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Fas Ligand System
Clinical Immunology Vol. 93, No. 1, October, pp. 65–74, 1999 Article ID clim.1999.4757, available online at http://www.idealibrary.com on
Apoptosis Provoked by the Oxidative Stress Inducer Menadione (Vitamin K 3) Is Mediated by the Fas/Fas Ligand System 1 Roberto Caricchio, 2,3 Dmitri Kovalenko,* ,2 William K. Kaufmann,* and Philip L. Cohen 3 Departments of Medicine and Microbiology/Immunology and *Departments of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
Fas (APO-1/CD95) is a member of the TNF/nerve growth factor receptor superfamily and is currently recognized as the principal cell surface receptor involved in the transduction of signals that induce apoptosis in lymphocytes and in a variety of tumor cells (3, 4). Crosslinking by its ligand (FasL) 4 rapidly induces the death-inducing signaling complex (5) and the activation of a cascade of interleukin-converting enzyme/ Ced3 proteases (6), now designated caspases (7). The Fas/FasL system plays crucial roles in homeostasis and self-tolerance (8, 9), in the activationinduced cell death of lymphocytes (10), and in the delimitation of immunoprivileged regions such as the eye and testis (11). Loss of function due to mutations in murine Fas ligand (gld), murine Fas (lpr), human Fas, or human FasL leads to lymphoproliferation, lymphadenopathy, and autoimmune diseases (12). The Fas/ FasL system is also implicated in the pathogenesis of other illnesses. Various tumor cells express FasL, potentially creating their own immunoprivileged sites (13). The deletion of CD4 1 T cells in human immunodeficiency virus-infected cells (14), cell-mediated immunity in acute graft-versus-host disease (15), and the destruction of myelin in multiple sclerosis all involve the Fas/FasL system (16). It has also recently been shown that gamma irradiation (17), UV irradiation (18), and drug-induced apoptosis of cancer cells (19) appear to involve the Fas/FasL system. Recent data indicate that apoptotic cell death takes place after oxidative stress (20). We wondered if menadione (vitamin K 3, VK 3), a potent oxidative stress inducer (21, 22), which induces cell cycle arrest and apoptosis (23, 24) and which has recently been realized to be a potential anticancer drug relatively free of adverse effects (23), might act via Fas/FasL. In the present study we demonstrate for the first time that VK 3-induced apoptosis is markedly reduced both in human T cell leukemia cell lines resistant to FasL killing and in cells from mice lacking functional FasL
Menadione, or vitamin K 3 (VK 3), a potent oxidative stress inducer, has been recently used as an effective and remarkably safe cytotoxic drug for treatment of several human tumors. VK 3 induces apoptotic cell death through a poorly understood mechanism. Here we show for the first time that VK 3-induced apoptosis requires the Fas/ FasL system. Spleen cells from both Fas- and FasL-deficient mice (C57BL/6-lpr and C57BL/ 6-gld, respectively) had much lower levels of VK 3 apoptosis in vitro compared to cells from control C57BL/6 mice. VK 3 cytotoxicity toward mouse splenocytes was also blocked with a Fas-Fc fusion protein. VK 3 induced apoptosis in Jurkat cells, coincident with an increase in both Fas and FasL expression. A FasL-resistant variant of these Jurkat cells was also resistant to VK 3induced apoptosis. Furthermore, because VK 3 effects were inhibited by glutathione, a potent antioxidant, oxidative stress was linked to the Fas/ FasL system. Moreover, since the Jurkat cell lines were p53 null, the activation of Fas/ FasL system after oxidative stress apparently acted through a p53-independent pathway. The therapeutic relevance of the K vitamins has been growing in recent years; our findings offer new insight for improving and expanding their applications. © 1999 Academic Press
Key Words: menadione; Fas; apoptosis; oxidative stress; lymphocytes. INTRODUCTION
Programmed cell death, or apoptosis, is a conserved and fundamental active cellular mechanism provoked by a range of physiological and pathological conditions (1). It is characterized by distinct morphological changes, including nuclear condensation and fragmentation, membrane blebbing, and formation of apoptotic bodies (2). 1
This study was supported by NIH Grants AR33887 and AR42573 to P.L.C. and CA59496 and CA42765 to W.K.K. R.C. is a fellow of the Arthritis Foundation. 2 The authors contributed equally to this article. 3 Current Address: Division of Rheumatology, University of Pennsylvania, 757 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104.
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Abbreviations used: FasL, Fas ligand; GSH, glutathione; VK 3, vitamin K 3; PI, propidium iodide; FSC, forward-angle light scatter; SSC, side scatter. 65
1521-6616/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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or FasR (B6/gld and B6/lpr, respectively). Moreover VK 3 itself induces FasL and Fas expression and VK 3induced apoptosis can be partially blocked by a neutralizing Fas-Fc fusion protein. Together the data suggest that a functional Fas/ FasL system is required for VK 3-induced apoptosis and that, because VK 3 is a potent oxidative stress inducer, the Fas/FasL system plays a major role in oxidative stress-induced apoptosis. MATERIAL AND METHODS
Mice. C57BL/6 (B6), C57BL/6-lpr/lpr (B6/lpr), and C57BL/6-gld/gld (B6/gld) mice were raised in our colony. Cell lines. The Jurkat human T cell leukemia subclone E6-1 (E6-1 S), constitutively expressing Fas and sensitive to killing by anti-Fas, was kindly provided by Dr. Philip D. Fernsten (University of North Carolina, Chapel Hill, NC). The E6-1 R2, a FasL-resistant variant of E6-1 S, was selected as described elsewhere (18). Drug. Menadione sodium bisulfite (VK 3) was obtained from Sigma (St. Louis, MO). VK 3 was dissolved in sterile distilled water to produce a stock solution (15 mM), which was stored at 270°C and diluted before use. Cell isolation and culture. Mouse spleen cells were isolated by disrupting the splenic capsule between frosted ends of glass slides and washing twice with HBSS containing 15 mM Hepes (University of North Carolina Cancer Center Tissue Culture Facility, Chapel Hill, NC). Erythrocytes were lysed with ammonium chloride for 5 min at 4°C. Mouse splenocytes and E6-1 S and E6-1 R2 cells were cultured at a concentration of 1 3 10 6/ml in RPMI 1640, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 10% fetal bovine serum, and nonessential amino acids (University of North Carolina Cancer Center Tissue Culture Facility), at 37°C in a 5% CO 2/95% air humidified atmosphere. In some experiments E6-1 S and E6-1 R2 cells were treated with ceramide (100 mM, 12 h; Biomol, Plymouth Meeting, PA), doxorubicin (0.25 mg/ml, 24 h; Sigma), cycloheximide (30 mg/ml, 12 h; Sigma), or staurosporine (1 mM, 12 h; Sigma). Cell growth inhibition test. The E6-1 S clone and its variants were cultured in complete RPMI at a concentration of 5 3 10 5 cells/ml in presence of VK 3 alone (15 mM) or with reduced glutathione (GSH, 2 mM, Sigma). After 18 h cells were harvested and counted with a Coulter counter (Coulter Electronics, Inc., Hialeah, Florida). Cell staining for surface markers and apoptosis. For cell staining, fluoresceinated human anti-Fas (clone DX2, isotype; mouse IgG1k) and isotype-
matched FITC-labeled mouse antibody controls were obtained from PharMingen (San Diego, CA). For DNA staining, after permeabilization with 70% ethanol, 0.1 ml of 1 mg/ml RNase A (Sigma) was added per sample, followed by 0.2 ml of 100 mg/ml propidium iodide (PI) (Sigma). Cells were incubated for 20 min in the dark at 4°C and were analyzed with a FACScan (Becton Dickinson) with Cytomation data acquisition software (Fort Collins, CO) for green and red fluorescence. Detection of apoptotic cells was also made according to the forward-angle light scatter (FSC) and side scatter (SSC) profile (25, 26) after paraformaldehyde fixation. The percentage of specific apoptosis was calculated as 100 3 (experimental 2 spontaneous apoptosis)/experimental apoptosis. At least 30,000 events were collected per sample in all experiments. Northern Blot and Caspase-3 activation. For Northern blot analysis, cytoplasmic RNA was extracted from E6-1 S cells using the RNeasy mini kit according to the instructions of the supplier (Qiagen Inc., Santa Clarita, CA). Cytoplasmic RNA (10 mg) was then applied to a 1% agarose gel and size-fractioned by electrophoresis in the presence of 2.0 M formaldehyde, transferred to a nylon membrane, and hybridized with a radiolabeled Fas cDNA (kindly provided by Dr. Keith Elkon, Cornell University, NY). After stringent washing, the membrane was exposed to X-ray film. To assay CPP32 (caspase-3) protease activity in E6-1 S and E61 R2 after VK 3 treatment, the ApoAlert CPP32 assay kit (Clontech, Palo Alto, CA) was used according to instructions. Western blotting for FasL. Samples of 4 3 10 6 E6-1 S cells were washed in PBS and lysed for 20 min on ice in 50 mM Hepes, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 10 mM NaF, 1 mM EDTA, 2 mM Na orthovanadate, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation for 15 min at 14,000 rpm, supernatant protein was determined by BCA* protein assay reagent (Pierce, Rockford, IL) using bovine serum albumin standards. Thirty micrograms of proteins in Laemmli buffer was separated per lane on 12% SDS–PAGE. Western blots were probed with rabbit polyclonal anti-FasL (Calbiochem, Cambridge, MA). Bound antibodies were detected with goat antirabbit– horseradish peroxidase conjugate using an enhanced chemiluminescence system (Renaissance, NEN Life Science Products, Boston, MA). RESULTS
Cells from mice lacking Fas or FasL expression (B6/ lpr and B6/gld) show reduced apoptosis after VK 3 treatment. In order to test the hypothesis that VK 3induced apoptosis requires a functional Fas/FasL system, spleen cells from 3-month-old B6/lpr and B6/
INVOLVEMENT OF Fas/ FasL SYSTEM IN MENADIONE-INDUCED APOPTOSIS
gld mice, which possess inactivating mutations in the genes encoding Fas and FasL, respectively, and from B6 wild type mice were isolated and cultured for different periods of time in presence of 1.5 or 3 mM VK 3. As shown in Fig. 1A, cells from both B6/lpr and B6/gld mice had much lower percentages of specific apoptosis compared to the B6 wild-type cells after both 8 and 18 h of culture. These results confirmed that expression of both Fas and FasL was necessary for efficient induction of apoptosis by VK 3 treatment. VK 3-induced apoptosis is blocked with a Fas-Fc fusion protein. To assess more directly the role of the Fas/FasL system in VK 3-induced apoptosis, a Fas-Fc fusion protein was added to VK 3-treated normal spleen cells. This Fas-human IgG 1 Fc fusion protein was successful in inhibiting Fas-mediated apoptosis of thymocytes. When thymocytes where incubated with antiFas and 100 mg/ml of Fas-Fc, apoptosis was inhibited by ;75% (data not shown; Refs. 17 and 27). After treatment with 3 mM VK 3, spleen cells from normal mice were incubated with Fas-Fc at different concentrations, and apoptosis was detected according to the FSC vs SSC profile after 18 h of culture. We found significant inhibition of apoptosis dependent on the concentration of the Fas-Fc fusion protein, confirming that VK 3-induced apoptosis was, in part, Fas mediated; the control, a human IgG 1 myeloma protein, had no effect (Fig. 1B). E6-1 R2 clones are specifically resistant to FasL-induced apoptosis. To demonstrate resistance to FasLinduced apoptosis, the E6-1 S and E6-1 R2 cell lines were cultured for 5 h with the 3T3-FasL cell line and were then stained with PI. DNA area histograms are shown in Fig. 2. The E6-1 S cells were sensitive to FasL killing. E6-1 R2 cells, in contrast, failed to undergo apoptosis when cultured with FasL 3T3 cells; furthermore, no caspase-3 activation was detected in the mutant clones, suggesting that the defect in this clone lies in upstream caspases (data not shown). E6-1 S and E6-1 R2 cell lines were stained with anti-Fas–FITC and the isotype control. Figure 2 shows that uncloned E6-1 R2 cells generally expressed Fas. In subsequent experiments, only Fas-expressing mutant clones derived from the E6-1 R2 (c1, c13, c21) cells were used (Fig. 3A). FasL expression was also tested in both wild-type and mutant clones and found comparable (data not shown). To ask whether there was specific resistance to FasL killing, E6-1 S and E6-1 R2 clones were treated with ceramide or doxorubicin, which are known to induce apoptosis in a Fas-dependent manner. Cells were resistant to these agents, yet the clones retained susceptibility to apoptosis induced by staurosporine and cycloheximide, which are known to induce apoptosis in a Fas-independent fashion (Fig. 3B).
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E6-1 R2 clones are also resistant to VK 3-induced apoptosis. To test whether the Fas/Fas-L system might mediate VK 3-induced apoptosis, E6-1 S cells and three Fas-expressing mutant clones resistant to FasL were cultured for different periods of time with 15 mM VK 3 (Fig. 4A). Apoptosis was detected according to the FSC vs SSC profile (25, 26). In the initial 12 h of culture, little apoptosis was detected in any of the clones tested. At 18 and 24 h of exposure of VK 3 a remarkable difference was noticed: E6-1 S showed marked apoptosis yet there was almost complete resistance to this effect in three mutant clones. Apoptosis was detected by assaying caspase-3 protease activity, a key early event in apoptosis. As shown in Fig. 4B, the E6-1 S cells but not E6-1 R2 showed a marked increase of caspase-3 activation after VK 3 treatment. These data were consistent with involvement of the Fas/FasL system in VK 3-induced apoptosis. Reduced glutathione inhibits VK 3-induced apoptosis. In order to demonstrate in our system that VK 3 was inducing cell growth inhibition and consequent apoptosis by generating oxidative stress, we used reduced glutathione as a source of antioxidant. As expected, VK 3 treatment completely inhibited cell growth in the E6-1 S cells but not in the E6-1 R2 FasL-resistant cells (Fig. 4C). On the contrary the presence of GSH in the medium almost completely reversed the effect of VK 3, apparently as a result of the inhibition of oxidative stress (Fig. 4C). In these experiments apoptosis was confirmed according to FSC vs SSC profile (data not shown). Fas and FasL increase in Jurkat T cells after VK 3 treatment. Since we showed that a functional Fas/ FasL system was required for VK 3-induced apoptosis in E6-1 cells, we wondered if Fas and FasL might increase after VK 3 treatment. To test Fas expression, the E6-1 S cell line was treated with 15 mM VK 3 for 7 h. Total RNA was isolated and Fas mRNA measured by Northern blot. Figure 5A shows that Fas increases twoto threefold after treatment, with no change in expression of the GAPDH housekeeping gene. We wondered if VK 3 treatment also induced cell surface and soluble FasL. E6-1 S cells were treated with 15 mM VK 3 for 12 h and then FasL was measured by Western blot. As shown in Fig. 5B, we could detect a clear FasL increase after VK 3 treatment. Moreover, sFasL release and cytotoxicity after VK 3 treatment was tested and confirmed in E6-1 S cells by detecting the ability of VK 3 treated E6-1 S cells to induce E6-1 S apoptosis as measured by the JAM test (28) (data not shown). DISCUSSION
We have shown that VK 3 -induced apoptosis requires a functional Fas/ FasL system. Cells from mice
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FIG. 1. (A) Mice lacking functional FasL (B6/gld) or Fas expression (B6/lpr) are resistant to VK 3-induced apoptosis. Spleen cells from B6/lpr, B6/gld, and B6 wild type were cultured for 8 and 18 h in the presence of 3 or 1.5 mM of VK 3. Then specific apoptosis was measured according to the FSC vs SSC profile. B6 wild type had a much higher percentage of specific apoptosis compared to both B6/lpr and B6/gld. Each bar is representative of three different experiments. Mean 1 SD is shown. (B) Inhibition of VK 3-mediated apoptosis with a neutralizing Fas-Fc fusion protein. The percentage of apoptosis detected with the FSC vs SSC profile is shown (mean 1 SD). Spleen cells from B6 wild-type mice were treated with VK 3 (3 mM) and cultured with Fas-Fc fusion protein or control at different concentrations for 18 h. Cells were harvested, and the FSC vs SSC profile was assessed. A significant inhibition of apoptosis was found with the Fas-Fc fusion protein in a dose-dependent manner (closed bars). In contrast, no difference was noted with the control (human IgG 1 myeloma protein and open bars). These results are representative of three experiments.
lacking functional FasL (B6/gld) or Fas expression (B6/lpr) showed much lower levels of apoptosis induced by VK 3 in vitro compared to cells from wild-
type B6 mice, and mutant T cell lines resistant to FasL killing showed little or no apoptosis after VK 3 treatment.
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FIG. 2. E6-1 R2 clones are resistant to FasL-induced apoptosis. The E6-1 R2 cell lines were originally generated by continuous culture with the 3T3-FasL-transfected cell line. Cells were cultured for 5 h with 3T3-FasL cell line and were then stained with PI. DNA area histograms are shown. E6-1 R2 cells failed to undergo apoptosis when cultured with FasL-transfected 3T3 cells (right). The E6-1 S, as expected, was very sensitive to the FasL killing (left). E6-1 S and E6-1 R2 were stained with anti-Fas–FITC and the isotype control (bottom).
Vitamin K is a generic term for compounds that include phylloquinone (vitamin K 1), menaquinone (vitamin K 2), and menadione (vitamin K 3) (23). Both VK 1 and VK 2 are natural K vitamins and act as cofactors for prothrombin and other VK-dependent coagulation factors (29). The synthetic compound VK 3 has been shown to inhibit the growth of a variety of animal and human tumor cell lines such as cervical carcinoma, breast carcinoma, lymphoma, and leukemia both in vitro and in vivo (23, 24, 30, 31). Phase I and II clinical trial results are encouraging especially because of low VK 3 toxicity, low multiple drug resistant independence and few side effects (24). Moreover VK 3 has been shown in mice to potentiate the effect of classical protocols of human cancer radiotherapy (32). Along with cell growth inhibition, previous work shows that VK 3-induced cell death is by apoptosis (30, 33), at least within certain doses; excessive doses in-
duce necrosis rather than apoptosis (23, 34). We have confirmed this result in the Jurkat subclone E6-1, derived from a human acute T cell leukemia. We detected the characteristic cell shrinkage and moreover the activation of caspase-3, a critical component of the apoptotic apparatus (6). Furthermore, we were able to induce apoptosis in both E6-1 and in spleen cells from B6 mice within the micromolar range, a level that is relevant for therapy in vivo (24). Several different stimuli such as TCR ligation, UV and gamma irradiation, and drugs are able to induce apoptosis in T lymphocytes (35); many of these stimuli are thought to induce apoptosis upregulating FasL through the activation of transcription factors such as nuclear factor of activating T cells (36) and nuclear factor kB (NF-kB) (37). Moreover, stress-induced apoptosis in T lymphocytes has been linked to the upregulation of FasL through the activation of stress-
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FIG. 3. (A) Fas expression on E6-1 R2 mutant clones. E6-1 S and E6-1 R2 clones c1, c13, and c21 were stained with FITC conjugated anti-Fas or isotype control and fixed in paraformaldehyde. Fas expression in wild type and mutant is similar. (B) E6-1 R2 clones are sensitive to Fas-independent killing. E6-1 S and E6-1 R2 clones were cultured in the presence of ceramide (100 mM, 12 h), doxorubicin (0.25 mg/ml, 24 h), cycloheximide (30 mg/ml, 12 h), or staurosporine (1 mM, 12 h). Cells were harvested and apoptosis was detected according to FSC vs SSC profile. Treatment with ceramide or doxorubicin (Fas-dependent killing) was unable to induce apoptosis in the mutant clones. In contrast, cycloheximide or staurosporine (Fas-independent killing) induced similar degrees of apoptosis in both wild-type and resistant clones similar to that seen with FasL.
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FIG. 4. (A) E6-1 R2 clones are resistant to VK 3-induced apoptosis. E6-1 S and three different clones resistant to FasL killing (E6-1 R2c1, E6-1 R2c13, and E6-1 R2c21) were treated with 15 mM VK 3 and cultured for 6, 12, 18, and 24 h. Cells were harvested and apoptosis was detected according to FSC vs SSC profile. No difference was noticed at time 0 or after 6 and 12 h. A dramatic increase in apoptosis was detectable after 18 and 24 h in the E6-1 S but not in the resistant clones (;65 vs ;15%). (B) Caspase-3 activation after VK 3 treatment. E6-1 S and E6-1 R2c21 clone were treated with 15 mM VK 3, and after culturing for 11 h, caspase-3 protease activity was measured. As shown in the figure, the E6-1 S cells showed a dose-dependent caspase-3 activity; in contrast, the resistant clone had only little activation at the maximum dose. (C) Antioxidant glutathione inhibits VK 3 cell growth arrest and consequent apoptosis. The main mechanism by which VK 3 induces cell growth arrest and apoptosis is believed to be through induction of oxidative stress. In order to show that in our system, cells were cultured at a concentration of 5 3 10 5 cells/ml and treated with VK 3 (15 mM) alone or in the presence of reduced GSH (2 mM). After 18 h of culture cells were harvested and counted with a Coulter counter. VK 3 cell growth inhibition was detected only in the E6-1 S clone and not in the FasL resistant FasL clone. On the contrary, the presence of reduced GSH completely reversed the effect of VK 3.
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FIG. 5. (A) Fas increases in Jurkat T cells after VK 3 treatment. Total RNA was isolated from cells (E6-1 S) treated with 15 mM VK 3 for 7 h and then hybridized with 32P-labeled Fas and GAPDH probes. Top lane shows increased Fas expression with VK 3 treatment. GAPDH expression was used as control (bottom lane). (B) FasL is increased after VK 3 treatment. FasL was detected by Western blot in the E6-1 S cell line after treatment with VK 3. Nontreated cells showed little FasL expression; on the contrary, after VK 3 treatment there was a clear FasL increase. Arrows indicate nonspecific binding as control for protein loading. 3T3-NIH FasL-transfected cell line and the empty vector transfected cell line are shown as controls.
activated protein kinase family (38). Finally a c-mycdependent cell death pathway in Fas/FasL apoptosis has been also described in activated T lymphocytes (39). Ligation of the T-cell receptor induces the expression of FasL (40), and this induction is dependent on c-myc expression (39). c-myc may function as a transcription factor to drive apoptosis when low amounts of survival factors are present (41) and this c-myc-induced cell death may be mediated by the cell surface interactions of Fas with its ligand (42). An early event following VK 3 treatment is the induction of genes such as c-myc, and it has been suggested that a block in cell cycle progression might be necessary for VK 3-induced apoptosis (23, 24). In light of our findings it is possible that induction of c-myc after VK 3 treatment may also facilitate the increase of both FasL (and also Fas), confirming once again the linkage between c-myc and the Fas/FasL system. Another important aspect of VK 3 is that its induction of oxidative stress is mediated mainly through the production of active oxygen species (21, 22, 43, 44). Moreover, glutathione, a potent antioxidant factor, completely inhibits VK 3 cytotoxicity (22, 23) as we
showed in our experiments. Oxidative stress is also involved in Fas-mediated apoptosis (45, 46). For instance, it has been shown that reactive oxygen intermediates are able to induce FasL mRNA expression (47) and that glutathione rapidly decreases during apoptosis induced by crosslinking of Fas receptor (48). Our present data provide further evidence for the relationship between oxidative stress and the Fas/FasL system. VK 3 induced FasL protein and Fas mRNA, and the integrity of the Fas/FasL system was needed for VK 3-induced apoptosis, since a cell line resistant to FasL killing and mice lacking functional FasL or expression of Fas were also resistant to VK 3 cytotoxic effects. Nevertheless, a redundant pathway could be present for VK 3-induced apoptosis since in our experiments we observed a residual degree of apoptosis in the mutant clones after VK 3 treatment. The p53 protein has been shown to be required for inducing cell death after oxidative damage (49) and several investigators have shown that p53 activation induces Fas expression (50, 51). Since the cell lines that we used were p53 null (52) (and data not shown), we infer that the activation of Fas/FasL system after oxidative stress might be possible through a p53-independent pathway. VK 3 has been emerging in the past few years as a useful tool for studying oxidative stress and more importantly as a potential anticancer drug. Its use of the Fas/FasL system is parallel to our recent observation of the involvement of this pathway in apoptosis induced by ionizing radiation (17). The reported clinical enhancement by VK 3 of radiotherapy effects on certain tumors (32) may reflect a synergism based on the convergence of radiotherapy and VK 3 at the level of Fas/ FasL. REFERENCES 1. Cohen, J. J., Apoptosis and its regulation. Adv. Exp. Med. Biol. 406, 11–20, 1996. 2. Hetts, S. W., To die or not to die: An overview of apoptosis and its role in disease. JAMA 279, 300 –307, 1998. 3. Rudin, C. M., and Thompson, C. B., Apoptosis and disease: Regulation and clinical relevance of programmed cell death. Annu. Rev. Med. 48, 267–281, 1997. 4. Rowan, S., and Fisher, D. E., Mechanisms of apoptotic cell death. Leukemia 11, 457– 465, 1997. 5. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M., FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85, 817– 827, 1996. 6. Salvesen, G. S., and Dixit, V. M., Caspases: Intracellular signaling by proteolysis. Cell 91, 443– 446, 1997. 7. Chinnaiyan, A. M., and Dixit, V. M., The cell-death machine. Curr. Biol. 6, 555–562, 1996.
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Apoptosis Provoked by the Oxidative Stress Inducer Menadione (Vitamin K 3) Is Mediated by the Fas/Fas Ligand System 1 Roberto Caricchio, 2,3 Dmitri Kovalenko,* ,2 William K. Kaufmann,* and Philip L. Cohen 3 Departments of Medicine and Microbiology/Immunology and *Departments of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
Fas (APO-1/CD95) is a member of the TNF/nerve growth factor receptor superfamily and is currently recognized as the principal cell surface receptor involved in the transduction of signals that induce apoptosis in lymphocytes and in a variety of tumor cells (3, 4). Crosslinking by its ligand (FasL) 4 rapidly induces the death-inducing signaling complex (5) and the activation of a cascade of interleukin-converting enzyme/ Ced3 proteases (6), now designated caspases (7). The Fas/FasL system plays crucial roles in homeostasis and self-tolerance (8, 9), in the activationinduced cell death of lymphocytes (10), and in the delimitation of immunoprivileged regions such as the eye and testis (11). Loss of function due to mutations in murine Fas ligand (gld), murine Fas (lpr), human Fas, or human FasL leads to lymphoproliferation, lymphadenopathy, and autoimmune diseases (12). The Fas/ FasL system is also implicated in the pathogenesis of other illnesses. Various tumor cells express FasL, potentially creating their own immunoprivileged sites (13). The deletion of CD4 1 T cells in human immunodeficiency virus-infected cells (14), cell-mediated immunity in acute graft-versus-host disease (15), and the destruction of myelin in multiple sclerosis all involve the Fas/FasL system (16). It has also recently been shown that gamma irradiation (17), UV irradiation (18), and drug-induced apoptosis of cancer cells (19) appear to involve the Fas/FasL system. Recent data indicate that apoptotic cell death takes place after oxidative stress (20). We wondered if menadione (vitamin K 3, VK 3), a potent oxidative stress inducer (21, 22), which induces cell cycle arrest and apoptosis (23, 24) and which has recently been realized to be a potential anticancer drug relatively free of adverse effects (23), might act via Fas/FasL. In the present study we demonstrate for the first time that VK 3-induced apoptosis is markedly reduced both in human T cell leukemia cell lines resistant to FasL killing and in cells from mice lacking functional FasL
Menadione, or vitamin K 3 (VK 3), a potent oxidative stress inducer, has been recently used as an effective and remarkably safe cytotoxic drug for treatment of several human tumors. VK 3 induces apoptotic cell death through a poorly understood mechanism. Here we show for the first time that VK 3-induced apoptosis requires the Fas/ FasL system. Spleen cells from both Fas- and FasL-deficient mice (C57BL/6-lpr and C57BL/ 6-gld, respectively) had much lower levels of VK 3 apoptosis in vitro compared to cells from control C57BL/6 mice. VK 3 cytotoxicity toward mouse splenocytes was also blocked with a Fas-Fc fusion protein. VK 3 induced apoptosis in Jurkat cells, coincident with an increase in both Fas and FasL expression. A FasL-resistant variant of these Jurkat cells was also resistant to VK 3induced apoptosis. Furthermore, because VK 3 effects were inhibited by glutathione, a potent antioxidant, oxidative stress was linked to the Fas/ FasL system. Moreover, since the Jurkat cell lines were p53 null, the activation of Fas/ FasL system after oxidative stress apparently acted through a p53-independent pathway. The therapeutic relevance of the K vitamins has been growing in recent years; our findings offer new insight for improving and expanding their applications. © 1999 Academic Press
Key Words: menadione; Fas; apoptosis; oxidative stress; lymphocytes. INTRODUCTION
Programmed cell death, or apoptosis, is a conserved and fundamental active cellular mechanism provoked by a range of physiological and pathological conditions (1). It is characterized by distinct morphological changes, including nuclear condensation and fragmentation, membrane blebbing, and formation of apoptotic bodies (2). 1
This study was supported by NIH Grants AR33887 and AR42573 to P.L.C. and CA59496 and CA42765 to W.K.K. R.C. is a fellow of the Arthritis Foundation. 2 The authors contributed equally to this article. 3 Current Address: Division of Rheumatology, University of Pennsylvania, 757 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104.
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Abbreviations used: FasL, Fas ligand; GSH, glutathione; VK 3, vitamin K 3; PI, propidium iodide; FSC, forward-angle light scatter; SSC, side scatter. 65
1521-6616/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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or FasR (B6/gld and B6/lpr, respectively). Moreover VK 3 itself induces FasL and Fas expression and VK 3induced apoptosis can be partially blocked by a neutralizing Fas-Fc fusion protein. Together the data suggest that a functional Fas/ FasL system is required for VK 3-induced apoptosis and that, because VK 3 is a potent oxidative stress inducer, the Fas/FasL system plays a major role in oxidative stress-induced apoptosis. MATERIAL AND METHODS
Mice. C57BL/6 (B6), C57BL/6-lpr/lpr (B6/lpr), and C57BL/6-gld/gld (B6/gld) mice were raised in our colony. Cell lines. The Jurkat human T cell leukemia subclone E6-1 (E6-1 S), constitutively expressing Fas and sensitive to killing by anti-Fas, was kindly provided by Dr. Philip D. Fernsten (University of North Carolina, Chapel Hill, NC). The E6-1 R2, a FasL-resistant variant of E6-1 S, was selected as described elsewhere (18). Drug. Menadione sodium bisulfite (VK 3) was obtained from Sigma (St. Louis, MO). VK 3 was dissolved in sterile distilled water to produce a stock solution (15 mM), which was stored at 270°C and diluted before use. Cell isolation and culture. Mouse spleen cells were isolated by disrupting the splenic capsule between frosted ends of glass slides and washing twice with HBSS containing 15 mM Hepes (University of North Carolina Cancer Center Tissue Culture Facility, Chapel Hill, NC). Erythrocytes were lysed with ammonium chloride for 5 min at 4°C. Mouse splenocytes and E6-1 S and E6-1 R2 cells were cultured at a concentration of 1 3 10 6/ml in RPMI 1640, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 10% fetal bovine serum, and nonessential amino acids (University of North Carolina Cancer Center Tissue Culture Facility), at 37°C in a 5% CO 2/95% air humidified atmosphere. In some experiments E6-1 S and E6-1 R2 cells were treated with ceramide (100 mM, 12 h; Biomol, Plymouth Meeting, PA), doxorubicin (0.25 mg/ml, 24 h; Sigma), cycloheximide (30 mg/ml, 12 h; Sigma), or staurosporine (1 mM, 12 h; Sigma). Cell growth inhibition test. The E6-1 S clone and its variants were cultured in complete RPMI at a concentration of 5 3 10 5 cells/ml in presence of VK 3 alone (15 mM) or with reduced glutathione (GSH, 2 mM, Sigma). After 18 h cells were harvested and counted with a Coulter counter (Coulter Electronics, Inc., Hialeah, Florida). Cell staining for surface markers and apoptosis. For cell staining, fluoresceinated human anti-Fas (clone DX2, isotype; mouse IgG1k) and isotype-
matched FITC-labeled mouse antibody controls were obtained from PharMingen (San Diego, CA). For DNA staining, after permeabilization with 70% ethanol, 0.1 ml of 1 mg/ml RNase A (Sigma) was added per sample, followed by 0.2 ml of 100 mg/ml propidium iodide (PI) (Sigma). Cells were incubated for 20 min in the dark at 4°C and were analyzed with a FACScan (Becton Dickinson) with Cytomation data acquisition software (Fort Collins, CO) for green and red fluorescence. Detection of apoptotic cells was also made according to the forward-angle light scatter (FSC) and side scatter (SSC) profile (25, 26) after paraformaldehyde fixation. The percentage of specific apoptosis was calculated as 100 3 (experimental 2 spontaneous apoptosis)/experimental apoptosis. At least 30,000 events were collected per sample in all experiments. Northern Blot and Caspase-3 activation. For Northern blot analysis, cytoplasmic RNA was extracted from E6-1 S cells using the RNeasy mini kit according to the instructions of the supplier (Qiagen Inc., Santa Clarita, CA). Cytoplasmic RNA (10 mg) was then applied to a 1% agarose gel and size-fractioned by electrophoresis in the presence of 2.0 M formaldehyde, transferred to a nylon membrane, and hybridized with a radiolabeled Fas cDNA (kindly provided by Dr. Keith Elkon, Cornell University, NY). After stringent washing, the membrane was exposed to X-ray film. To assay CPP32 (caspase-3) protease activity in E6-1 S and E61 R2 after VK 3 treatment, the ApoAlert CPP32 assay kit (Clontech, Palo Alto, CA) was used according to instructions. Western blotting for FasL. Samples of 4 3 10 6 E6-1 S cells were washed in PBS and lysed for 20 min on ice in 50 mM Hepes, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 10 mM NaF, 1 mM EDTA, 2 mM Na orthovanadate, 1 mM DTT, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation for 15 min at 14,000 rpm, supernatant protein was determined by BCA* protein assay reagent (Pierce, Rockford, IL) using bovine serum albumin standards. Thirty micrograms of proteins in Laemmli buffer was separated per lane on 12% SDS–PAGE. Western blots were probed with rabbit polyclonal anti-FasL (Calbiochem, Cambridge, MA). Bound antibodies were detected with goat antirabbit– horseradish peroxidase conjugate using an enhanced chemiluminescence system (Renaissance, NEN Life Science Products, Boston, MA). RESULTS
Cells from mice lacking Fas or FasL expression (B6/ lpr and B6/gld) show reduced apoptosis after VK 3 treatment. In order to test the hypothesis that VK 3induced apoptosis requires a functional Fas/FasL system, spleen cells from 3-month-old B6/lpr and B6/
INVOLVEMENT OF Fas/ FasL SYSTEM IN MENADIONE-INDUCED APOPTOSIS
gld mice, which possess inactivating mutations in the genes encoding Fas and FasL, respectively, and from B6 wild type mice were isolated and cultured for different periods of time in presence of 1.5 or 3 mM VK 3. As shown in Fig. 1A, cells from both B6/lpr and B6/gld mice had much lower percentages of specific apoptosis compared to the B6 wild-type cells after both 8 and 18 h of culture. These results confirmed that expression of both Fas and FasL was necessary for efficient induction of apoptosis by VK 3 treatment. VK 3-induced apoptosis is blocked with a Fas-Fc fusion protein. To assess more directly the role of the Fas/FasL system in VK 3-induced apoptosis, a Fas-Fc fusion protein was added to VK 3-treated normal spleen cells. This Fas-human IgG 1 Fc fusion protein was successful in inhibiting Fas-mediated apoptosis of thymocytes. When thymocytes where incubated with antiFas and 100 mg/ml of Fas-Fc, apoptosis was inhibited by ;75% (data not shown; Refs. 17 and 27). After treatment with 3 mM VK 3, spleen cells from normal mice were incubated with Fas-Fc at different concentrations, and apoptosis was detected according to the FSC vs SSC profile after 18 h of culture. We found significant inhibition of apoptosis dependent on the concentration of the Fas-Fc fusion protein, confirming that VK 3-induced apoptosis was, in part, Fas mediated; the control, a human IgG 1 myeloma protein, had no effect (Fig. 1B). E6-1 R2 clones are specifically resistant to FasL-induced apoptosis. To demonstrate resistance to FasLinduced apoptosis, the E6-1 S and E6-1 R2 cell lines were cultured for 5 h with the 3T3-FasL cell line and were then stained with PI. DNA area histograms are shown in Fig. 2. The E6-1 S cells were sensitive to FasL killing. E6-1 R2 cells, in contrast, failed to undergo apoptosis when cultured with FasL 3T3 cells; furthermore, no caspase-3 activation was detected in the mutant clones, suggesting that the defect in this clone lies in upstream caspases (data not shown). E6-1 S and E6-1 R2 cell lines were stained with anti-Fas–FITC and the isotype control. Figure 2 shows that uncloned E6-1 R2 cells generally expressed Fas. In subsequent experiments, only Fas-expressing mutant clones derived from the E6-1 R2 (c1, c13, c21) cells were used (Fig. 3A). FasL expression was also tested in both wild-type and mutant clones and found comparable (data not shown). To ask whether there was specific resistance to FasL killing, E6-1 S and E6-1 R2 clones were treated with ceramide or doxorubicin, which are known to induce apoptosis in a Fas-dependent manner. Cells were resistant to these agents, yet the clones retained susceptibility to apoptosis induced by staurosporine and cycloheximide, which are known to induce apoptosis in a Fas-independent fashion (Fig. 3B).
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E6-1 R2 clones are also resistant to VK 3-induced apoptosis. To test whether the Fas/Fas-L system might mediate VK 3-induced apoptosis, E6-1 S cells and three Fas-expressing mutant clones resistant to FasL were cultured for different periods of time with 15 mM VK 3 (Fig. 4A). Apoptosis was detected according to the FSC vs SSC profile (25, 26). In the initial 12 h of culture, little apoptosis was detected in any of the clones tested. At 18 and 24 h of exposure of VK 3 a remarkable difference was noticed: E6-1 S showed marked apoptosis yet there was almost complete resistance to this effect in three mutant clones. Apoptosis was detected by assaying caspase-3 protease activity, a key early event in apoptosis. As shown in Fig. 4B, the E6-1 S cells but not E6-1 R2 showed a marked increase of caspase-3 activation after VK 3 treatment. These data were consistent with involvement of the Fas/FasL system in VK 3-induced apoptosis. Reduced glutathione inhibits VK 3-induced apoptosis. In order to demonstrate in our system that VK 3 was inducing cell growth inhibition and consequent apoptosis by generating oxidative stress, we used reduced glutathione as a source of antioxidant. As expected, VK 3 treatment completely inhibited cell growth in the E6-1 S cells but not in the E6-1 R2 FasL-resistant cells (Fig. 4C). On the contrary the presence of GSH in the medium almost completely reversed the effect of VK 3, apparently as a result of the inhibition of oxidative stress (Fig. 4C). In these experiments apoptosis was confirmed according to FSC vs SSC profile (data not shown). Fas and FasL increase in Jurkat T cells after VK 3 treatment. Since we showed that a functional Fas/ FasL system was required for VK 3-induced apoptosis in E6-1 cells, we wondered if Fas and FasL might increase after VK 3 treatment. To test Fas expression, the E6-1 S cell line was treated with 15 mM VK 3 for 7 h. Total RNA was isolated and Fas mRNA measured by Northern blot. Figure 5A shows that Fas increases twoto threefold after treatment, with no change in expression of the GAPDH housekeeping gene. We wondered if VK 3 treatment also induced cell surface and soluble FasL. E6-1 S cells were treated with 15 mM VK 3 for 12 h and then FasL was measured by Western blot. As shown in Fig. 5B, we could detect a clear FasL increase after VK 3 treatment. Moreover, sFasL release and cytotoxicity after VK 3 treatment was tested and confirmed in E6-1 S cells by detecting the ability of VK 3 treated E6-1 S cells to induce E6-1 S apoptosis as measured by the JAM test (28) (data not shown). DISCUSSION
We have shown that VK 3 -induced apoptosis requires a functional Fas/ FasL system. Cells from mice
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FIG. 1. (A) Mice lacking functional FasL (B6/gld) or Fas expression (B6/lpr) are resistant to VK 3-induced apoptosis. Spleen cells from B6/lpr, B6/gld, and B6 wild type were cultured for 8 and 18 h in the presence of 3 or 1.5 mM of VK 3. Then specific apoptosis was measured according to the FSC vs SSC profile. B6 wild type had a much higher percentage of specific apoptosis compared to both B6/lpr and B6/gld. Each bar is representative of three different experiments. Mean 1 SD is shown. (B) Inhibition of VK 3-mediated apoptosis with a neutralizing Fas-Fc fusion protein. The percentage of apoptosis detected with the FSC vs SSC profile is shown (mean 1 SD). Spleen cells from B6 wild-type mice were treated with VK 3 (3 mM) and cultured with Fas-Fc fusion protein or control at different concentrations for 18 h. Cells were harvested, and the FSC vs SSC profile was assessed. A significant inhibition of apoptosis was found with the Fas-Fc fusion protein in a dose-dependent manner (closed bars). In contrast, no difference was noted with the control (human IgG 1 myeloma protein and open bars). These results are representative of three experiments.
lacking functional FasL (B6/gld) or Fas expression (B6/lpr) showed much lower levels of apoptosis induced by VK 3 in vitro compared to cells from wild-
type B6 mice, and mutant T cell lines resistant to FasL killing showed little or no apoptosis after VK 3 treatment.
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FIG. 2. E6-1 R2 clones are resistant to FasL-induced apoptosis. The E6-1 R2 cell lines were originally generated by continuous culture with the 3T3-FasL-transfected cell line. Cells were cultured for 5 h with 3T3-FasL cell line and were then stained with PI. DNA area histograms are shown. E6-1 R2 cells failed to undergo apoptosis when cultured with FasL-transfected 3T3 cells (right). The E6-1 S, as expected, was very sensitive to the FasL killing (left). E6-1 S and E6-1 R2 were stained with anti-Fas–FITC and the isotype control (bottom).
Vitamin K is a generic term for compounds that include phylloquinone (vitamin K 1), menaquinone (vitamin K 2), and menadione (vitamin K 3) (23). Both VK 1 and VK 2 are natural K vitamins and act as cofactors for prothrombin and other VK-dependent coagulation factors (29). The synthetic compound VK 3 has been shown to inhibit the growth of a variety of animal and human tumor cell lines such as cervical carcinoma, breast carcinoma, lymphoma, and leukemia both in vitro and in vivo (23, 24, 30, 31). Phase I and II clinical trial results are encouraging especially because of low VK 3 toxicity, low multiple drug resistant independence and few side effects (24). Moreover VK 3 has been shown in mice to potentiate the effect of classical protocols of human cancer radiotherapy (32). Along with cell growth inhibition, previous work shows that VK 3-induced cell death is by apoptosis (30, 33), at least within certain doses; excessive doses in-
duce necrosis rather than apoptosis (23, 34). We have confirmed this result in the Jurkat subclone E6-1, derived from a human acute T cell leukemia. We detected the characteristic cell shrinkage and moreover the activation of caspase-3, a critical component of the apoptotic apparatus (6). Furthermore, we were able to induce apoptosis in both E6-1 and in spleen cells from B6 mice within the micromolar range, a level that is relevant for therapy in vivo (24). Several different stimuli such as TCR ligation, UV and gamma irradiation, and drugs are able to induce apoptosis in T lymphocytes (35); many of these stimuli are thought to induce apoptosis upregulating FasL through the activation of transcription factors such as nuclear factor of activating T cells (36) and nuclear factor kB (NF-kB) (37). Moreover, stress-induced apoptosis in T lymphocytes has been linked to the upregulation of FasL through the activation of stress-
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FIG. 3. (A) Fas expression on E6-1 R2 mutant clones. E6-1 S and E6-1 R2 clones c1, c13, and c21 were stained with FITC conjugated anti-Fas or isotype control and fixed in paraformaldehyde. Fas expression in wild type and mutant is similar. (B) E6-1 R2 clones are sensitive to Fas-independent killing. E6-1 S and E6-1 R2 clones were cultured in the presence of ceramide (100 mM, 12 h), doxorubicin (0.25 mg/ml, 24 h), cycloheximide (30 mg/ml, 12 h), or staurosporine (1 mM, 12 h). Cells were harvested and apoptosis was detected according to FSC vs SSC profile. Treatment with ceramide or doxorubicin (Fas-dependent killing) was unable to induce apoptosis in the mutant clones. In contrast, cycloheximide or staurosporine (Fas-independent killing) induced similar degrees of apoptosis in both wild-type and resistant clones similar to that seen with FasL.
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FIG. 4. (A) E6-1 R2 clones are resistant to VK 3-induced apoptosis. E6-1 S and three different clones resistant to FasL killing (E6-1 R2c1, E6-1 R2c13, and E6-1 R2c21) were treated with 15 mM VK 3 and cultured for 6, 12, 18, and 24 h. Cells were harvested and apoptosis was detected according to FSC vs SSC profile. No difference was noticed at time 0 or after 6 and 12 h. A dramatic increase in apoptosis was detectable after 18 and 24 h in the E6-1 S but not in the resistant clones (;65 vs ;15%). (B) Caspase-3 activation after VK 3 treatment. E6-1 S and E6-1 R2c21 clone were treated with 15 mM VK 3, and after culturing for 11 h, caspase-3 protease activity was measured. As shown in the figure, the E6-1 S cells showed a dose-dependent caspase-3 activity; in contrast, the resistant clone had only little activation at the maximum dose. (C) Antioxidant glutathione inhibits VK 3 cell growth arrest and consequent apoptosis. The main mechanism by which VK 3 induces cell growth arrest and apoptosis is believed to be through induction of oxidative stress. In order to show that in our system, cells were cultured at a concentration of 5 3 10 5 cells/ml and treated with VK 3 (15 mM) alone or in the presence of reduced GSH (2 mM). After 18 h of culture cells were harvested and counted with a Coulter counter. VK 3 cell growth inhibition was detected only in the E6-1 S clone and not in the FasL resistant FasL clone. On the contrary, the presence of reduced GSH completely reversed the effect of VK 3.
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FIG. 5. (A) Fas increases in Jurkat T cells after VK 3 treatment. Total RNA was isolated from cells (E6-1 S) treated with 15 mM VK 3 for 7 h and then hybridized with 32P-labeled Fas and GAPDH probes. Top lane shows increased Fas expression with VK 3 treatment. GAPDH expression was used as control (bottom lane). (B) FasL is increased after VK 3 treatment. FasL was detected by Western blot in the E6-1 S cell line after treatment with VK 3. Nontreated cells showed little FasL expression; on the contrary, after VK 3 treatment there was a clear FasL increase. Arrows indicate nonspecific binding as control for protein loading. 3T3-NIH FasL-transfected cell line and the empty vector transfected cell line are shown as controls.
activated protein kinase family (38). Finally a c-mycdependent cell death pathway in Fas/FasL apoptosis has been also described in activated T lymphocytes (39). Ligation of the T-cell receptor induces the expression of FasL (40), and this induction is dependent on c-myc expression (39). c-myc may function as a transcription factor to drive apoptosis when low amounts of survival factors are present (41) and this c-myc-induced cell death may be mediated by the cell surface interactions of Fas with its ligand (42). An early event following VK 3 treatment is the induction of genes such as c-myc, and it has been suggested that a block in cell cycle progression might be necessary for VK 3-induced apoptosis (23, 24). In light of our findings it is possible that induction of c-myc after VK 3 treatment may also facilitate the increase of both FasL (and also Fas), confirming once again the linkage between c-myc and the Fas/FasL system. Another important aspect of VK 3 is that its induction of oxidative stress is mediated mainly through the production of active oxygen species (21, 22, 43, 44). Moreover, glutathione, a potent antioxidant factor, completely inhibits VK 3 cytotoxicity (22, 23) as we
showed in our experiments. Oxidative stress is also involved in Fas-mediated apoptosis (45, 46). For instance, it has been shown that reactive oxygen intermediates are able to induce FasL mRNA expression (47) and that glutathione rapidly decreases during apoptosis induced by crosslinking of Fas receptor (48). Our present data provide further evidence for the relationship between oxidative stress and the Fas/FasL system. VK 3 induced FasL protein and Fas mRNA, and the integrity of the Fas/FasL system was needed for VK 3-induced apoptosis, since a cell line resistant to FasL killing and mice lacking functional FasL or expression of Fas were also resistant to VK 3 cytotoxic effects. Nevertheless, a redundant pathway could be present for VK 3-induced apoptosis since in our experiments we observed a residual degree of apoptosis in the mutant clones after VK 3 treatment. The p53 protein has been shown to be required for inducing cell death after oxidative damage (49) and several investigators have shown that p53 activation induces Fas expression (50, 51). Since the cell lines that we used were p53 null (52) (and data not shown), we infer that the activation of Fas/FasL system after oxidative stress might be possible through a p53-independent pathway. VK 3 has been emerging in the past few years as a useful tool for studying oxidative stress and more importantly as a potential anticancer drug. Its use of the Fas/FasL system is parallel to our recent observation of the involvement of this pathway in apoptosis induced by ionizing radiation (17). The reported clinical enhancement by VK 3 of radiotherapy effects on certain tumors (32) may reflect a synergism based on the convergence of radiotherapy and VK 3 at the level of Fas/ FasL. REFERENCES 1. Cohen, J. J., Apoptosis and its regulation. Adv. Exp. Med. Biol. 406, 11–20, 1996. 2. Hetts, S. W., To die or not to die: An overview of apoptosis and its role in disease. JAMA 279, 300 –307, 1998. 3. Rudin, C. M., and Thompson, C. B., Apoptosis and disease: Regulation and clinical relevance of programmed cell death. Annu. Rev. Med. 48, 267–281, 1997. 4. Rowan, S., and Fisher, D. E., Mechanisms of apoptotic cell death. Leukemia 11, 457– 465, 1997. 5. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M., FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85, 817– 827, 1996. 6. Salvesen, G. S., and Dixit, V. M., Caspases: Intracellular signaling by proteolysis. Cell 91, 443– 446, 1997. 7. Chinnaiyan, A. M., and Dixit, V. M., The cell-death machine. Curr. Biol. 6, 555–562, 1996.
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