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Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio
Correspondence: Joan M. Cook-Mills, Ph.D., Department of Pathology and Laboratory Medicine, University of Cincinnati, 231 Bethesda Ave., Cincinnati, OH 45267-0529. E-mail: joan.cook{at}uc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: annexin V • DNA fragmentation • MC540 • propidium iodide • caspase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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A variety of these cellular changes are used as indicators of the
apoptotic process. Some assays commonly used to quantify apoptosis are
decreased cell-membrane lipid-packing as assessed by incorporation of
the lipophilic dye, MC540 [2
]; accumulation of
phosphatidylserine on the outer cell membrane as evaluated by annexin
V-fluorescein isothiocyanate (FITC) binding [3
];
appearance of hypodiploid nuclei examined by propidium iodide (PI)
labeling of DNA in ethanol-fixed cells [4
]; DNA
fragmentation as determined by a colorimetric fragmentation assay
[5
]; and cell-membrane permeability to PI
[2
]. Several studies have shown that as cells progress
through the apoptotic process, there is a temporal acquisition of these
apoptotic characteristics [6
, 7
]. Mower et
al. [2
] described three sequential stages of apoptosis
in resting, murine splenic B cells in which stage 1 was characterized
by increased MC540 binding to the outer cell membrane and decreased
cell volume, stage 2 was distinguished by endonucleosomal cleavage of
DNA, and stage 3 was classified by plasma-membrane permeability to PI.
Reid et al. [8
] demonstrated that MC540 incorporation
into the cell membrane preceded DNA fragmentation of 
-irradiated
splenic B cells isolated from DBA/2 mice. Studies by Martin et al.
[9
] demonstrated that externalization of anionic
phosphatidylserine to the outer membrane leaflet occurred prior to
cell-membrane permeability to PI in human neutrophils and murine T
cells regardless of the method for induction of apoptosis. Furthermore,
Frey [10
] shows that labeling cells with MC540 and
annexin V occurs after labeling cells with LDS-751 or fluorescein
diacetate, and in some other cell lines, there is no change in MC540 or
annexin-V binding. However, Frey [10
] does not report
the time for acquisition of these indicators in relationship to
acquisition of DNA fragmentation. Frey’s studies suggest that
multi-parameter analysis is important for detecting induction of
apoptosis. It is not known whether the acquisition of multiple
apoptotic parameters occurs in the same sequence for different
apoptotic cells.
The study of the regulation of the acquisition of these apoptotic
parameters by caspases has been limited. It has been demonstrated that
inhibition of caspases by the general caspase inhibitor,
benzoyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk),
completely blocks accumulation of hypodiploid nuclei in
anti-immunoglobulin (Ig)M-treated WEHI-231 cells as detected by PI
labeling [11
]. However, in these cells, inhibition of a
terminal caspase of the caspase cascade, caspase 3, by Ac-DEVD-aldehyde
(CHO) had no effect on acquisition of PI-labeled hypodiploid nuclei
[11
]. Pretreatment of -irradiated leukemic T cells
(MOLT-4 cells) with DEVD-fmk, a caspase 3 inhibitor, inhibited the
acquisition of annexin V-FITC binding and PI permeability
[12
]. In contrast, Drenou et al. [13
]
showed no z-VAD-fmk or DEVD-fmk inhibition of HLA-DR-mediated death of
human B cells as determined by annexin V-FITC binding and PI
permeability. In these cells, caspase inhibitors also do not block DNA
fragmentation [14
]. DEVD-fmk also does not block DNA
fragmentation in anti-Ig-treated, transformed B cells (WEHI-231 cells)
[11
]. It remains unclear whether in nontransformed
caspase-dependent apoptotic cells DNA fragmentation and the membrane
changes are linked to a common signal-transduction pathway such as the
caspase cascade.
In summary, previous studies [2 ] indicate that there appears to be a defined order in the acquisition of apoptotic characteristics by apoptotic cells in which the sequence is labeling by MC540 and annexin V-FITC, DNA fragmentation, and then plasma-membrane permeability to PI. However, no one has shown the order for acquisition of all of these apoptotic characteristics in multiple cell types under different modes for induction of apoptosis. In addition, it remains unclear whether both the DNA degradation and membrane changes within an apoptotic leukocyte are linked to the caspase pathway. We demonstrate here, for the first time, that the order in which apoptotic characteristics are acquired by myeloid and lymphoid cells is cell type-specific, and for the same cell type, it differs with the mode of induction of apoptosis. The order for acquisition of the apoptotic characteristics is not linked to caspase activity. These changes can occur independent of caspase activation. In nontransformed cells, the membrane changes occur independent of caspase activity, whereas DNA degradation was caspase-dependent.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Reagents
Affinity-purified, goat anti-mouse Ig F(ab')2 (IgA,
IgG, and IgM, H+L) was obtained from Organon Teknika Corporation (West
Chester, PA). z-VAD-fmk, z-Asp-Glu-Val-Asp-fmk (z-DEVD-fmk), and
z-Phe-Ala-fmk (z-FA-fmk) were from Enzyme Systems Products (Livermore,
CA).
Leukocyte culture
Spleen cells were isolated from BALB/c mice (Harlan Industries),
the red blood cells were removed by hypotonic shock with distilled
water for 3 s, and the spleen lymphocytes (>90% viability) were
cultured in medium as previously described [15
]. S49.1
cells [BALB/c mouse-derived, T-cell lymphoma from American Type
Culture Collection (ATCC), Manassas, VA] were cultured in Dulbecco’s
modified Eagle’s medium (DMEM)-growth medium [DMEM medium (Cellgro
from Fisher, Cincinnati, OH) plus 10% heat-inactivated fetal calf
serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin,
and 50 µg/ml gentamicin]. WEHI-231 cells (BALB/c mouse-derived,
B-cell lymphoma from ATCC) were maintained in DMEM-growth medium
supplemented with 50 µM 2-mercaptoethanol (2-ME). The 32D cells
(pre-myeloid cell line from David Askew, University of Cincinnati, OH)
were maintained in DMEM-growth medium supplemented with 10%
WEHI-3-conditioned medium as a source of interleukin-3 (IL-3)
[15
].
Induction of apoptosis
Spleen cells, S49.1 cells, and WEHI-231 cells were irradiated
with 500, 750, and 750 rads of -irradiation, respectively, and
placed in culture medium [5
]. WEHI-231 cells
(2x106/ml) were also induced to undergo apoptosis by
cross-linking cell-surface immunoglobulins by the addition of 4 µg/ml
goat anti-mouse Ig F(ab')2 in DMEM-culture medium
[16
, 17
]. After 24 h at 37°C, the
WEHI-231 cells were washed and placed in DMEM-culture medium. 32D cell
apoptosis was induced by washing the cells to remove IL-3 and
incubating in culture medium without IL-3 [18
].
Spontaneous spleen-cell death was induced by placing spleen cells in
culture medium [15
].
MC540 flow cytometric assay [8
]
Leukocytes (1x106 cells) were centrifuged for 5 min
at 200 g, resuspended in 0.16 µg/ml MC540 (Sigma Chemical
Co., St. Louis, MO) in phosphate-buffered saline (PBS) for 20 min in
the dark at room temperature, washed twice in PBS, and analyzed by flow
cytometry.
Annexin V-FITC flow cytometric assay (Trevigen Apoptotic Cell
System Annexin V-FITC, Trevigen, Inc., Gaithersburg, MD)
Leukocytes (1x106 cells) were washed with PBS
(4°C) and suspended in 100 µl annexin V incubation reagent (4°C)
containing annexin V-conjugate for 15 min in the dark at room
temperature. Binding buffer (400 µl 1x) was added, and then the
cells were analyzed by flow cytometry.
% of PI-labeled hypodiploid nuclei [4
]
Leukocytes (2x106 cells) were washed with PBS
(4°C), fixed in 2 ml 70% ethanol (4°C) for 30 min, washed in 10 ml
PBS (4°C), suspended in DNA staining reagent [PBS; 0.1 mM
ethylenediaminetetraacetate (EDTA); 0.05 mg/ml RNase A (50 units/mg,
protease- and DNase-free, Boehringer-Mannheim, Indianapolis, IN); pH
7.4] containing 50 µg/ml PI (Sigma) at room temperature for 15 min,
and then analyzed by flow cytometry.
Cell membrane permeability [2
]
Leukocytes (1x106 cells) were suspended in 1 ml
0.2% glucose in PBS at room temperature. DNA staining reagent (5
µl), as described above, was added to the cells immediately before
flow cytometry analysis of each sample.
DNA fragmentation assay [5
]
WEHI-231 cells (6x106 anti-Ig-treated or control
cells) were centrifuged at 200 g for 10 min. The cells were
lysed at room temperature for 20 min with 400 µl lysing solution
comprised of 0.2% Triton X-100, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5. To
pellet the intact DNA, the lysates were centrifuged at 13,000
g. The supernatant containing the fragmented DNA was
transferred to a separate tube. Additional lysing solution (400 µl)
was added to the pellet of intact DNA. To precipitate the DNA, 200 µl
of 25% trichloroacetic acid (TCA) was added, and the samples were
incubated at 4°C overnight. The TCA was removed, and 80 µl of 5%
TCA was added to the DNA to facilitate hydrolysis at 90°C for 10 min.
Diphenylamine (DPA) reagent (160 µl) comprised of 1.5% sulfuric
acid, 0.2% acetaldehyde, and 98% glacial acetic acid was added to
supernatant and pellet samples, and they were then incubated at room
temperature overnight for color development by an unknown mechanism.
The OD600 was determined using a microtiter plate reader.
The % of DNA fragmented =
ODsupernatant/(ODpellet+ODsupernatant).
Enzyme assay for caspase 3 activity
Apoptosis was induced in WEHI-231 cells (1x106
cells) by anti-Ig treatment and immediately followed by the addition of
z-DEVD-fmk (40 µM) or the peptide control z-FA-fmk (40 µM). After
18 h, the cleavage of the peptide substrate DEVD-7-amino-4-methyl
coumarin (DEVD-AMC) was measured in a fluorometric assay (Fluorometric
CaspACE Assay System, Promega Corp., Madison, WI) according to
manufacturer’s instructions. Briefly, cells were lysed for 1 h on
ice with hypotonic lysis buffer consisting of 25 mM HEPES (pH 7.5), 5
mM MgCl2, 5 mM EDTA, 5 mM dithiothreitol (DTT), 2 mM
phenylmethylsulfonylfluoride, 10 µg/ml pepstatin A (Sigma), and 10
µg/ml leupeptin (Sigma). Cell extract and substrate (2.5 mM) were
then combined in a standard reaction mixture of caspase assay buffer
[12.5 mM HEPES (pH 7.5), 31.25% w/v sucrose, and 0.3125% w/v
3-[(3-cholamido-propyl)-dimethamminio]-1propane-sulfonate] plus 100
mM DTT for 1 h at 30°C. The specific cleavage of DEVD-AMC was
monitored for AMC liberation using a fluorescent plate reader with 355
nm excitation and 460 nm emission wavelengths. Fluorescence units were
converted to pmol of AMC liberated per minute per 1 x
106 cells using a standard curve of AMC versus
fluorescence.
Data analysis
Data were analyzed by analysis of variance (ANOVA) and multiple
comparison tests (Sigma Stat, SPSS Inc., Chicago, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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-irradiated WEHI-231 cells, the order for acquisition of apoptotic
characteristics was annexin V-FITC labeling and MC540 labeling and then
PI-permeability, PI-labeled hypodiploid nuclei, and DNA fragmentation.
Hence, these transformed leukocytes do not follow the same order for
acquiring apoptotic features.
To determine whether the order for acquisition of these apoptotic
characteristics was the same among lymphoid cells, three lymphoid cell
types (spleen cells, WEHI-231 B lymphoma cells, and S49.1 T lymphoma
cells) were irradiated and examined for acquisition of apoptotic
characteristics. These lymphoid cell types differed in their order for
acquisition of apoptotic characteristics (Table 1)
. The -irradiated
spleen cells had the following order for accumulation of 50% maximum
of the apoptotic characteristics: DNA fragmentation and PI-labeled
hypodiploid nuclei, followed by PI-permeability and annexin V-FITC and
MC540 labeling. In comparison, the -irradiated S49.1 T cells first
acquired 50% maximum of MC540 labeling, annexin V-FITC labeling, and
PI-labeled hypodiploid nuclei, and then acquired PI-permeability and
DNA fragmentation. As stated previously, the -irradiated WEHI-231
cells had their own order for accumulation of these apoptotic features.
Thus, although the same method was used to induce apoptosis in these B
and T lymphocytes, each lymphoid cell type possessed its own sequential
pattern for obtaining the examined apoptotic characteristics.
We next determined whether the order was specific for a cell type by
examining different methods for induction of apoptosis in WEHI-231 B
lymphoma cells as well as murine splenocytes. When anti-Ig-treated
WEHI-231 cells were compared with -irradiated WEHI-231 cells, the
cells acquired apoptotic characteristics in a distinct order (Table 1)
.
The anti-Ig-treated WEHI-231 cells obtained 50% maximum of the
apoptotic features in the following order: DNA fragmentation, annexin
V-FITC and PI permeability, and then MC540 and PI-labeled hypodiploid
nuclei. In contrast, the order for -irradiated WEHI-231 cells was
annexin V-FITC and MC540 labeling and then PI permeability, PI-labeled
hypodiploid nuclei, and DNA fragmentation. Similar to the
anti-Ig-treated and irradiated WEHI-231 cells, spleen cells undergoing
spontaneous cell death during in vitro culture displayed apoptotic
characteristics in a different order from those that were irradiated.
Spontaneously dying spleen cells acquired apoptotic features in the
following order: DNA fragmentation, PI permeability and PI-labeled
hypodiploid nuclei, and lastly, MC540 and annexin V-FITC labeling. This
contrasts with irradiated splenocytes, in which DNA fragmentation and
PI-labeled hypodiploid nuclei preceded permeability to PI, annexin
V-FITC, and MC540 labeling. Thus, WEHI-231 cells and mouse spleen cells
differ in their order for 50% maximum accumulation of the apoptotic
characteristics when induced to undergo apoptosis by different methods.
The only consistencies in the order for accumulation of apoptotic characteristics by the cell types in Table 1 were detection of 50% maximum MC540 incorporation at the same time or after 50% maximum annexin V-FITC binding. In addition, DNA fragmentation occurred at the same time or before PI labeling of hypodiploid nuclei. Although the DNA fragmentation assay and labeling of hypodiploid nuclei detect DNA degradation, the time differed for these parameters in some cells. These differences in time for PI-labeled hypodiploid nuclei and DNA fragmentation are likely a result of the fact that these assays have different units of measurement. PI-labeled hypodiploid nuclei detects the percent of cells, whereas the DNA fragmentation assay detects the percent of DNA in a population of cells that is fragmented.
Differential caspase dependence for apoptotic cell-membrane changes
and DNA degradation
The independent orders for acquisition of the apoptotic features
could result from independent or divergent signal-transduction
pathways. Therefore, it was determined whether the apoptotic
characteristics were linked to a signal-transduction pathway that
induces DNA degradation, the caspase cascade. First, it was determined
whether the caspase 3 inhibitor z-DEVD-fmk was functional by examining
its ability to block caspase activity in anti-Ig-treated WEHI-231 cells
by a fluorometric enzyme assay for caspase 3 activity. In these
WEHI-231 cells, z-DEVD-fmk blocked anti-Ig-stimulated caspase activity
compared with the control peptide z-FA-fmk (Table 2
). However, in the anti-Ig-treated WEHI-231 cells, z-DEVD-fmk did
not block induction of DNA degradation (unpublished results), as
recently shown by Bras et al. [11
]. Nevertheless,
z-DEVD-fmk was functional because it blocked caspase activity. Next, we
examined the effect of the caspase inhibitors on nontransformed cells.
The effect of capsase inhibitors on acquisition of PI labeling of
hypodiploid nuclei and membrane changes in apoptotic spleen cells was
examined at the time for 50% maximum accumulation of the apoptotic
parameters. When -irradiated spleen cells and spontaneously dying
spleen cells were examined, z-VAD-fmk and z-DEVD-fmk blocked
acquisition of hypodiploid nuclei as detected by labeling with PI at
5.5 h after irradiation (Fig. 3
). z-FA-fmk, the control peptide, did not block acquisition of the
apoptotic parameters (Fig. 3) . It is interesting that treatment with
the caspase inhibitors did not block membrane permeability to PI (11.5
h) or cell labeling with annexin V-FITC (12.5 h) or MC540 (13 h; Fig. 3
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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The different orders for acquisition of the five apoptotic characteristics analyzed in this study may differ because the signals for the generation of apoptotic features may be independent of one another. If they are independent, then acquisition of the apoptotic features may occur at different rates in different cells. There is evidence that changes in phosphatidylserine in the outer membrane can occur independent of DNA fragmentation. In the absence of extracellular calcium, the accumulation of phosphatidylserine is blocked, and there is still DNA fragmentation in apoptotic Jurkat T cells and HL-60 monocytic cells [19 ]. Mechanisms for DNA fragmentation during apoptosis have been examined extensively and are most commonly dependent on caspases [20 , 21 ]. However, caspases are not always required for activation of apoptosis [22 ], and activation of caspases does not always lead to cell death [23 ]. For example, inhibition of caspase 3 activity in anti-Ig-induced WEHI-231 cells does not prevent the accumulation of DNA fragmentation [11 ], and caspases can be highly active during proliferation of human and murine T cells [23 ]. The mechanism for apoptotic membrane changes and whether caspases regulate the cell membrane changes characterized by annexin V-FITC labeling, MC540 labeling, or membrane permeability to PI are poorly understood. It has been shown that caspases are required for an increase in phosphatidylserine in the outer membrane of anti-Fas-treated Jurkat T cells and tumor necrosis factor (TNF)-induced U937 monocytic cells because inhibitors of caspases blocked the induction of annexin V-FITC labeling and DNA fragmentation [24 ]. In contrast, in human peripheral blood CD19+ B cells, annexin V-FITC binding and PI permeability were not inhibited in HLA-DR-mediated, caspase-independent death by the caspase inhibitors z-VAD-fmk or DEVD-fmk [13 , 14 ]. These data suggest that in some cells, membrane and nuclear changes are dependent on caspases, whereas in other cell types, membrane and nuclear changes are independent of caspases. Little data exist that determine whether several apoptotic membrane changes and nuclear changes are linked to caspases in the same cell type after activation of apoptosis. In our studies examining the order for acquisition of apoptotic characteristics, we demonstrate herein that caspases blocked DNA degradation in spleen cells under two conditions for induction of apoptosis, but importantly, these inhibitors did not block three membrane changes. This suggests that in these nontransformed cells, the mechanism for induction of membrane changes is independent of caspases or is divergent upstream of caspase activation of DNA fragmentation. Future studies will focus on mechanisms for induction of membrane changes.
The time for detection of DNA degradation by PI labeling of hypodiploid nuclei and DNA fragmentation differed for some cells. DNA fragmentation was acquired before PI labeling of hypodiploid nuclei for receptor-related apoptosis (lack of IL-3R stimulation for 32D cells, Ig cross-linking for WEHI-231 cells, and lack of receptor activation in spontaneously dying spleen cells). Although both assays detect DNA degradation, PI labeling detects the percent of cells with DNA degradation, whereas the DNA fragmentation assay detects the percent of DNA that was fragmented in the population of cells. A small amount of DNA degradation/cell may not be detected by PI labeling but may be detected in an assay for percent of DNA that was fragmented in a pool of cells if many cells had a small amount of DNA degradation. In contrast to receptor-related apoptosis, the DNA fragmentation and PI labeling of hypodiploid nuclei occurred simultaneously for the irradiated spleen cells and WEHI-231 cells, whereas for irradiated S49 cells, 50% DNA fragmentation occurred after PI labeling of hypodiploid nuclei. For irradiated cells, irradiation causes direct, radiation-induced changes and caspase-induced changes in DNA [25 , 26 ], increasing the changes in the DNA. The differences in direct-radiation damage to the DNA may influence the sensitivity for detection by PI labeling of hypodiploid nuclei versus DNA fragmentation in irradiated apoptotic cells.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGEMENTS |
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Received July 17, 1999; revised April 23, 2001; accepted April 24, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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