of tumor cell lines. Based on our previous report that cultured human monocyte–derived dendritic cells were capable of mediating the apoptotic death of tumor cell lines in vitro (8), we analyzed whether bone marrow–derived murine dendritic cells generated in GM-CSF + IL-4 supported cultures were similarly capable of killing murine tumor cells. Approximately 30 x 106 CD11c+ (MACS isolated; 90-95% pure) dendritic cells per BALB/c donor mouse were obtained reproducibly on day 7 (immature dendritic cell) and day 9 (spontaneously matured; i.e., SM-DC) of culture. SM-DCs expressed markedly higher levels of MHC class II molecules (I-Ad) and costimulatory molecules (CD80 and CD86) than their immature day 7 counterparts (Fig. 1A).



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  		Figure 1. SM-DCs induce a greater degree of apoptosis in A20 B-lymphoma cells than immature dendritic cells. Bone marrow–derived dendritic cells were generated using a bulk-culture method (20). On days 7 and 9, immature dendritic cells and SM-DCs, respectively, were purified using CD11c MACS beads. Dendritic cells were stained with specific antibodies against (A) MHC class II and costimulatory molecules or (B) FasL, and flow cytometry was done. The results are presented as (A) overlays of single-color histograms obtained with isotype-matched control (open histograms) and specific antibodies (filled histograms) and (B) single-color histograms with MFI indicated in each histogram panel. C, immature dendritic cells and SM-DCs were cocultured with A20 tumor cells at a dendritic cell/tumor cell ratio of 2.5:1 or 5:1 for 4 hours. Cells were stained with PE-conjugated anti-B220 antibodies and FITC-conjugated pan-caspase inhibitor z-VAD-FMK and analyzed by fluorescence-activated cell sorting. Tumor cell population was gated on B220 positive staining. Percentage of cytotoxicity (top right quadrant) was calculated based on the fraction of apoptotic tumor cells (B220+VAD-FMK+) divided by total number of tumor cells (B220+). Representative of three independent experiments.

 
Because our recent studies showed a role for TNF family ligands expressed on human immature dendritic cells in the direct anticancer effector function mediated by these cells (9), we assessed the potential role of the murine homologues in dendritic cell–mediated killing of the A20 B-lymphoma cell line. As it has been reported that activated B cells express abundant membrane-bound Fas and are sensitive to Fas-mediated apoptosis (23), we initially targeted an analysis of the FasL-Fas pathway. Compared with expression on the FasL-transfected L5178Y (L5178.FasL; positive control) cell line, FasL expression on dendritic cells was clearly evident, albeit at a lower level (Fig. 1B). Notably, SM-DCs expressed higher levels of FasL than immature dendritic cells, indicating that SM-DCs might be more effective at inducing the apoptotic death of Fas+ tumor cells. To test this hypothesis directly, we incubated A20 tumor cells with either immature dendritic cell or SM-DCs for 4 hours at 37°C at a dendritic cell/tumor cell ratio of 2.5:1 or 5:1. Cells were then stained with antibodies against the B-cell marker B220 to distinguish tumor cells from dendritic cells, in addition to the fluorescent caspase substrate z-VAD-FMK to detect apoptotic cells. As shown in Fig. 1C, in the absence of dendritic cells, only 5.4% of A20 cells were determined to undergo spontaneous apoptosis. However, cocultures of dendritic cells and A20 cells contained significantly greater frequencies of apoptotic A20 cells. Of interest, killing was dendritic cell dose dependent and regardless of the E/T ratio evaluated, SM-DCs were more cytotoxic against A20 cells than immature dendritic cells. Because effector cell function seemed to correlate with the relative expression level of membrane-bound FasL, these data suggest an important functional role for FasL in dendritic cell–mediated tumor cell apoptosis.

To provide further support for the role of the Fas-FasL pathway in dendritic cell–mediated tumoricidal activity, we analyzed A20 tumor cells for their level of expression of membrane-bound Fas by flow cytometry. We detected uniform, high levels of Fas expression on the surface of A20 cells (Fig. 2A). However, because expression of Fas is necessary but not sufficient for cells to undergo Fas-mediated apoptosis (23), we also determined whether ligation of Fas could trigger A20 cells to become apoptotic (in 24-hour MTT assays) after addition of various concentrations of agonist anti-Fas antibody (Jo2) to cultures. As shown in Fig. 2B, the anti-Fas antibody induced A20 cell death in a dose-dependent manner, whereas the isotype-matched control antibody had little effect on A20 viability. Similarly, nearly 30% of A20 cells became apoptotic after a 4-hour coincubation with L5178Y.FasL cells but not control L5178Y cells (Fig. 2C). These data show that A20 tumor cells were sensitive to the apoptosis mediated through FasL-Fas pathway induced by antibody agonist, FasL+ transfected cell lines or FasL+ dendritic cells.



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  		Figure 2. Dendritic cell–mediated A20 tumor apoptosis is mediated at least partially through the FasL/Fas pathway. A, A20 cells express membrane-bound Fas. Cell surface expression of Fas was assessed using a three-step flow cytometry technique, with the results depicted as overlays of isotype-matched control (open histogram) and Fas antibody (filled histograms) staining profiles. B, agonist anti-Fas antibody (Jo2) induces apoptosis in A20 tumor cells. A20 cells were incubated with anti-Fas antibody at the indicated concentrations for 24 hours at 37°C and cytotoxicity was evaluated by MTT assay. Wells with medium alone were used as background control (BG) and wells for total viability/spontaneous death of untreated cells (TS) contained only medium and A20 cells. Experimental (Ex) wells contained A20 cells and the indicated doses of anti–Fas Ab (Jo2). Percentage of cytotoxicity was calculated using the following formula: % cytotoxicity = [(TS-BG) – (Ex-BG)] / (TS –BG) x 100%. C, ligation of FasL by Fas triggers apoptosis in A20 cells. FasL-transfected (L5178Y.mFasL) or mock-transfected L5178Y cells (L5178Y) were coincubated with A20 tumor cells at an E/T ratio of 5:1 for 4 hours. Cells were stained with a PE-labeled B220-specific mAb and FITC-labeled caspase activation marker z-VAD-FMK. Dendritic cell cytotoxicity was calculated as described above. D, bone marrow–derived dendritic cells were generated from H-2d wild type (wtDC) or FasL-deficient gld mice (gldDC). Dendritic cells were then coincubated with A20 tumor cells at a dendritic cell/tumor ratio of 5:1 for 4 hours. Cells were stained and analyzed as described above. Representative of two independent experiments.

 
As a further demonstration of the relevance of FasL in dendritic cell–mediated tumor killing and to determine whether additional Fas-independent pathways were also involved in this effector mechanism, we assessed the tumoricidal activity of bone marrow–derived dendritic cells generated from FasL-deficient mice. Whereas dendritic cells generated from wild-type animals efficiently induced the apoptosis of A20 cells, dendritic cells derived from FasL-deficient mice had ~3-fold less tumor cell killing capacity (Fig. 2D). These data indicate that FasL plays a dominant role in dendritic cell–mediated tumor apoptosis and that Fas-independent pathway(s) may also partially contribute to this killing activity.

Pretreatment of A20 tumor cells with a nitric oxide donor increases their sensitivity to dendritic cell–mediated, Fas-dependent apoptosis. Because A20 cells were moderately sensitive to Fas-dependent killing mediated by dendritic cells, we next sought to determine whether tumor cells could become conditionally sensitized to such killing using pharmacologic agents. In particular, we chose to attempt to alter the balance of antiapoptotic/proapoptotic protein expression in tumor cells by either up-regulating the expression of proapoptotic proteins or down-regulating antiapoptotic proteins, or both, to theoretically make the cells more susceptible to dendritic cell–mediated apoptosis. We attempted to modulate this balance by affecting the functional activity of protein phosphatases that regulate the levels of proapoptotic/antiapoptotic protein expression based on their state of phosphorylation, making them targets for E3 ligases and consequently, the proteasome (24). Because NO is known to oxidize critical cysteine residues within protein phosphatase catalytic sites, we pretreated A20 cells with the NO donor compound PAPA-NO for 1 hour and maintained the cells in fresh media for an additional 18 hours. The cells were then stained with FITC-conjugated pan-caspase inhibitor z-VAD-FMK to determine the level of cellular apoptosis. As shown in Fig. 3A, as a single agent, PAPA-NO induced apoptosis in A20 cells in a dose-dependent manner. More importantly, at low concentrations (i.e., <100 µg/mL), PAPA-NO was nontoxic and did not promote significant A20 apoptosis by itself. However, the NO-conditioned tumor cells seemed to become sensitized to apoptosis mediated by anti-Fas agonist antibody (Fig. 3B) or L5178Y.FasL cells (Fig. 3C).



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  		Figure 3. A20 cells treated with low doses of PAPA-NO are sensitized to Fas-mediated apoptotic death. A, NO donors induced A20 cell apoptosis in a dose-dependent manner. A20 tumor cells were preincubated with PAPA-NO at the indicated concentrations for 1 hour at 37°C. Medium was then replaced and the tumor cells maintained in culture overnight. Cells were then stained with VAD-FMK-FITC to detect apoptosis. Representative of two independent experiments. B, NO-treated A20 cells were sensitized to anti-Fas antibody–induced apoptosis. Tumor cells were treated with PAPA-NO at the indicated concentrations for 1 hour at 37°C and then incubated in fresh media in the presence or absence of anti-Fas antibodies overnight. The MTT assay was used to determine cytotoxicity. Representative of three independent experiments. C, NO-treated A20 cells were sensitized to FasL-mediated apoptosis. A20 cells were treated with NO donors as described in (A). Eighteen hours posttreatment, FasL- or mock-transfected L5178Y cells were added to culture for 4 hours and cytotoxicity was evaluated as described above. Representative of two independent experiments.

 
We next determined whether NO-pretreated A20 cells were also sensitized to dendritic cell–mediated apoptosis. Figure 4A provides the cumulative results derived from three independent experiments done. We observed that although treatment with the NO donor was nontoxic to A20 cells, it greatly enhanced their sensitivity to immature dendritic cell–mediated apoptosis. Tumor cells pretreated with 25 and 50 µg/mL of PAPA-NO showed significant differences in their apoptotic frequencies versus the nontreated tumor control group (P ≤ 0.05). Similar results were also obtained using SM-DCs as effector cells (Fig. 4B). Given minor concerns that the small contaminant CD11c-negative cell populations might be at least partially responsible for mediating tumor cell apoptosis, we did an additional density gradient centrifugation step using metrizamide before CD11c MACS bead selection. This resulted in CD11c+ dendritic cell purities exceeding 99%, with no significant change in tumoricidal function mediated by these effector cells (data not shown).



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  		Figure 4. NO pretreatment significantly sensitizes A20 tumor cells to both immature dendritic cell (DC)– and SM-DC-mediated apoptosis. A20 cells were pretreated with PAPA-NO as described in Fig. 3 and then cocultured with immature dendritic cell (A) or SM-DC (B) for 4 hours in cytotoxicity assays. The statistical analyses of three independent experiments are provided in (A). B, representative of two independent experiments. C, bulk splenocytes were cultured at 37°C in the presence of 10 µg/mL LPS. Three days later, cells were harvested and treated with or without PAPA-NO at a final concentration of 25 µg/mL before being cocultured with SM-DCs as described above. Cells were gated on B220+ events and the data presented as overlays of histograms obtained with B cell only culture (open histograms) and B-DC coculture (filled histograms). Representative of two independent experiments.

 
Given additional concerns that such preconditioning could also affect the sensitivity of normal tissue targets to dendritic cell–mediated killing, we analyzed whether SM-DCs could promote the apoptotic death of normal B cells or B cells pretreated with a source of NO. B-cell blasts were generated from BALB/c splenocytes cultured with lipopolysaccharide (LPS) for 3 days. As shown in Fig. 4C, SM-DCs did not induce apoptosis in normal B-cell blasts and B-cell pretreatment with PAPA-NO did not increase the observed activated caspase staining when compared with controls.

Sensitization of A20 cells to dendritic cell–mediated apoptosis by nitric oxide involves both Fas-dependent and Fas-independent pathways. Because dendritic cell killing of A20 cells seemed at least partially mediated through a Fas-dependent pathway, we initially hypothesized that this would also be the case for NO-treated tumor cells. Indeed, the capacity of dendritic cells generated from FasL-deficient mice to kill NO-treated A20 cells was greatly impaired when compared with dendritic cells derived from wild type mice (Fig. 5A). These results suggest that the Fas/FasL-mediated pathway contributes partially to the observed tumoricidal activity associated with dendritic cells. At the same time, these data also suggest that FasL-independent pathway(s) are also significantly involved in dendritic cell–mediated killing of A20 tumor cells. The most likely potential candidates would be other TNF family members, such as TNF{alpha} and TRAIL, based on our previous results published for human dendritic cells (9).



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  		Figure 5. Treatment of A20 tumor cells with PAPA-NO enhances their sensitivity to dendritic cell–induced apoptosis through both Fas-dependent and Fas-independent pathways. A, A20 tumor cells were pretreated with PAPA-NO at the indicated concentrations before being coincubated with dendritic cells that were generated from either wild-type mice (wtDC) or FasL-deficient mice (gldDC). Dendritic cell cytotoxicity against tumor cells was analyzed as described in the Fig. 2 legend. B, dendritic cells generated from the bone marrow of FasL-deficient mice were cultured for 1 hour in the presence or absence of 20 µg/mL of anti-TNF antibody and/or anti-TRAIL antibody, or isotype control antibody. Dendritic cells were then added to A20 cell cultures that has been pretreated with 50 µg/mL of PAPA-NO and analyzed for dendritic cell–mediated cytotoxicity as shown in (A). Data of density plots have been converted into histograms. Representative of two independent experiments.

 
To test these possibilities, we did blocking experiments to interfere with the residual tumor cell killing associated with FasL-deficient dendritic cells using antagonist antibodies. Dendritic cells generated from FasL-deficient mice were preincubated with individual or combinations of antibodies specific for either TNF{alpha} or TRAIL, before addition to NO-pretreated A20 tumor cell cultures. As shown in Fig. 5B, disruption of either TNF/TNF-R or TRAIL/TRAIL-R interactions served to further (partially) inhibit dendritic cell–mediated killing of NO-treated A20 cells. In this setting, anti-TNF{alpha} antibody seemed less effective than anti-TRAIL antibodies in blocking the residual killing mediated by FasL-deficient dendritic cells. Indeed, because the simultaneous disruption of all three ligand/receptor (FasL/Fas, TNF/TNF-R, and TRAIL/TRAIL-R) pairs seemed to inhibit dendritic cell–mediated killing of NO-treated A20 cells to a degree comparable with that noted for dendritic cells whose FasL and TRAIL interactions were simultaneously disrupted, TNF{alpha} likely plays a very minor role in this model of apoptosis.

Therefore, these data suggest that pretreatment of A20 with NO donors sensitizes these target cells not only to FasL-induced apoptosis but also to (at least) TRAIL-mediated apoptosis. However, the minor but detectable level of apoptosis observed under conditions in which the Fas, TNF, and TRAIL pathways were coordinately blocked may suggest the further minor participation of additional molecule(s) in dendritic cell tumoricidal activity.

Treatment of tumor cells with nitric oxide donors preferentially accelerates survivin degradation through a proteasome-dependent pathway in association with enhanced sensitivity to dendritic cell. To further investigate the mechanism(s) by which NO donors sensitize A20 tumor cells to dendritic cell–mediated killing, we analyzed the NO-treated tumor cells for alterations in their expression of proapoptotic and antiapoptotic proteins. One of the most well known protein families that regulate cell survival and apoptosis is the Bcl-2 family, in particular, Bcl-2 and Bcl-XL (25). The protective function of these proteins is, in great part, due to the formation of inactivating heterodimers with the proapoptotic protein Bax. An imbalance among these proteins, in favor of the proapoptotic activities, might sensitize tumor cells to apoptosis. However, we did not observe any differences in the normalized (versus ß-actin) expression levels of the Bcl-2, Bcl-XL, or Bax proteins within 18 hours of NO treatment (Fig. 6A). In contrast, we noted that expression of another antiapoptotic protein, survivin, which belongs to the IAP family, was markedly down-regulated in an NO donor dose-dependent manner (Fig. 6A).



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  		Figure 6. NO donor pretreatment preferentially accelerates survivin protein turnover in A20 cells. A, whole cell lysates of PAPA-NO treated or untreated A20 tumor cells were prepared for Western blot analyses using anti-survivin, anti-Bcl-2, anti-Bcl-xL, and anti-Bax antibodies. B, A20 cells were treated with 300 µg/mL of PAPA-NO. Media were then changed and tumor cells maintained in CM in the presence or absence of 0.2 or 1 µmol/L lactacystin (LC) for 4 hours. Whole cell lysates were prepared for immunoblot analyses using anti-survivin antibody, as described in the Materials and Methods. Normalized by expression of ß-actin and representative of three (A) or two (B) independent experiments.

 
Because the degradation of survivin is ubiquitin-proteasome dependent (22), we assessed whether the NO-induced effects on tumor cell survivin expression could be inhibited by the proteasome inhibitor lactacystin. A20 cells that were treated with NO donors alone exhibited a 50% reduction in survivin expression by 4 hours posttreatment with lactacystin completely blocking this process (Fig. 6B). Indeed, even control A20 tumor cells treated with lactacystin displayed a slight elevation in their expression of survivin thus indicating that constitutive survivin turnover is blocked by this agent.

Pretreatment of tumor cells with nitric oxide donor results in increased dendritic cell uptake and cross-presentation of tumor antigen to specific T cells. We further hypothesized that increased ability of dendritic cell to mediate the apoptotic death of NO-treated tumor cells might lead to a more efficient protocol for apoptotic body uptake and consequent cross-presentation of tumor antigens in vitro. To properly evaluate uptake of apoptotic body by dendritic cells using a flow cytometry–based analysis, we chose to prelabel tumor cells for 30 minutes with the cell-permeable, UV-fluorescent nucleic acid dye Hoechst 33342. After staining with Hoechst 33342, tumor cells were washed and cultured in media for an additional 3 hours to allow for any unbound dye to diffuse from the cells. To validate that the staining procedure did not promote tumor cell apoptosis directly, labeled tumor cells were cultured overnight and stained with propidium iodide (PI) to evaluate viability. At a final concentration of 4 µg/mL Hoechst 33342, 100% of tumor cells were stained and this signal was maintained for at least 24 hours, without any alteration in cell viability. Moreover, labeling was stable because the supernatant harvested from labeled cells was not able to consequently label fresh tumor cells (data not shown).

To evaluate apoptotic body uptake by dendritic cells, we treated tumor cells with PAPA-NO, stained them with Hoechst 33342 before addition of dendritic cells, and 24 hours later, harvested these cultures for flow cytometric analyses. Cells were counterstained with anti-mouse B220 mAb (to distinguish A20 cells from dendritic cell) and PI. Viable dendritic cells (PI–B220–) were gated and analyzed for uptake of Hoechst 33342+ apoptotic bodies. As shown in Fig. 7A, dendritic cells cocultured with NO-treated A20 cells exhibited much higher MFI than those cultured with untreated tumor cells. This suggests that more apoptotic tumor cells were taken up by dendritic cells per unit time if the tumor cells were pretreated with the NO donor compound.



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  		Figure 7. PAPA-NO pretreatment enhances dendritic cell–mediated apoptotic body uptake and cross-presentation of OVA to tumor-specific T cells. A, pretreatment of A20 tumor cells with PAPA-NO enhanced dendritic cell uptake of apoptotic tumor cells. A20 tumor cells were treated with 25 µg/mL of PAPA-NO as described above and stained with 4 µg/mL Hoechst 33342 for 30 minutes. Cells were washed and maintained in culture for additional 3 hours before washing again and adding dendritic cells at a dendritic cell/tumor ratio of 5:1. After 24 hours, the cells were harvested and stained with anti-mouse B220 mAb and PI before flow cytometry analysis. Viable dendritic cells (B220-PI-) were gated for analysis and MFI of Hoechst 33342 staining is reported. B, pretreatment of EG7 (OVA+; H-2b) cells with PAPA-NO sensitized them to dendritic cell (H-2d)-mediated apoptosis. EG7 tumor cells were treated with 25 µg/mL PAPA-NO and coincubated with dendritic cells, as described above. The cells were then stained with anti-mouse H-2Kd mAb and VAD-FMK-FITC to distinguish EG7 (Kb) and dendritic cell (Kd) and to determine viability, respectively, in the flow cytometry assays. C, NO donor pretreatment increases uptake of tumor apoptotic body by dendritic cells. EG7 tumor cells were treated with PAPA-NO and stained with 1 µg/mL Hoechst 33342 before addition of dendritic cells, as described in Materials and Methods. Twenty-four hours later, cells were stained with anti-mouse H-2Kd mAb and PI. The dendritic cell populations were gated on H-2Kd-positive and PI-negative events and MFI of Hoechst 33342 staining was evaluated. D, cross-presentation of tumor antigen to specific T cells was enhanced if EG7 cells were pretreated with PAPA-NO. EL4 and EG7 tumor cells were pretreated with PAPA-NO at the indicated concentrations before the addition of dendritic cells. After 24, 36, or 48 hours, DO11.10 T-cell hybridoma was added to the culture and IL-2 production (per 106 DO11.10 cells) within 24 hours was tested by ELISA. Positive controls included dendritic cell pulsed with the OVA323-339 peptide, with negative controls including dendritic cell pulsed with the MCS peptide and cohorts including the OVA– EL4 tumor cell line. Representative of two independent experiments. *, P < 0.05 versus corresponding data from untreated (i.e., 0 µg/mL NO) controls.

 
We next asked whether this NO-dependent uptake of tumor cells would result in the improved cross-presentation of tumor-associated antigens to specific T cells. To test this, we used an OVA model antigen system; H-2b OVA-expressing EL4 (EG7) tumor cells and the DO11.10 T cell hybridoma that recognizes the OVA323-339 peptide epitope presented in the context of I-Ad. As was the case for A20 B lymphoma cells, both EL4 and its derivative T-lymphoma cell line EG7 were sensitized to dendritic cell–mediated killing by PAPA-NO pretreatment (Fig. 7B; data not shown), providing support for the generality of this mechanism. Also similar to A20 cells, uptake of apoptotic EG7 cells by dendritic cells was also increased if these tumor cells were pretreated with PAPA-NO (Fig. 7C).

To access cross-presentation of tumor antigens to T cells, H-2b EG7 (OVA+) or EL4 (OVA–) tumor cells were pretreated with PAPA-NO, before addition of H-2d dendritic cells to culture. To allow for optimal antigen uptake, processing, and presentation, we coincubated the dendritic cell tumor mix for 24 to 48 hours, before addition of I-Ad-restricted DO11.10 T hybridoma cells. IL-2 production by the hybridoma cells was used as an indicator for T-cell activation. As shown in Fig. 7D, T cells stimulated with dendritic cells loaded with synthetic OVA323-339 peptide produced high levels of IL-2, whereas no response was detected for dendritic cells pulsed with the control MCS peptide. Similarly, minimal IL-2 was produced when these T cells were cultured with dendritic cells and EL4 (OVA–) tumor cells. On the contrary, when the EG7 (OVA+) tumor cells were pretreated with 10 or 25 µg/mL of PAPA-NO, IL-2 production from responder T cells was elevated to a level comparable with that of the dendritic cell + OVA323-339 peptide cohort and this level of production was significantly greater than that observed for T cells + dendritic cell + untreated EG7 cells (with maximal effects observed after 36-48 hours processing periods). To further exclude the possibility of dendritic cell uptake of soluble OVA protein elaborated from EG7 cells, we also established transwell cultures in parallel in which untreated or treated EG7 were added to upper wells, whereas dendritic cells and T cells were loaded into the bottom wells. Under these conditions, IL-2 was not detected in cultures at any time point evaluated (data not shown), indicating that the activation of T cells in Fig. 7D was primarily due to the direct cross-presentation of OVA peptide by H-2d dendritic cell uptake of apoptotic body, which is accentuated by tumor cell pretreatment with the NO donor compound.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current study, our central hypothesis was that apoptotic signals mediated via TNF family ligand/receptor complexes allow for dendritic cell–mediated tumor cell apoptosis, thereby defining a simplified cellular pathway capable of supporting the cross-priming of specific antitumor T cells. Our data provide novel information that multiple TNF family ligands participate in murine dendritic cell–mediated tumoricidal activity against B-lymphoma cells in vitro and that this innate dendritic cell function can be further enhanced by pretreating tumor cells with the phosphatase inhibitor NO to preferentially extinguish tumor cell expression of the antiapoptotic protein survivin.

Previous reports have shown that FasL may be used by murine dendritic cells to kill susceptible target cells, such as T cells (4, 26). Here, we report that multiple TNF family ligands, FasL and TRAIL (and possibly TNF{alpha}), participate in dendritic cell tumoricidal activity. Among the molecules that are involved, FasL clearly played a dominant role, because FasL-deficient dendritic cells were reduced over 65% in their killing capacity. Approximately 85% of the dendritic cell–mediated cytotoxicity was ablated when all three ligand/receptor (i.e., FasL, TRAIL, and TNF{alpha}) pairs were simultaneously disrupted. However, a low but detectable level of residual tumor cell apoptosis still remained under such conditions, suggesting the minor involvement of an as yet undefined molecule(s). Whereas we were unable to assess its importance in the current study, one obvious candidate in this regard is lymphotoxin-{alpha}/ß, which belongs to the same TNF ligand superfamily and has been reported to be used by human dendritic cells to kill tumor cell lines in vitro (9).

Fas-induced apoptosis of B cells has been extensively evaluated in the past (27). Naive murine B cells express low levels of Fas, but upon activation, expression of Fas is markedly up-regulated (23). Notably, this is not sufficient to enhance B-cell sensitivity to Fas-induced apoptosis. Engagement of B-cell receptor or expression of antiapoptotic proteins may induce resistance to Fas-mediated B-cell death (27). Expression of p53 may also be required for B cells to undergo apoptosis. Cross-linking CD40 induces normal B cells to proliferate and differentiate but causes many tumor cell lines to undergo apoptosis. Murine B lymphoma lines that contain mutated p53, such as A20 cells, are induced to undergo apoptosis upon CD40 cross-linking, whereas other lines that express wild-type p53 are comparatively resistant (28). In this context, it is perhaps not surprising that we observed that murine bone marrow–derived dendritic cells were selectively cytotoxic against A20 B lymphoma cells and were nontoxic to normal naive B cells or LPS-activated B cells (either not treated or pretreated with NO donor compounds). This provides translational support that adoptively transferred dendritic cells may selectively kill B lymphoma cells without damaging their normal counterparts in vivo.

Several groups have also shown that treatment of tumor cells with ionizing radiation, chemotherapeutic, or other pharmacologic agents can sensitize them to TNF family ligand–induced apoptosis (29–32). Our findings extend this work by suggesting that the pretreatment of tumor cells with a source of NO markedly increases their apoptotic sensitivity to dendritic cells that express (at least) three TNF family ligands including FasL, TRAIL, and TNF{alpha}. Because these molecules are coordinately used by CTLs (33, 34) and NK cells (21) to kill tumor cells, the in vivo treatment of tumor lesions with locoregional NO would be anticipated to sensitize tumor cells to not only dendritic cell– but also to specific CTL- and NK-mediated apoptosis, particularly under proinflammatory, delayed-type hypersensitivity-type conditions.

The mechanism by which NO sensitizes tumor cells to dendritic cell–mediated killing was also investigated. Among the antiapoptotic/proapoptotic proteins analyzed, survivin was found to be uniquely down-regulated after NO treatment. Survivin is a recently identified antiapoptotic protein that is aberrantly expressed in cancer cells but is not expressed by normal, differentiated adult tissues (35). Consistent with the homeostatic turnover/degradation of survivin being regulated by the ubiquitin-proteasome pathway (22), we observed that the proteasome inhibitor lactacystin blocked survivin degradation in NO-treated A20 cells. Based on our current data and previous reports (36, 37), we hypothesize that the mechanism of NO-induced survivin degradation and tumor sensitization involves the following pathway: (a) treatment of tumor cells with an NO donor compound rapidly inhibits the function of protein phosphatase (b) thus leading to the sustained activation of protein kinases, such as p34(cdc2)-cyclin B1, that are capable of phosphorylating survivin (36); (c) enhanced phosphorylation of survivin facilitates its ubiquitination and (d) its consequent proteasome-dependent degradation. As a result of altering the balance between antiapoptotic and proapoptotic proteins towards a more proapoptotic phenotype, the death signals delivered by dendritic cells, under such sensitized conditions, are enhanced. From an immunotherapeutic point of view, NO-facilitated survivin degradation also provides an experimental basis for the design of new combinational therapy strategies in which a "sensitizing agent" and autologous dendritic cells could be sequentially injected intratumorally. This would promote tumor cell death in an immunologically important manner yielding enhanced dendritic cell–mediated cross-priming of specific T cells in vivo as supported in principle by the results of in vitro experiments reported in Fig. 7.

Previous reports suggested the NO has a dichotomous nature. Instead of enhancing apoptosis, some groups showed that NO could inhibit apoptosis induced by different stimuli (38–41). These differences in behavior may be explained by the chemistry of NO. After the treatment of NO donor, released NO is thought to either react directly with radicals, or form intermediates that can facilitate nitrosylation reaction (42). Because various NO donors may induce either direct or indirect effects, this could yield different end effects on treated cells. For instance, it has been reported that pretreatment of lung fibroblast cells with PAPA-NO and DEA-NO enhanced tumor sensitivity to cisplatin-mediated death, whereas other NO donors failed to do so (43). Hence, these distinct effects may be ultimately useful in defining clinical strategies for the use of NO donor compounds in cancer patients.

Because survivin is a shared tumor-associated antigen that is differentially expressed in a variety of malignancies and can be recognized by specific CTLs (44), it is also possible that proteasome-derived survivin peptides could be expressed at higher stochastic levels on NO-treated tumor cells, making them better targets for specific T-cell clearance. We are currently evaluating the ability of survivin peptide-specific CTLs to differentially recognize control versus NO-treated tumor cells in vitro.

Somewhat in contrast to our previous studies of human dendritic cells (8, 9), phenotypically mature murine dendritic cells (i.e., SM-DCs) rather than immature murine dendritic cells mediated superior levels of tumor cell killing. Because the lytic capacity of SM-DCs seemed comparable with that of LPS-matured dendritic cells (data not shown), the manner in which dendritic cells become "mature" may not profoundly influence this functional aspect of these APCs.

Of translational importance, SM-DCs and immature dendritic cells seemed equally capable of engulfing apoptotic tumor bodies after first mediating their demise (data not shown). In addition, our data has shown that sensitization of tumor cells to dendritic cell–mediated killing by NO donor treatment also leads to enhanced apoptotic body uptake and cross-presentation of tumor-associated antigens to tumor-specific T cells. This provides confidence that the adoptive transfer of SM-DCs ± NO intratumorally may lead to improved cross-presentation of tumor-derived epitopes to T cells in vivo. The coapplication of NO in situ may also affect dendritic cell function however, as suggested by an equivocal literature. Hence, although one group has showed that glioma induces apoptosis in dendritic cells via inducible NO synthase (45), others have presented contradictory data that NO-treated dendritic cells not only reduced the growth of the highly tumorigenic and poorly immunogenic B16 melanoma but also caused tumor regression and improved animal survival (46). Clearly, the direct testing of such combinational therapies in murine tumor models warrants further evaluation.

In summary, our study shows that murine dendritic cells mediate tumor cell apoptosis and that such lysis may be increased by tumor cell pretreatment with NO-elaborating agents in association with the depletion of tumor cell survivin protein levels. These findings provide a better understanding of important connections between the innate and adaptive immune effector functions of dendritic cells and provide support for the development and preclinical testing of combinational immunotherapy approaches integrating tumor cell apoptosis sensitization regimen(s) and intratumoral dendritic cell administration.


   Acknowledgments
 
Grant support: NIH grant R01 CA63350 (W.J. Storkus).

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.

We thank Drs. William Chambers and Amy Wesa for their careful review and critique of this article during its preparation, Dr. Hideho Okada for his generosity in providing the mFasL-transfected L5178Y and control L5178Y cell lines, Dr. Louis Falo for providing the EL4 and EG7 cell lines and the DO11.10 T cell hybridoma, Dr. Per Basse (Department of Pathology, University of Pittsburgh, Pittsburgh, PA) for providing Hoechst 33342 nucleic acid dye, and Dr. Lawrence Keefer for providing the NO donor compound PAPA-NO.

Received 2/24/05. Revised 6/16/05. Accepted 7/ 1/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Steinman RM. The dendritic cells system and its role in immunogenicity. Annu Rev Immunol 1991;9:271–96.[CrossRef][Medline]
  2. Albert ML, Sauter B, BharDwaj N. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 1998;392:86–9.[CrossRef][Medline]
  3. Hoffmann TK, Meidenbauer N, Dworacki G, Kanaya H, Whiteside TL. Generation of tumor-specific T lymphocytes by cross-priming with human dendritic cells ingesting apoptotic tumor cells. Cancer Res 2000;60:3542–9.[Abstract/Free Full Text]
  4. Suss G, Shortman K. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. J Exp Med 1996;183:1789–96.[Abstract/Free Full Text]
  5. Fanger NA, Maliszewski CR, Schooley K, Griffith TS. Human dendritic dells mediate cellular apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). J Exp Med 1999;190:1155–64.[Abstract/Free Full Text]
  6. Liu S, Yu Y, Zhang M, Wang W, Cao X. The involvement of TNF-{alpha}-related apoptosis-inducing ligand in the enhanced cytotoxicity of IFN-ß-stimulated human dendritic cells to tumor cells. J Immunol 2001;166:5407–15.[Abstract/Free Full Text]
  7. Yang R, Xu D, Zhang A, Gruber A. Immature dendritic cells kill ovarian carcinoma cells by a Fas/FasL pathway, enabling them to sensitize tumor-specific CTLs. Int J Cancer 2001;94:407–13.[CrossRef][Medline]
  8. Janjic BM, Lu G, Pimenov A, Whiteside TL, Storkus WJ, Vujanovic NL. Innate direct anticancer effector function of human immature dendritic cells. I. Involvement of an apoptosis-inducing pathway. J Immunol 2002;168:1823–30.[Abstract/Free Full Text]
  9. Lu G, Janjic BM, Janjic J, Whiteside TL, Storkus WJ, Vujanovic NL. Innate direct anticancer effector function of human immature dendritic cells. II. Role of TNF, lymphotoxin-{alpha} (1) ß (2), Fas ligand, and TNF-related apoptosis-inducing ligand. J Immunol 2002;168:1831–9.[Abstract/Free Full Text]
  10. Tolba KA, Bowers WJ, Muller J, et al. Herpes simplex virus (HSV) amplicon-mediated codelivery of secondary lymphoid tissue chemokine and CD40L results in augmented antitumor activity. Cancer Res 2002;62:6545–51.[Abstract/Free Full Text]
  11. Tonks NK, Neel BG. From form to function: signaling by protein tyrosine phosphatases. Cell 1996;87:365–8.[CrossRef][Medline]
  12. Li S, Whorton AR. Regulation of protein tyrosine phosphatase 1B in intact cells by S-nitrosothiols. Archives of Biochemistry and Biophysics 2002;410:269–79.
  13. Haendeler J, Hoffmann J, Rahman S, Zeiher AM, Dimmeler S. Regulation of telomerase activity and anti-apoptotic function by protein-protein interaction and phosphorylation. FEBS Lett 2003;536:180–6.[CrossRef][Medline]
  14. Takakura K, Beckman JS, MacMillan-Crow LA, Crow JP. Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite. Archives of Biochemistry and Biophysics 1999;369:197–207.[CrossRef][Medline]
  15. Tamura Y, Simizu S, Osada H. The phosphorylation status and anti-apoptotic activity of Bcl-2 are regulated by ERK and protein phosphatase 2A on the mitochondria. FEBS Lett 2004;569:249–55.[CrossRef][Medline]
  16. Chang YC, Lee YS, Tejima T, et al. Mdm2 and bax, downstream mediators of the p53 response, are degraded by the ubiquitin-proteasome pathway. Cell Growth Differ 1998;9:79–84.[Abstract]
  17. Jesenberger V, Jentsch S. Deadly encounter: ubiquitin meets apoptosis. Nat Rev Mol Cell Biol 2002;3:112–21.[CrossRef][Medline]
  18. Zhang HG, Wang J, Yang X, Hsu HC, Mountz JD. Regulation of apoptosis proteins in cancer cells by ubiquitin. Oncogene 2004;23:2009–15.[CrossRef][Medline]
  19. Fujii S-i, Shimizu K, Shimizu T, Lotze MT. Interleukin-10 promotes the maintenance of antitumor CD8+ T-cell effector function in situ. Blood 2001;98:2143–51.[Abstract/Free Full Text]
  20. Son Y, Egawa S, Tatsumi T, Redlinger REJ, Kalinski P, Kanto T. A novel bulk-culture method for generating nature dendritic cells from mouse bone marrow cells. J Immunol Methods 2002;262:145–57.[CrossRef][Medline]
  21. Kashii Y, Giorda R, Herberman RB, Whiteside TL, Vujanovic NL. Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells. J Immunol 1999;163:5358–66.[Abstract/Free Full Text]
  22. Zhao J, Tenev T, Martins LM, Downward J, Lemoine NR. The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner. J of Cell Science 2000;113:4363–71.[Abstract]
  23. Watanabe D, Suda T, Nagata S. Expression of Fas in B cells of mouse germinal center and Fas-dependent killing of activated B cells. International Immunology 1995;7:1949–56.[Abstract/Free Full Text]
  24. Tanaka K, Kawakami T, Tateishi K, Yashiroda H, Chiba T. Control of I{kappa}B{alpha} proteolysis by the ubiquitin-proteasome pathway. Biochimie 2001;83:351–6.[Medline]
  25. Juin P, Geneste O, Raimbaud E, Hickman JA. Shooting at survivors: Bcl-2 family members as drug targets for cancer. Biochimica et Biophysica Acta 2004;1644:251–60.[Medline]
  26. Lu L, Qian S, Hershberger PA, Rudert WA, Lynch DH, Thomson AW. Fas ligand (CD95L) and B7 Expression on dendritic cells provide counter-regulatory signals for T cell survival and proliferation. J Immunol 1997;158:5676–84.[Abstract]
  27. Mizuno T, Zhong X, Rothstein TL. Fas-induced apoptosis in B cells. Apoptosis 2003;8:451–60.[CrossRef][Medline]
  28. Hollmann AC, Gong Q, Owens T. CD40-mediated apoptosis in murine B-lymphoma lines containing mutated p53. Exp Cell Res 2002;280:201–11.[CrossRef][Medline]
  29. Woo SH, Park IC, Park MJ, et al. Arsenic trioxide sensitizes CD95/Fas-induced apoptosis through Ros-mediated upregulation of CD95/Fas by NF-kB activation. Int J Cancer 2004;112:596–606.[CrossRef][Medline]
  30. Shi R, Ong C, Shen H. Luteolin sensitizes tumor necrosis factor-a-induced apoptosis in human tumor cells. Oncogene 2004;23:7712–21.[CrossRef][Medline]
  31. Huerta-Yepez S, Vega M, Jazirehi H, Hongo F, Cheng G, Bonavida B. Nitric oxide sensitizes prostate carcinoma cell lines to TRAIL-mediated apoptosis via inactivation of NF-kB and inhibition of Bcl-xL expression. Oncogene 2004;23:4993–5003.[CrossRef][Medline]
  32. Fulda S, Debatin KM. Sensitization for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol. Cancer Res 2004;64:337–46.[Abstract/Free Full Text]
  33. Dorothee G, Vergnon I, Menez J, et al. Tumor-infiltrating CD4+ T lymphocytes express APO2 ligand (APO2L)/TRAIL upon specific stimulation with autologous lung carcinoma cells: role of IFN-{alpha} on APO2L/TRAIL expression and -mediated cytotoxicity. J Immunol 2002;169:809–17.[Abstract/Free Full Text]
  34. Kagi D, Vignaux F, Ledermann B, et al. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 1994;265:528–30.[Abstract/Free Full Text]
  35. Altieri DC. The molecular basis and potential role of survivin in cancer diagnosis and therapy. Trends Mol Med 2001;7:542–7.[CrossRef][Medline]
  36. O'Connor DS, Grossman D, Plescia J, et al. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc Natl Acad Sci U S A 2000;97:13103–7.[Abstract/Free Full Text]
  37. Chao JI, Kuo PC, Hsu TS. Down-regulation of survivin in nitric oxide-induced cell growth inhibition and apoptosis of the human lung carcinoma cells. J Biol Chem 2004;279:20267–76.[Abstract/Free Full Text]
  38. Kim Y-M, Talanian RV, Billiar TR. Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms. J Biol Chem 1997;272:31138–48.[Abstract/Free Full Text]
  39. Li J, Bombeck CA, Yang S, Kim Y-M, Billiar TR. Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes. J Biol Chem 1999;274:17325–33.[Abstract/Free Full Text]
  40. De Nadai C, Sestili P, Cantoni O, et al. Nitric oxide inhibits tumor necrosis factor-{alpha}-induced apoptosis by reducing the generation of ceramide. PNAS 2000;97:5480–5.[Abstract/Free Full Text]
  41. Kim Y-M, Kim T-H, Seol D-W, Talanian RV, Billiar TR. Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome c release. J Biol Chem 1998;273:31437–41.[Abstract/Free Full Text]
  42. Wink DA, Grisham M, Mitchell JB, Ford PC. Direct and indirect effects of nitric oxide. Biologically relevant chemical reactions in biology of NO. Methods Enzymol 1996;268:12–31.[CrossRef][Medline]
  43. Wink DA, Cook JA, Christodoulou D, et al. Nitric oxide and some nitric oxide donor compounds enhance the cytotoxicity of cisplatin. Nitric Oxide: Biology and Chemistry 1997;1:88–94.
  44. Schmidt SM, Schag K, Muller MR, et al. Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells. Blood 2003;102:571–6.[Abstract/Free Full Text]
  45. Yang T, Witham TF, Villa L, et al. Glioma-associated hyaluronan induces apoptosis in dendritic cells via inducible nitric oxide synthase: implications for the use of dendritic cells for therapy of gliomas. Cancer Res 2002;62:2583–91.[Abstract/Free Full Text]
  46. Perrotta C, Falcone S, Capobianco A, et al. Nitric oxide confers therapeutic activity to dendritic cells in a mouse model of melanoma. Cancer Res 2004;64:3767–71.[Abstract/Free Full Text]



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