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Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health (C.L.M., J.A.C.), Research Triangle Park, North Carolina 27709; and Curriculum in Toxicology, University of North Carolina (C.L.M.), Chapel Hill, North Carolina 27599
Address all correspondence and requests for reprints to: Dr. John A. Cidlowski, P.O. Box 12233 MD E2–02, Research Triangle Park, North Carolina 27709. E-mail: cidlowski{at}niehs.nih.gov
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One feature observed in all known examples of apoptosis is the loss of cell volume, apparently as a result of ions leaving the cell (3, 4, 5, 6, 7). This characteristic has been well documented in spontaneously dying rat thymocytes as well as thymocytes treated with glucocorticoids (8) and is thought to be due to the loss of intracellular potassium (3, 4). In addition to cell shrinkage, during apoptosis, phosphatidylserine residues reorient to the outside of the cell, enabling the dying cell and apoptotic bodies to be recognized and removed by macrophages (9). Chromatin condensation and DNA cleavage are also common late features of apoptosis (10, 11) and were observed in thymocytes after in vivo administration of glucocorticoids to rats (1).
The aforementioned morphological and biochemical changes in apoptosis are also accompanied by activation of the caspase cascade. Activation of a series of caspases has been described in numerous examples of apoptosis (12, 13, 14). Glucocorticoids have been shown to activate caspases in human transformed cell lines (15, 16); however, the regulation of caspase activity has not been studied in rat thymocytes. Caspase activation is thought to be a central mediator in the production of the apoptotic phenotype (17), presumably by dismantling cellular substrates (18, 19, 20, 21). Finally, apoptosis induced by a number of stimuli is also accompanied by the loss of mitochondrial potential (22, 23), which is thought to produce signals for the activation of downstream caspases.
The regulation of the pathways that lead to these biochemical and morphological manifestations of apoptosis may vary depending on the initiating signal. Thymocytes undergo apoptosis in response to a number of factors, including glucocorticoids (24, 25), the calcium ionophore A23187 (26), and T cell receptor ligation (27). Moreover, simply placing thymocytes in culture is a sufficient signal to rapidly induce cell death in the isolated thymocytes. This spontaneous death is presumably due to the withdrawal of survival factors, and the kinetics of its appearance suggest that thymocytes are poised to die in response to an apoptotic stimulus. Spontaneous death induces many of the same biochemical and morphological features (8) as glucocorticoid-induced death. However, it is not known whether spontaneous death and the biochemical features associated with it are regulated by gene expression or caspase activity.
In contrast to spontaneous cell death, which occurs simply by the withdrawal of survival factors, glucocorticoids are a death factor that upsets the balance between signals and results in apoptosis. In previous studies it has been shown that glucocorticoid-induced apoptosis is dependent on protein and RNA synthesis (11, 28, 29). These studies were based on analysis of viability and DNA fragmentation, both of which are late markers of apoptosis. In this report, we assess whether glucocorticoid regulation of upstream components of the apoptotic pathway are also dependent on gene regulation to determine whether glucocorticoids induced a protein(s) that triggers all subsequent features of cell death, or whether each component of the pathway is differentially regulated. The results of these experiments suggest that glucocorticoids induce the expression of a protein(s) that triggers the subsequent events of apoptosis, whereas survival factor withdrawal leads to spontaneous death in the absence of gene expression or caspase activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Male Sprague Dawley rats (2–3 months old) were used in all
experiments. The animals were bilaterally adrenalectomized by the
provider at least 5 days before use, maintained under controlled
conditions of temperature (25 C) and lighting, and allowed free access
to food and saline. All experimental protocols were approved by the
animal review committee at the institute and were performed in
accordance with the guidelines set forth in the NIH Guide for the Care
and Use of Laboratory Animals published by the USPHS. Animals were
killed by decapitation, and the thymus was removed.
Thymocyte cultures
Thymocytes were prepared from freshly isolated thymus as
previously described (28, 30, 31). Thymocytes were dispersed by gentle
homogenization in a Kontes no. 22 glass/glass homogenizer (Kontes Co.,
Vineland, NJ), filtered, washed in cold PBS, and counted on a
hemocytometer. Cells were cultured at 5 x
106 cells/ml in RPMI 1640 medium supplemented
with 10% heat-inactivated FCS, 4 mM glutamine, 100 U/ml
penicillin, and 75 U/ml streptomycin sulfate. Cells were incubated at
37 C in 5% CO2 for 0, 2, 4, and 6 h before
harvest.
Viability and annexin V staining
Cellular viability and cellular membrane changes
(phosphatidylserine expression on the outer leaflet of the plasma
membrane) were evaluated concurrently using a TACS annexin
V-fluorescein isothiocyanate (FITC) kit (Trevigen, Gaithersburg, MD).
Briefly, 1 ml cells was pelleted and resuspended in 89 µl 1 x
binding buffer (10 mM HEPES, pH 7.4;0.15
M NaCl; 5 mM KCl; 1 mM
MgCl2; and 1.8 mM CaCl2), 1 µg/ml
propidium iodide (PI), and annexin V-FITC. Cells were incubated for 15
min before the addition of 400 µl 1 x binding buffer and flow
cytometry. Ten thousand cells were counted on a Becton Dickinson and Co. FACSort equipped with CellQuest software (Becton Dickinson and Co., San Jose, CA). PI staining and annexin V-FITC
staining were measured on an FL-3 (650 nm) vs. FL-1 (530 nm)
plot, respectively. Gating on cells with positive PI staining with
CellQuest software identified the cells with a loss of viability.
Similarly, gating on cells with positive annexin V staining identified
the cells that had externalized phosphatidylserine residues.
Caspase activity assay
Caspase-3-like activity was measured using a fluorometric assay
as previously described (3, 31, 32, 33). Briefly, cytoplasmic extracts were
prepared by resuspending thymocytes in 10 mM
MgCl2 and 0.25% Nonidet P-40. Debris was
pelleted at 100,000 x g for 30 min, and supernates
were placed on ice. Ten to 50 µg extract [measured by the method of
Bradford (34)] were preincubated (10 mM
dithiothreitol, 50 mM HEPES, 10% sucrose, and
0.1%
(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, pH
7.5 (CHAPS) with 200 µM of the noncompetitive
inhibitor DEVD-fmk. Parallel samples were prepared without inhibitor.
Substrate (DEVD-afc) was then added to all tubes (200
µM, final concentration). Samples were
incubated for 5 min at 30 C, and their fluorescence at 505
nM was measured (excitation at 400
nM). Samples were incubated for an additional
hour, and fluorescence was again measured. A standard curve of
fluorescence vs. free 7-amino-4-trifluoromethylcoumarin was
then used to calculate the specific activity of caspase-3-like enzymes
in each sample.
Determination of cell size
Forward and side light scattering properties of cells were
analyzed by flow cytometry (Becton Dickinson and Co.
FACSort equipped with CellQuest software). Apoptotic cells show a
decrease in forward light-scattering properties and a concomitant
increase in side light-scattering properties. A gate was created based
on these parameters, and for all experiments, the percentage of cells
in this region was used to determine the number of cells with a loss of
cell volume as a percentage of the total number of cells.
Determination of mitochondrial membrane potential
Changes in mitochondrial membrane potential were examined by
flow cytometry (Becton Dickinson and Co. FACSort equipped
with CellQuest software) using JC-1 (Molecular Probes, Inc., Eugene, OR). JC-1 was added to 1 ml cultured cells to a
final concentration of 10 µM 30 min before harvest and
analysis by flow cytometry. Ten thousand cells were then examined by
flow cytometry on an FL-1 (530 nm) vs. FL-2 (585 nm) plot.
JC-1 aggregates, which represent intact mitochondrial membrane
potential, are identified by an increase in FL-2 fluorescence. Cells
with a loss of mitochondrial potential were identified by gating on a
population with a decrease in FL-2 fluorescence, which indicates a
decrease in JC-1 aggregates.
Analysis of DNA content
The DNA content of each sample was determined as previously
described (3, 8). Briefly, 1 ml pelleted cells was resuspended in cold
70% ethanol to a volume of approximately 2 ml. Samples were stored
overnight at 4 C. Fixed cells were washed once in PBS and then stained
in 1 ml 20 µg/ml PI and 1 mg/ml ribonuclease in PBS for 20 min. Cells
were examined with flow cytometry (Becton Dickinson and Co. FACSort equipped with CellQuest software) by gating on an
area vs. width plot to exclude debris. Ten thousand cells
were counted within the gate for each treatment.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). DEX augments this loss
of viability. Subsequently, to determine whether the loss of viability
is regulated by gene expression, we assessed the effects of
cycloheximide, a protein synthesis inhibitor, and actinomycin D, a RNA
synthesis inhibitor, on glucocorticoid-induced and spontaneous cell
death. The loss of viability induced by DEX was blocked completely by
cycloheximide and partially by actinomycin D (Fig. 1, A and B). These
data are in accordance with previous studies that showed that
glucocorticoid-induced thymocyte death requires de novo gene
expression (11, 29). In contrast, spontaneous cell death was not
dependent upon new gene expression, suggesting that cells contain
sufficient machinery to die without additional macromolecular
synthesis.
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). To determine whether this effect is
dependent upon gene regulation or caspase activity, we assessed the
ability of cycloheximide, actinomycin D, and z-VAD-fmk (a pan-caspase
inhibitor) to inhibit spontaneous and DEX-induced DNA fragmentation by
flow cytometry. In spontaneously dying cells, there is an increase in
DNA fragmentation by 4 h and, as with the observed effects on
viability, DEX augments this effect. Inhibition of protein or RNA
synthesis blocks the effect of DEX, but does not abrogate the DNA
fragmentation associated with spontaneous death (Fig. 2, A and B).
Inhibition of caspase activity, however, completely inhibits both
DEX-induced DNA fragmentation and spontaneous DNA fragmentation to the
same levels as those seen in freshly isolated thymocytes (Fig. 2C).
These data demonstrate that there is a common pathway shared by
spontaneous and DEX-induced cell death that leads to DNA degradation
and that this pathway is controlled by caspases. However, the upstream
events that lead to activation of this common pathway differ between
spontaneous and DEX-induced cell death. Specifically,
glucocorticoid-induced activation of the DNA degradation pathway
requires de novo gene expression, whereas spontaneous
apoptosis does not.
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). In the absence of DEX, the
spontaneously dying cells activate caspase-3-like enzymes to levels
10-fold greater than those found in freshly isolated cells. DEX induces
further caspase activity in a dose-dependent fashion, with an
ED50 of about 1.5 nM and a
maximum dose of 100 nM. These values are
consistent with the binding of DEX to its cognate receptor (36). These
results suggest that both survival factor withdrawal-induced and
DEX-stimulated pathways of apoptosis are mediated through the
activation of caspase-3-like enzymes.
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). However, two natural
glucocorticoids, cortisol and corticosterone, significantly increased
caspase activity above spontaneous levels, indicating that the
activation of caspases in isolated thymocytes is specific to
glucocorticoids and cannot be activated by sex steroids under our
experimental conditions.
Next, we investigated the effect of a glucocorticoid receptor
antagonist, RU486, on the activation of caspase-3-like activity in both
spontaneous and DEX-induced death to elucidate whether growth factor
deprivation was activating the glucocorticoid receptor in the absence
of RU486. RU486 did not inhibit activation of caspase-3-like activity
in survival factor withdrawal-induced death. These findings confirmed
that activation of caspase-3-like activity in survival factor
withdrawal-induced death occurs independently of the glucocorticoid
receptor or its activation by placement of cells in culture. To
determine whether the ability of glucocorticoids to activate
caspase-3-like activity in thymocytes was dependent upon their
interaction with the glucocorticoid receptor, we tested the ability of
the receptor antagonist, RU486, to block DEX-induced caspase-3-like
enzyme activation (Fig. 3C). RU486 effectively blocked DEX-induced
caspase activation in thymocytes, which demonstrates that the ability
of glucocorticoids to activate caspases is dependent upon the
interaction with the glucocorticoid receptor.
Finally, to determine whether activation of caspase-3-like activity in
spontaneous and glucocorticoid-induced death is dependent upon de
novo gene expression, we assessed caspase-3-like activity in the
presence of cycloheximide or actinomycin D (Fig. 3D). Cycloheximide and
actinomycin D did not prevent the increase in caspase-3-like activity
observed in spontaneously dying cells. However, both cycloheximide and
actinomycin D reduced DEX-induced caspase-3-like activity to the same
levels of activation as those found in spontaneously apoptosing cells.
These results demonstrate that the activation of caspase activity in
spontaneous and glucocorticoid-induced apoptosis occurs by two distinct
pathways. In spontaneous death, gene regulation is not required for
caspase activation, whereas in glucocorticoid-induced death, gene
regulation lies upstream of caspase activation.
Effect of caspase inhibition on glucocorticoid-induced and
spontaneous death
To determine whether glucocorticoid-induced or spontaneous death
was dependent upon caspase activity, we used the pan-caspase inhibitor
z-VAD to inhibit caspase activity within the cells and then measured
the loss of viability induced either spontaneously or by the addition
of DEX. Spontaneous cell death in the untreated thymocytes could not be
inhibited by z-VAD. However, z-VAD inhibited DEX-induced loss of
viability to the same levels as in spontaneously dying cells (Fig. 4). These data also show that in rat
thymocytes, inhibition of the caspase cascade blocks DEX-induced cell
death, but not spontaneous death. The fact that the spontaneous cell
death observed in the untreated group cannot be inhibited by
cycloheximide, actinomycin D, or z-VAD demonstrates that rat thymocytes
can die by an inherent caspase-independent pathway that does not
require macromolecular synthesis.
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).
Neither cycloheximide nor actinomycin D affected cell shrinkage of the
spontaneously dying thymocytes. Interestingly, z-VAD delayed cell
shrinkage in the spontaneously dying population for the first 4 h,
which is consistent with our previous results (8), suggesting that
there is a caspase-independent loss of cell volume in the spontaneously
dying cells. These data suggest that although cell shrinkage in the
growth factor-deprived population is not dependent upon caspase
activity, caspase activity can modulate the pathway that leads to cell
shrinkage. Both cycloheximide and z-VAD abrogate the effect of DEX on
cell shrinkage by reducing the percentage of shrunken cells to the same
levels as the spontaneously dying population. Actinomycin D also
inhibits DEX-induced cell shrinkage, but not as efficiently as
inhibition of protein synthesis or caspase activity.
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, spontaneous cell death was characterized by a time-dependent loss of
mitochondrial potential, an effect that was augmented by DEX. The loss
of mitochondrial potential in spontaneously dying cells was independent
of both protein and RNA synthesis (Fig. 6, A and B) as well as of
caspase activity (Fig. 6C). However, the DEX-induced loss of
mitochondrial potential was blocked by coadministration of
cycloheximide, indicating that the DEX-induced loss of mitochondrial
potential is dependent upon protein synthesis. Actinomycin D and z-VAD,
however, were less effective than cycloheximide at rescuing the
DEX-induced loss of mitochondrial potential. Overall, these results
suggest that the component of glucocorticoid-induced apoptosis that is
dependent upon protein synthesis is functioning upstream of any changes
in the mitochondria. In addition, we have shown that in rat thymocytes,
the glucocorticoid-induced loss of mitochondrial potential is probably
occurring upstream of caspase activation.
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, in spontaneously dying cells, there is
a time-dependent increase in the exposure of phosphatidylserine
residues to the exterior of the cell, and glucocorticoids will enhance
this effect (Fig. 7). The macromolecular synthesis inhibitors
cycloheximide and actinomycin D block the glucocorticoid-induced
increase in phosphatidylserine exposure, but they do not block the
increase associated with spontaneous cell death. Similarly, inhibition
of caspase activity by z-VAD blocks phosphatidylserine externalization
in glucocorticoid-induced death. However, in spontaneous cell death,
z-VAD retards the kinetics of phosphatidylserine residue exposure, but
does not block the spontaneous increase in phosphatidylserine
externalization. These data suggest that although the membrane changes
associated with apoptosis are not dependent upon caspase activity,
caspase activity can modulate this pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our initial observation that cell death induced by glucocorticoids was gene expression and caspase dependent, whereas spontaneous cell death was not, directed our attention toward these two independent modes of cell death in thymocytes. To further elucidate the mechanistic differences between these two pathways, we evaluated the effect of macromolecular synthesis inhibitors and a caspase inhibitor on different biochemical end points that are characteristic of apoptosis. First, we assessed the effect of macromolecular synthesis inhibitors and caspase inhibitors on DNA fragmentation. Previous studies have shown that macromolecular synthesis is required for glucocorticoid-induced DNA fragmentation (11, 28, 29). However, it was not known whether it was required for spontaneous DNA fragmentation. We observed that inhibition of protein and RNA synthesis could not block DNA fragmentation associated with spontaneous cell death. These data were consistent with our observation that in growth factor withdrawal-induced death, neither caspase-3-like activity nor loss of viability was prevented by macromolecular synthesis inhibitors. DNA fragmentation was blocked by z-VAD in both spontaneous and glucocorticoid-induced death. This is consistent with other reports in the literature, which show that DNA fragmentation lies downstream of caspase activation (18, 19, 20). In addition, these data demonstrate that thymocytes can die in the absence of DNA degradation, which has been reported in other model systems (41, 42).
As activation of caspase-3-like activity has been shown to induce DNA fragmentation (18, 19, 20), we next considered the regulation of caspase-3-like activity in both spontaneous and glucocorticoid-induced apoptosis. The effect of glucocorticoids on caspase-3-like activity was steroid specific and receptor mediated, which is consistent with the previous observation made by Ingle (24) that steroid-induced thymocyte death was unique to glucocorticoids. Further experiments revealed that the effects of glucocorticoids on caspase-3-like activity were also dependent upon de novo gene expression. We observed that untreated thymocytes undergoing spontaneous apoptosis in culture have a significant increase in caspase-3-like activity over that in freshly isolated thymocytes, and that this increase occurred independently of gene expression, which was consistent with our observation that spontaneous cell death does not require de novo gene expression. However, inhibition of caspase activity did not protect the cells from spontaneous death. Therefore, although spontaneous cell death involves caspase activation, caspase activity is not required for spontaneous cell death.
To further characterize the differences between glucocorticoid-induced and spontaneous apoptosis, we analyzed plasma membrane changes, cell shrinkage, and mitochondrial potential changes. Macromolecular synthesis inhibitors blocked glucocorticoid-induced changes in these parameters to the same level as in time-matched untreated cells. However, they did not inhibit glucocorticoid-induced changes in these parameters to the same levels as in freshly isolated thymocytes. Macromolecular synthesis inhibitors also did not block the plasma membrane changes, cell shrinkage, and loss of mitochondrial potential associated with spontaneous cell death. These observations confirmed that the manifestation of these biochemical endpoints is dependent upon gene expression in glucocorticoid-induced cell death, but not in spontaneous death.
Inhibition of caspase activity in glucocorticoid-treated and spontaneously dying cells did not effect the changes in mitochondrial potential, an observation consistent with other studies that show changes in mitochondrial potential occur upstream of caspase activation (43). Caspase inhibition stalled the exposure of phosphatidylserine residues on the outside of the cell as well as the loss of cell volume in spontaneously dying cells, but did not completely inhibit either of these end points. This demonstrates that caspases can modulate these pathways, but are not required for phosphatidylserine exposure or the loss of cell volume. In addition, these data suggest that there is a slower, caspase-independent pathway that can lead to spontaneous cell death and that this pathway includes cell shrinkage, the loss of mitochondrial potential, and the exposure of phosphatidylserine residues, but does not include caspase activation and DNA fragmentation.
The ratio of survival factors to death factors dictates the fate of a cell. Thymocytes are sensitive to survival factor withdrawal and undergo spontaneous apoptosis when placed in culture. Thymocytes are also susceptible to death-inducing agents such as glucocorticoids. Although apoptosis induced by the two different mechanisms share some common biochemical and morphological features, we have demonstrated that in thymocytes, there are two distinct pathways. The loss of viability and the manifestation of all of the biochemical features of glucocorticoid-induced apoptosis that we studied were dependent upon de novo gene expression, suggesting that glucocorticoids do not individually regulate multiple steps in the apoptotic pathway. This demonstrates that gene regulation is an early step in the glucocorticoid-induced pathway after the interaction of steroid with glucocorticoid receptor and that gene regulation serves as a molecular switch that controls the progression of glucocorticoid-induced apoptosis. In contrast, spontaneous death bypasses this particular molecular switch and proceeds independently of gene expression. Eventually, the glucocorticoid pathway engages a death program similar to that initiated by survival factor withdrawal. This represents a convergence point for these two pathways. Together, the data in this study delineate a network of signals that mediate spontaneous apoptotic cell death and glucocorticoid-induced apoptotic cell death.
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Acknowledgments |
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Footnotes |
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Received August 23, 1999.
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