| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Department of Veterinary and Animal Sciences and
Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, MA 01003;
Department of Biology, Tufts University, Medford, MA 02155;
Department of Biology, Amherst College, Amherst, MA 01002; and
¶ Center for Cancer Research, Massachusetts General Hospital, Boston, MA 02114
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The involvement of intracellular redox status and ROS in T cell and thymocyte apoptosis has been suggested by the following correlative observations: 1) depletion of molecular oxygen inhibits apoptosis (3, 4), 2) production of ROS and/or depletion of intracellular antioxidants are observed upon induction of apoptosis (5, 6, 7), 3) treatment of cells with ROS induces apoptosis (8), and 4) overexpression of an antioxidant or elevated levels of intracellular antioxidants inhibits apoptosis (3, 8, 9, 10). Taken together, these data strongly suggest that the intracellular oxidative status influences cellular susceptibility to apoptosis. However, where and how the production of ROS occurs, and how the oxidative activity of ROS plays a role in the signaling processes that induce thymocyte apoptosis still remains unclear.
It has been demonstrated that the early processes of apoptosis are reversible, however, at some point during apoptosis, cells can no longer be rescued (11). Growing evidence shows that many of the death-inducing signaling pathways converge on the mitochondria, where the decision and the commitment to die, by either an apoptotic or necrotic pathway, are made (12, 13). When death-inducing signals reach the mitochondria, a series of cell-death related events mediated by mitochondria take place. These cell-death-related events are mediated by mitochondria, namely the permeability transition (PT) of the inner membrane, the loss of the inner mitochondrial membrane potential (
m), and cytochrome c (Cyt c) release collectively bring the cell to a point of no return in the apoptotic pathway. Once Cyt c is released into the cytoplasm, it forms a complex with ATP, apoptosis-activating factor-1, and procaspase-9 called the apoptosome, which cleaves procaspase-9 to caspase-9 (14, 15). The caspase-9 then cleaves procaspase-3, its immediate downstream substrate, initiating the cascade of protease activation in the execution phase of apoptosis.
In addition to the mitochondria, one of the key regulators of lymphocyte apoptosis is the proteasome (16, 17), a large proteolytic multienzyme complex residing in the cytoplasm and nucleus. We have previously observed that the inhibition of proteasome activities results in a reduction of caspase-3 activity. This finding suggests that the proteasome may exert its regulatory role upstream of cell death events mediated by mitochondria during thymocyte apoptosis. Therefore, it is possible that the proteasome may also play a role in regulating oxidative stress originating from mitochondria during apoptosis. ROS production at sites other than the mitochondria has been shown to play a role in some forms of apoptosis (18). However, it is likely that the majority of ROS are produced by mitochondria, and accumulating evidence suggests that the regulation of inner and outer membrane permeability, electron transport, and transmembrane transportation of ions and molecules such as Cyt c seems crucial for the maintenance of the mitochondrial homeostasis, as well as for the regulation of apoptosis (19, 20, 21, 22).
In this study, we define the contribution of molecular oxygen and H2O2 to the apoptotic processes, as well as the site of H2O2 production during DEX-induced thymocyte apoptosis. Additionally, we demonstrate the regulatory role of the proteasome in modulating cell death events mediated by mitochondria and H2O2 production.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Dexamethasone (DEX), N-acetyl-L-cysteine (NAC), antimycin A, rotenone, and H2O2 were purchased from Sigma-Aldrich (St. Louis, MO). Lactacystin was purchased from Kamiya Biomedical (Seattle, WA). Anti-Cyt c, anti-CD3, and anti-CD28 mAbs were obtained from BD PharMingen (San Diego, CA). Chloromethyl-dichloro-dihydrofluorescein-diacetate (CM-H2DCFDA) and H2DCFDA-di(acetoxymethyl ester) were purchased from Molecular Probes (Eugene, OR).
Ex vivo thymocyte culture
Thymocytes were obtained from 3- to 5-wk-old female BALB/c mice. Thymocytes were cultured at 37°C in 5% CO2 in RPMI 1640 medium containing 10% FBS. All mice were maintained in the animal facility at the University of Massachusetts (Amherst, MA).
In vivo treatment of BALB/c mice with NAC and DEX
NAC solution was prepared at the concentration of 32 mg/ml in PBS (pH 7.2). DEX solution was prepared at the concentration of 1 mg/ml in 16% ethanol/PBS. Female BALB/c mice at 4 wk of age were weighed and given NAC injections (8 mg NAC/20-g mouse) at 6 h and 1 h before a DEX injection (250 µg DEX/20-g mouse). Mice in the control group were given an equivalent volume of PBS or 16% ethanol/PBS. Thymocytes were isolated from all mice at 10–16 h after the DEX injection and assayed for apoptosis.
Treatment of thymocytes with NAC
Thymocytes were incubated with 60 mM NAC starting 1 h before the induction of apoptosis and throughout the experiment. PBS was added to the control cultures.
Induction of apoptosis by DEX, TCR-mediated signaling, or H2O2
Apoptosis in thymocytes was induced by 5 µM DEX or 40 µM H2O2. The final dilutions were prepared from the following stock solutions: 2.5 mM DEX in 70% ethanol and 8.8 M H2O2 (30% solution w/w) in PBS. To the control cultures, 70% ethanol or PBS was added. To induce apoptosis via TCR, thymocytes were cultured in a tissue culture plate that was coated with anti-CD3 and anti-CD28 mAbs at 10 mg/ml each.
Treatments of thymocytes with mitochondrial electron transport chain inhibitors
Mitochondrial electron transport chain inhibitors were added to thymocyte cultures 30 min before the induction of apoptosis. The final concentrations of each inhibitor used for the experiments were: 10 µM rotenone and 1 µM antimycin A. In the control cultures, the appropriate solvents were added at volumes equal to the experimental.
Treatment of thymocyte with proteasome inhibitors
Lactacystin was added to thymocyte cultures at 8 µM 1 h before the induction of apoptosis. The stock solution was prepared as a 20-mM solution in H2O.
Detection of H2O2 in thymocytes
For the detection of H2O2, thymocytes were preincubated with 5 µM CM-H2DCFDA or 1 µM H2DCFDA-di(acetoxymethyl ester) in medium for 30 min at 37°C in a 5% CO2 incubator before any other treatment. These dyes get oxidized by intracellular H2O2 and become fluorescent. After appropriate treatments were performed, cells were harvested and assayed for the H2O2-associated fluorescence by flow cytometry.
Detection of thymocyte apoptosis by Annexin VFITC staining
Apoptosis was detected by the binding of Annexin VFITC to phosphatidylserine exposed on the outside of the plasma membrane of apoptotic cells. At designated time points, thymocytes were harvested and stained with Annexin VFITC for 15 min at room temperature in dark. The fluorescent signals were detected by flow cytometry.
Detection of m
At each time point, thymocytes were incubated with 50 nM 3,3' dihexyloxacarbocyanine iodide (DiOC6) in medium for 15 min at 37°C in a 5% CO2 incubator. After incubation, cells were washed once and resuspended in cold PBS/BSA. Stained cells were immediately analyzed by flow cytometry.
Detection of caspase-3 activity; PhiPhiLux assay
One hour before the desired time point, 5 x 106 thymocytes per sample were pelleted and resuspended in 50 µl of RPMI medium containing 9 nM PhiPhiLuxG2D2 or G1D2 (OncoImmunin, Gaithersburg, MD) and 10% FBS. After a 1-h incubation at 37°C in a 5% CO2 incubator, the samples were analyzed immediately by flow cytometry. Uncleaved PhiPhiLuxG2D2 and PhiPhiLuxG1D2 emit background fluorescence at 552 and 505 nm, respectively, and cleaved PhiPhiLuxvG2D2 and PhiPhiLuxG1D2 emit fluorescence at 580 and 530 nm, respectively.
Preparation of cytoplasmic, nuclear, and mitochondrial fractions
Preparation of all fractions was performed as described by Andrews et al. (23) with some modifications. Briefly, thymocytes were resuspended in ice cold buffer A (10 mM HEPES-potassium hydroxide (pH 7.9 at 4°C), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 mM leupeptin, 0.2 mM pefablock), and incubated for 35 min on ice for hypotonic cell lysis. After incubation, nuclei were pelleted at 1000 x g for 15 min at 4°C, and the supernatant was separated and centrifuged at 16,000 x g for 30 min at 4°C. This high-speed supernatant was stored as the cytoplasmic fraction. The high-speed pellet was then lysed in mitochondrial lysis buffer (50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 50 mM NaCl, 1% Nonidet P-40), and stored as the mitochondrial extract. Protein concentrations of all the fractions were determined by using the BCA protein assay kit (Pierce, Rockford, IL) or the Bio-Rad protein assay kit (Hercules, CA). Each fraction was aliquoted at 70 µg of total protein per tube and stored at -70°C.
Western blot analysis of Cyt c
Western blot analysis of Cyt c was performed using appropriate cellular fractions prepared as previously described. Each cytoplasmic extract (70 µg of total protein per lane) was separated by electrophoresis on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were then blocked in Blotto (0.2% Tween 20, 10% nonfat milk, and 3% BSA in PBS) and probed with anti-Cyt c mAb at 1:250 in Blotto. Sheep anti-mouse Ab conjugated to streptavidin HRP was used as secondary Abat 1:10,000 in Blotto. Bands were visualized using the ECL Western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ) and exposed to x-ray film. The bands were quantified using Kodak 1D Image Analysis software (Rochester, NY).
Statistical analysis
Data shown in the graphs are expressed as the mean ± SD of three samples, and represent at least two independently performed experiments. Repeated measures analysis of variance and unpaired Student’s t test were performed using JMP IN Edition, version 3.1.5 statistical analysis software (SAS Institute, Cary, NC).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We first examined the involvement of molecular oxygen in DEX-induced thymocyte apoptosis. Thymocytes were isolated from BALB/c mice and incubated ex vivo in an anaerobic environment (95% N2, 5% CO2) for an hour before the induction of apoptosis by DEX. This anaerobic treatment of cells significantly reduced DEX-induced apoptosis (Fig. 1A). As shown in Fig. 1B, when thymocytes were treated with NAC, a thiol antioxidant, DEX-induced apoptosis was significantly reduced. These results confirmed our previous report (3) and strongly suggest that molecular oxygen and ROS play an important role in DEX-induced apoptosis ex vivo.
|
|
To elucidate the relationship between intracellular levels of H2O2 and apoptotic events in thymocytes, it was desirable to monitor intracellular levels of H2O2 during the course of thymocyte apoptosis. To measure intracellular levels of H2O2 during apoptosis, thymocytes were incubated with two H2DCFDA derivative dyes, CM-H2DCFDA or H2DCFDA-di(acetoxymethyl ester), whose fluorescence is proportional to the level of intracellular H2O2. The resulting fluorescence was then analyzed by flow cytometry. When thymocytes were induced to die by DEX, elevated intracellular levels of H2O2 were observed, and this increase was reduced by NAC treatment (Fig. 3A). NAC treatment also reduced the amount of apoptosis induced by DEX (Fig. 3B). In each case, thymocytes were also induced to die by anti-CD3 and anti-CD28 mAbs, and by an appropriate amount of exogenous H2O2 sufficient to cause apoptosis as a positive control for the H2DCFDA derivative dyes. In both cases, the exogenous H2O2- and the TCR-mediated signaling increased the intracellular levels of H2O2 and the amount of apoptosis, which were also attenuated by the NAC treatment (Fig. 3, A and B). These results suggest that the increased oxidative stress during thymocyte apoptosis is not unique to the DEX-mediated pathway. To preclude differences in dye loading, all thymocytes were loaded with dye in the same flask before any other treatments. Because the signal intensity of the dyes used for H2O2 measurement are affected by the pH of the cultures, the pH of each culture was measured before the cytometric analysis, and no detectable differences were observed (data not shown).
|
m depolarization, caspase-3 activation, and Cyt c release was investigated. As Fig. 3, C–E, illustrated, TCR-mediated signaling, exogenous H2O2, and DEX resulted in m depolarization, caspase-3 activation, and Cyt c release. However, when NAC was added to the cultures, these effects were significantly reduced (Fig. 3, C–E). Taken together, these results show that NAC treatment protects thymocytes from apoptosis by reducing the level of intracellular oxidative stress that promotes m depolarization, caspase-3 activation, and Cyt c release. H2O2 production and mitochondria
To locate the site of H2O2 production during DEX-induced thymocyte apoptosis, we used various mitochondrial electron transport chain inhibitors, and observed their effect on the production of H2O2. These inhibitors block electron transfer from one component to the next in the transport chain, and the site of blockage is specific for each inhibitor. The production of ROS in the mitochondria starts with the formation of a superoxide anion through the leak of an electron from the transport chain to a molecule of oxygen. Therefore, when an inhibitor of electron transportation blocks the electron flow, the production of ROS at sites downstream of the blockage is also decreased. When rotenone (complex I inhibitor) or antimycin A (complex III at Qi site) (26) were used, the production of H2O2 and the amount of apoptosis induced by DEX were significantly reduced, suggesting the formation of H2O2 is downstream of their blocking sites. In contrast, in the presence of exogenously introduced H2O2 both the amount of apoptosis and the production of H2O2 were enhanced (Figs. 4, A and B, and 5, A and B). To address the involvement of complex IV in the formation of H2O2, azide, a Cyt c oxidase (complex IV) inhibitor was used, which showed no effect in H2O2 production, or apoptosis (data not shown). Taken together, these results suggest that the site for DEX-induced H2O2 production is most likely at the ubiquinone cycle at complex III, because the reduction of H2O2 production was observed when the electron flow was reduced by rotenone before electrons entered the ubiquinone cycle, or by antimycin A at the ubiquinone cycle.
|
|
m, Cyt c release, and caspase-3 activation by DEX and exogenous H2O2. Increases in the levels of m depolarization, and activity of caspase-3 induced by DEX were attenuated by rotenone and antimycin A, while those induced by exogenous H2O2 were enhanced (Figs. 4, C and D, and 5, C and D). Antimycin A also attenuated DEX-induced Cyt c release by 97% (Fig. 5E). These observations correlated with the trend in changes in the levels of the intracellular H2O2 and the amount of apoptosis. When cells were treated with H2O2 and antimycin A, they died very quickly, and the cytoplasmic extracts from these cells did not contain enough protein for Cyt c Western blot analysis. Depriving cultured thymocytes of molecular oxygen should reduce the intracellular molecular oxygen, which may become superoxide anion after accepting the electron from the electron transport chain in mitochondria. We hypothesized that deprivation of oxygen from thymocyte cultures should enhance the protective effect of antimycin A in DEX-treated cells. Therefore, we treated thymocytes with antimycin A in an anaerobic environment (95% N2, 5% CO2). Treatment with antimycin A in an anaerobic environment resulted in an additive protective effect against DEX-induced production of H2O2 and apoptosis (Fig. 6, A and B). In this study, increased levels of intracellular H2O2 were observed when thymocytes were induced to die by the addition of exogenous H2O2, however, we could not tell whether this increase was by the diffusion of the added H2O2 or by the increased production at the mitochondria, especially because antimycin A did not show the inhibitory effect. As shown in Fig. 6A, the anaerobic conditions attenuated the enhanced H2O2 production seen in the cultures treated with exogenous H2O2, therefore confirming that the increase in intracellular H2O2 observed in cultures treated with exogenous H2O2 is not solely by result of diffusion into the cells of the added H2O2, but is also mitochondria- and molecular oxygen-dependent.
|
We have previously shown that proteasome activities are required for thymocytes to undergo apoptosis (16, 17). To determine whether the proteasome exerts its regulatory role upstream of the mitochondria, proteasome activities were inhibited using lactacystin, a proteasome-specific peptide inhibitor. The production of H2O2 as well as all other phenomena tested in the previous experiments were examined. As shown in Fig. 7, all the parameters measured were reduced by lactacystin treatment, suggesting that the proteasome regulates apoptotic signaling at or above the level of the mitochondria in DEX-induced thymocyte apoptosis. Additionally, the proteasome is also capable of regulating apoptotic events induced by exogenous H2O2 (Fig. 7), indicating its ability to respond to the changes in intracellular oxidative stress.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
m loss, Cyt c release, and caspase-3 activation, providing direct evidence that increased oxidative stress is a proapoptotic factor in thymocytes. In addition, the production of H2O2 was significantly reduced when the electron flow was reduced by rotenone before electrons entered the ubiquinone cycle, or by antimycin A at the ubiquinone cycle. These results determined that the site of H2O2 production during DEX-induced thymocyte apoptosis is at the ubiquinone cycle at complex III in mitochondria. This finding was not entirely unexpected, because the majority of ROS in living cells are produced by semiquinone anion species that occur as an intermediate in the ubiquinone cycle (2). At the ubiquinone cycle, ubiquinone carries two electrons to the Qo site, where one electron is transferred to cytochrome c1 of the complex III and eventually to Cyt c. Ubiquinone with one electron (semiubiquinone anion) is generated as an intermediate at the Qo site, and it passes the second electron to cytochrome b of complex III. Then, cytochrome b passes the electron to a fully oxidized ubiquinone at the Qi site, generating an ubisemiquinone anion, which antimycin A inhibits. Antimycin A can be used to induce apoptosis when used at a relatively high concentration that blocks electron flow completely, and this concentration of antimycin a also induces enhanced production of ROS, due to the increased reduction of electron carriers/complexes upstream of the blockage. In our study, antimycin A was titrated before each experiment to assure that the concentration used was inhibitory to H2O2 production as well as apoptosis. Enhanced H2O2 production and apoptosis were observed with 50 µM antimycin A (data not shown), which is 50 times more than the concentration used in the experiments, and therefore, we believe the concentration used in our experiments reduced the electron flow, and did not block it completely. The generation of the free radical semiquinone anion species during the ubiquinone cycle is a function of the rate of electron transport, therefore, when antimycin A is used to reduce the electron flow at the cycle, the production of ROS is reduced as well. Our study shows that a death-inducing signal can mediate increased production of H2O2 at the ubiquinone cycle, but the precise mechanisms involved in the signaling process remains to be elucidated.
We observed that while treatment with rotenone or antimycin A decreased the production of H2O2 in DEX-treated cells, it enhanced the H2O2 production upon addition of exogenous H2O2. Some evidence indicates that Cyt c works as an antioxidant by being released into the intermembrane space of mitochondria, thus making it available to accept an electron from ROS generated in the mitochondria (27). Therefore, it is possible that elevated intracellular H2O2 and increased mitochondrial oxidative stress induced by adding exogenous H2O2 draws Cyt c into the intermembrane space to control the oxidative stress. When oxidative stress is more than this defense mechanism can handle, the unsuccessful scavenging of elevated H2O2 by Cyt c may result in increased vulnerability for PT, Cyt c release to the cytoplasm, ROS generation at ubiquinone cycle, and apoptosis. In addition, a recent report showed that antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3 and directly inhibits the function of Bcl-xL, an anti-apoptotic Bcl-2 family member, thereby causing mitochondrial swelling and m depolarization (28). Thus, when cells are given H2O2 exogenously, Cyt c can be released into the intermembrane space of the mitochondria to control the oxidative stress, and an otherwise protective concentration of antimycin A can mediate the release of Cyt c through the inhibitory interaction with antiapoptotic Bcl-2 family members. As shown in our study, addition of exogenous H2O2 by itself can cause PT, Cyt c release, caspase-3 activation, and apoptosis, suggesting that the increased production of H2O2 in mitochondria during apoptosis is not only a result of the mitochondrial homeostasis disruption, but is also a cause of the disruption. This observation agrees with the report that the mitochondrial PT pore that mediates PT is sensitive to ROS (11). Therefore, the production of H2O2 and the mitochondrial homeostasis disruption may create a self-amplifying process in the mitochondria leading to apoptosis.
We used lactacystin to address the role of the proteasome in regulating cell death events mediated by mitochondria. Inhibiting proteasome activities attenuated cell death events mediated by mitochondria observed in this study, including the production of H2O2, providing evidence that the proteasome plays a regulatory role upstream of the mitochondria. Because our data showed that the proteasome was capable of regulating apoptotic events in response to changes in levels of intracellular oxidative stress, it is possible that the H2O2 generated in mitochondria creates a negative and/or positive feedback loop to the proteasome, which in turn affects the selective degradation of Bcl-2 family members in the mitochondria. The molecular mechanism(s) by which the proteasome senses oxidative stress remains to be elucidated. One possible mechanism in which proteasome regulates apoptosis is to degrade pro- and/or antiapoptotic proteins. Recent observations suggest that the proteasome regulates the expression levels of pro- and antiapoptotic Bcl-2 family members in mitochondria (29, 30). Although the precise molecular mechanisms are yet to be elucidated, data from numerous studies suggested that proapoptotic Bcl-2 family members such as Bid, Bax, and Bak mediate Cyt c release either by forming a channel/pore themselves and thus permeabilizing the mitochondrial outer membrane, or by interacting and regulating the voltage-dependent anion channel, a resident mitochondrial membrane pore (20, 21, 22, 31). The antiapoptotic Bcl-2 family members such as Bcl-2 and Bcl-xL may prevent apoptosis either by physically interacting with the proapoptotic Bcl-2 family members and inhibiting their action, or by regulating voltage-dependent anion channel (20, 21, 22, 31). Therefore, changes in the ratio of pro- and antiapoptotic Bcl-2 proteins could be the mechanism by which the proteasome acts to regulate apoptosis in thymocytes at or before the mitochondrial level. Most of the proapoptotic Bcl-2 members are localized to the cytoplasm or cytoskeleton before an apoptotic signal (32, 33, 34). Upon receiving such a signal, they undergo conformational changes that allow them to migrate, possibly by the dynein motor complex and integrate into the mitochondrial outer membrane (34). Therefore, it is possible that proteasomal activities are modifying the migration process of the proapoptotic Bcl-2 family members by degrading the cytoskeletal/motor proteins. Another example of proteasome-mediated degradation of antiapoptotic protein is the degradation of inhibitors of apoptosis proteins (IAPs) upon apoptotic stimuli, including DEX (35). IAPs inhibit activation and/or activities of caspases, and this degradation occurs very early in the apoptotic process through autoubiquitination (35). Slee et al. (36) have shown that in Jurkat cells, caspase-3 establishes a proapoptotic feedback loop to mitochondria through direct cleavage of Bid, causing further permeabilization of the outer membrane. Thus, it is possible that the proteasome may play a critical role in regulating apoptosis in T cells/thymocytes at several points in the cell death cascade.
Fig. 8 shows a summary of this study and a hypothesized regulatory role of H2O2 and proteasomes. We showed that the increased production of H2O2 is observed during DEX-, TCR- and exogenous H2O2-induced thymocytes apoptosis, and we identified the site of the production of H2O2 at ubiquinone cycle at complex III in the mitochondria. The deprivation of molecular oxygen and the treatment with NAC diminished the increases in the production of H2O2, loss of m, caspase-3 activation, Cyt c release, and amount of apoptosis, therefore suggesting that higher levels of intracellular H2O2 promotes these apoptotic events and increases cell susceptibility to apoptosis. We showed that inhibition of the proteasome resulted in a reduction of mitochondria-mediated cell death events induced by DEX and exogenous H2O2. This result indicates that the proteasome plays the following roles: 1) regulation of mitochondrial membrane permeabilization, possibly by degradation of Bcl-2 family members, and/or through degradation of IAPs, and 2) regulation of apoptosis in response to the intracellular oxidative stress, therefore establishing a feedback loop with the oxidative stress originating from mitochondria during apoptosis.
|
B and AP-1 (37). Multiple pathways and transcription factors can be activated or deactivated by the rise in intracellular H2O2 during DEX-induced thymocyte apoptosis, and while these direct mechanisms remain to be elucidated, our study has shed light on two important areas of oxidative stress signaling in thymocyte apoptosis: the role of molecular oxygen and H2O2, and the role of the proteasome in mediating and modulating the signaling pathway before a mitochondrial involvement.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Barbara A. Osborne, Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003. E-mail address: Osborne{at}vasci.umass.edu
3 Abbreviations used in this paper: ROS, reactive oxygen species; PT, permeability transition; Cyt c, cytochrome c; DEX, dexamethasone; NAC, N-acetyl-L-cysteine; CM-H2DCFDA, chloromethyl-dichlorodihydrofluorescein diacetate; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; m, mitochondrial membrane potential; IAP, inhibitor of apoptosis protein.
Received for publication September 9, 2002. Accepted for publication December 20, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. C. Jaramillo, J. B. Frye, J. D. Crapo, M. M. Briehl, and M. E. Tome Increased Manganese Superoxide Dismutase Expression or Treatment with Manganese Porphyrin Potentiates Dexamethasone-Induced Apoptosis in Lymphoma Cells Cancer Res., July 1, 2009; 69(13): 5450 - 5457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kubota, K. Kinoshita, K. Suetomi, A. Fujimori, and S. Takahashi Mcl-1 Depletion in Apoptosis Elicited by Ionizing Radiation in Peritoneal Resident Macrophages of C3H Mice J. Immunol., March 1, 2007; 178(5): 2923 - 2931. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Ranta, D. Avram, S. Berchtold, M. Dufer, G. Drews, F. Lang, and S. Ullrich Dexamethasone induces cell death in insulin-secreting cells, an effect reversed by exendin-4. Diabetes, May 1, 2006; 55(5): 1380 - 1390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Wang, N. Muller, K. G. McPherson, and H. M. Reichardt Glucocorticoids Engage Different Signal Transduction Pathways to Induce Apoptosis in Thymocytes and Mature T Cells J. Immunol., February 1, 2006; 176(3): 1695 - 1702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-H. Li, S.-L. Tzeng, Y.-W. Cheng, and J.-J. Kang Chloramphenicol-induced Mitochondrial Stress Increases p21 Expression and Prevents Cell Apoptosis through a p21-dependent Pathway J. Biol. Chem., July 15, 2005; 280(28): 26193 - 26199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Quadrilatero and L. Hoffman-Goetz N-acetyl-l-cysteine protects intestinal lymphocytes from apoptotic death after acute exercise in adrenalectomized mice Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2005; 288(6): R1664 - R1672. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Malone, Z. Wang, and C. W. Distelhorst The Glucocorticoid-induced Gene tdag8 Encodes a Pro-apoptotic G Protein-coupled Receptor Whose Activation Promotes Glucocorticoid-induced Apoptosis J. Biol. Chem., December 17, 2004; 279(51): 52850 - 52859. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Lepine, B. Lakatos, M.-P. Courageot, H. Le Stunff, J.-C. Sulpice, and F. Giraud Sphingosine Contributes to Glucocorticoid-Induced Apoptosis of Thymocytes Independently of the Mitochondrial Pathway J. Immunol., September 15, 2004; 173(6): 3783 - 3790. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Y. Almawi, O. K. Melemedjian, and M. M. A. Jaoude On the link between Bcl-2 family proteins and glucocorticoid-induced apoptosis J. Leukoc. Biol., July 1, 2004; 76(1): 7 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Hoffman-Goetz, J. Quadrilatero, J. Boudreau, and J. Guan Adrenalectomy in mice does not prevent loss of intestinal lymphocytes after exercise J Appl Physiol, June 1, 2004; 96(6): 2073 - 2081. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Cavigelli and M. K. McClintock Fear of novelty in infant rats predicts adult corticosterone dynamics and an early death PNAS, December 23, 2003; 100(26): 16131 - 16136. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
