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Bcl-2 Does Not Protect Burkitt’s Lymphoma Cells
From Oxidant-Induced Cell Death
By Yang-ja Lee and Emily Shacter
Bcl-2 is an oncogene that confers deregulated growth potential to B lymphocytes through its ability to inhibit apoptotic
cell death. A specific molecular activity for the Bcl-2 protein
has not been identified, but several lines of evidence have
supported a role in protection of cells from oxidative stress.
We investigated whether there is a correlation between expression of high levels of Bcl-2 and susceptibility of human
Burkitt’s lymphoma cell lines to H2O2 -induced killing. The
amount of H2O2 required to kill 50% of cells in 24 hours varied
widely in the seven different lymphoma cell lines that were
tested, ranging from 35 to 500 mmol/L H2O2 . However, expression of high levels of endogenous Bcl-2 did not protect
the cells from H2O2 -induced killing, even though it was effective in protecting the cells from apoptosis induced by agents
such as A23187. Thus, Bcl-2 was functional in preventing
apoptosis but did not act in an antioxidant capacity. The
results were confirmed using a Burkitt’s lymphoma cell line
overexpressing transfected bcl-2. The results may be explained by the observation that H2O2 was inefficient at inducing apoptosis in these mature B-cell lines. Nonapoptotic
death induced by H2O2 was not prevented by Bcl-2.
This is a US government work. There are no restrictions on
its use.
T
from killing induced by depletion of endogenous glutathione.
Other studies have supported these initial findings (reviewed
in Korsmeyer et al25). However, the theory that Bcl-2 acts
through an antioxidant mechanism has recently been challenged by studies showing that oxygen depletion has no
effect on the induction of apoptosis and that Bcl-2 protects
against apoptosis without inhibiting the production or activity of reactive oxygen compounds.26,27
Almost all of the investigations showing that Bcl-2 protects cells from oxidant-induced killing were performed with
cell lines transfected with an expression vector carrying an
exogenous bcl-2 gene.16,17,28-31 In this report, we address the
question of whether Burkitt’s lymphoma (BL) cell lines that
express different levels of endogenous Bcl-2 show a differential susceptibility to oxidant-induced killing. BL is a highgrade B-cell lymphoma that is distinguished by the presence
of chromosomal translocations resulting in activation of the
c-myc oncogene.32,33 This tumor is of germinal center origin,32 similar to follicular lymphoma, but is otherwise phenotypically and genotypically distinct.34,35 For example, unlike
follicular lymphoma, elevated Bcl-2 expression is not generally found in primary BL tumors, but it may appear in some
tumors upon relapse.36 Thus, activation of bcl-2 may lead to
tumor progression in BL, but it is not a feature of primary
BL development. The level of Bcl-2 expression varies among
established BL cell lines in tissue culture depending on the
stage at which it was introduced into culture and the EpsteinBarr virus (EBV) infection status37-40; infection of BL cell
lines with EBV can lead to induction of the bcl-2 oncogene
and to failure to undergo apoptosis.37,40 In contrast to bcl-2,
mutations in the p53 gene are a common feature of primary
BL tumors and cell lines.41,42
Using BL cell lines that were matched for their p53 and
EBV status, we found no correlation between the level of
expression of endogenous Bcl-2 and resistance to H2O2 induced killing. In fact, H2O2 was the only agent against
which a Bcl-2hi BL cell line was not resistant. The results
with endogenous Bcl-2 were confirmed using cells overexpressing transfected Bcl-2. The data argue against the generalization that Bcl-2 protects B cells from oxidant-induced
cell killing.
HE bcl-2 ONCOGENE is thought to confer oncogenic
potential to cells by inhibiting programmed cell death,
or apoptosis.1 It was first discovered by virtue of its translocation (t[14;18]) and elevated expression in follicular B-cell
lymphomas.2 Subsequent studies have shown that it is also
expressed during normal B- and T-cell development.3-5
Transfection and overexpression of the bcl-2 gene protects
a variety of different cell types from induction of apoptotic
cell death.6-9 Treatments that induce apoptosis and from
which Bcl-2 protects include calcium ionophores, serum and
growth factor depletion, glucocorticoids, and g-irradiation.7,8,10,11 The mechanism by which the Bcl-2 protein extends cell survival in the face of such a wide variety of
treatments is unclear. No biochemical activity has thus far
been ascribed to the oncoprotein and it has little or no significant homology with other known cellular proteins.12,13
Early reports indicated that Bcl-2 is localized to the mitochondrial inner membrane,6 suggesting that Bcl-2 might act
by modulating mitochondrial function. However, Bcl-2 is
also found in the endoplasmic reticulum and nuclear envelope.14,15
One theory of how Bcl-2 functions suggests that it acts
by protecting cells from oxidative stress. It may do this by
reducing cellular generation of reactive oxygen compounds
or by blocking the activity of these compounds after they
are formed.16,17 Oxidants such as superoxide and hydrogen
peroxide (H2O2 ) are generated by a variety of conditions,18
including activation of phagocytes during inflammation19,20
and from reperfusion of ischemic tissue.21,22 Intracellular oxidants are generated by electron leakage from the electron
transport chain23 and by the action of mixed function oxidases within the cell.22,24 Hockenbery et al17 found that overexpression of the Bcl-2 protein in a murine pro-B–cell line
protected those cells from killing induced by treatment with
high levels of reagent H2O2 . Kane et al16 found that overexpression of Bcl-2 in a neural cell line protected the cells
From the Laboratory of Immunology, Food and Drug Administration, Center for Biologics Evaluation and Research, Bethesda, MD,
Submitted June 26, 1996; accepted January 30, 1997.
Address reprint requests to Emily Shacter, PhD, FDA/CBER,
HFM-538, Bldg 29A, Room 2A-11, Bethesda, MD 20892-4555.
This is a US government work. There are no restrictions on its use.
0006-4971/97/8912-0026$0.00/0
MATERIALS AND METHODS
Cells. The Burkitt’s lymphoma cell lines used in this study were
provided by Dr Kishor Bhatia (National Cancer Institute, National
Blood, Vol 89, No 12 (June 15), 1997: pp 4480-4492
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BCL-2 AND OXIDANT-INDUCED CELL DEATH
4481
Institutes of Health, Bethesda, MD). Lymphoblastoid cell lines were
derived in the laboratory of Dr Giovanna Tosato (Center for Biologics Evaluation and Research, Food and Drug Administration,
Bethesda, MD). The mutation status of the p53 gene of these cell
lines is taken from previous reports.43-45 Cells were grown in RPMI
1640 containing 10% heat-inactivated fetal calf serum (FCS), 2
mmol/L L-glutamine, and 50 mmol/L b-mercaptoethanol at 377C in
5% CO2 in air.
Cell treatments. Exponentially growing cells (7.5 to 12 1 105
cells/mL) were harvested and resuspended in fresh growth media
(10% FCS media except for the serum depletion experiments) to
achieve a culture density 5 1 105 cells/mL. H2O2 was added to the
cell suspensions at the beginning of the experiments and the cells
were incubated for 6 to 48 hours as indicated in the text. The calcium
ionophore A23187 (Calbiochem, San Diego, CA) was dissolved in
dimethyl sulfoxide to make a 10 mmol/L stock solution that was
stored in the dark at 47C. It was added to cell suspensions (5 1 105
cell/mL) to achieve final concentrations of 0.5 to 2.0 mmol/L. gIrradiation was performed using a 137Cs irradiator (Nordion 137Cs
Gammacell Elite; Nordion International, Kanada, Ontario, Canada)
at a dose rate of 22.2 rads/s. Cells in fresh growth media (5 1 105
cells/mL) were irradiated at room temperature for 27 seconds (600
rads) or 54 seconds (1,200 rads) and incubated for 12 to 72 hours
before harvesting. For serum depletion experiments, cells were harvested, washed with phosphate-buffered saline (PBS), resuspended
in RPMI 1640 containing 0.2% or 1% heat-inactivated FCS, and
incubated for 24 to 72 hours as indicated in the text. Etoposide was
prepared as a 10 mg/mL stock solution in PBS. All incubations were
performed at 377C in 5% CO2 in air.
Viability assay. Cell viability was determined by trypan blue
exclusion. Cells that were treated with various agents and incubated
for the times indicated were suspended in an equal volume of 0.4%
trypan blue. Dead (blue) and live (clear) cells were counted using
a hemacytometer. The percentage of viability is defined as the number of live cells divided by the number of live and dead cells.
H2O2 assay. Cells were suspended at a density of 5 1 105 cells/
mL in PBS containing 1 mg/mL glucose and 0.2 mg/mL phenol
red.46 At time 0, H2O2 was added to each cell suspension and the
mixtures were incubated at 377C for 0 to 3 hours. Aliquots were
taken at timed intervals and the cells were removed immediately by
centrifugation. Horseradish peroxidase (50 mg/mL final concentration) was added to the cell supernatants and the samples were incubated at room temperature for approximately 5 minutes. NaOH was
then added to a final concentration of 20 mmol/L and the resulting
color was allowed to stabilize for at least 5 minutes. The absorbance
of reduced phenol red (A610 ) was measured on a Hewlett Packard
8452A diode array spectrophotometer (Hewlett Packard Inc, Palo
Alto, CA). The concentration of H2O2 was calculated based on standard curves using reagent H2O2 .
Western blotting analysis. Cells were harvested by centrifugation; washed once with cold PBS; resuspended in lysis buffer containing 62.5 mmol/L Tris (pH 6.8), 2% sodium dodecyl sulfide
(SDS), 10% glycerol, and 1 mmol/L diethylenetriaminepentaacetic
acid; and heated for 12 minutes at 1007C. The total cell lysates
(Ç50,000 cells per lane; 5 to 20 mg protein) were subjected to
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (4% to 12%
gradient SDS-polyacrylamide gels; Novex, San Diego, CA).47 The
protein bands were then transferred electrophoretically48 to Immobilon membranes (Millipore, Bedford, MA). After electrotransfer, the
membrane was blocked for 1 hour in 3% bovine serum albumin at
room temperature (for monoclonal antibody treatments) or overnight
at 47C in 5% skim milk (for polyclonal antibody treatments). Primary
antibodies were incubated overnight at 47C (for monoclonals) or for
1 hour at room temperature (for polyclonals). Anti–Bcl-2 (monoclonal mouse antibody to a synthetic peptide sequence comprising
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amino acids 41-54 of human Bcl-2; DAKO, Carpinteria, CA) was
used at 1:10,000, anti-Bax (rabbit polyclonal antibody against amino
acids 11-30 of human Bax; Santa Cruz Biotechnology, Santa Cruz,
CA) was used at 1:4,000, and anti–Bcl-x (rabbit polyclonal antibody
against amino acids 2-19 of human Bcl-x; Santa Cruz Biotechnology) was used at 1:400. Immunoreactivity was visualized using peroxidase detection systems and chemiluminescence (Renaissance kit;
Dupont NEN, Boston, MA). X-ray films were scanned (Microtek
Scanmaker, Microte K Inc, Redondo Beach, CA) and analyzed using
the Macintosh desitometry program IMAGE (National Institutes of
Health, Bethesda, MD).
Assessment of apoptosis by agarose gel electrophoresis. The
fragmentation of total cellular DNA was analyzed by the procedure
of Smith et al.49 The extracts from 106 cells were subjected to electrophoresis (30 V, 12 to 15 mA, 16 hours) on 2% (wt/vol) agarose gels
containing 0.1 mg/mL ethidium bromide. DNA was visualized by
fluorescence under UV light. Molecular weight standards (HaeIIIdigested f X174; New England Biolabs, Beverly, MA) were run in
adjacent lanes.
Assessment of apoptosis by TUNEL (terminal deoxynucleotidyl
transferase-mediated dUTP-X nick end labeling) and flow cytometry.
The TUNEL assay used in this study was adapted from Gold et al,50
with little modification. Cell samples (2 1 106/mL) were fixed by
mixing with PBS containing 2.5% formaldehyde and incubating for
30 minutes at room temperature. The fixed cells were washed with
fluorescence-activated cell sorting (FACS) buffer (PBS containing
0.5% bovine serum albumin and 0.01% sodium azide) and permeabilized with 0.1% Triton X-100/0.1% sodium citrate for 2 minutes
on ice. After washing with FACS buffer, the permeabilized cells
were resuspended in apoptosis cell labeling mixture consisting of
11 terminal deoxynucleotidyl transferase (TdT) buffer, 25 U of
TdT enzyme, 250 mmol/L CoCl2 , 0.6 mmol/L fluorescein-dUTP, and
deoxynucleotide mixture (dATP, dGTP, and dCTP at 2 mmol/L and
dTTP at 1.4 mmol/L) and then incubated at 377C for 1 hour. All
reagents were purchased from Boehringer Mannhein (Indianapolis,
IN). Reactions were stopped by adding 1 mL FACS buffer. The
cells were washed once more, resuspended in 1 mL FACS buffer,
and analyzed (10,000 cells per sample) on a FACScan (Becton Dickinson, San Jose, CA) using CELLQUEST flow cytometric analysis
software.51 The fluorescein-dUTP incorporated into the cells was
detected using a 530/30-nm bandpass filter (FL1 channel). The percentage of apoptosis was calculated as the number of cells in the
high fluorescence intensity population (reflecting fluorescein dUTP
incorporation) divided by the total number of cells analyzed. In the
absence of TdT, there was no shift in fluorescent staining upon
treatment of the cells with any agent. Forward and side light scatter
(FSC and SSC) were also measured for each sample and analyzed
according to Dive et al.52
Morphologic assessment of apoptosis using Hoechst/propidium
iodide nuclear staining and fluorescence microscopy. Cells (Ç5 1
106 cells/mL) were incubated for 15 minutes at 377C with Hoechst
33342 dye (5 mg/mL in PBS), centrifuged, washed once in PBS,
and then resuspended at an approximate density of 2.5 1 107 cells/
mL. Propidium iodide (50 mg/mL from a 1 mg/mL stock in PBS)
was added just before microscopy. Cells were visualized using a
Nikon Optiphot microscope equipped with a fluorescent light source
and a UV-2A filter cube (Nikon, Japan); (excitation wavelength, 330
to 380 nm; barrier filter, 420 nm). Cell morphology was scored as
follows. (1) Viable cells had blue-stained normal, smooth nuclei. (2)
Viable, apoptotic cells had blue-stained nuclei with multiple bright
specks of condensed chromatin. (3) Nonviable, necrotic cells had
red-stained, smooth nuclei that were roughly the same size as normal
(control) nuclei. (4) Nonviable, apoptotic cells had red-stained nuclei
with either multiple bright specks of fragmented chromatin or one
or more spheres of condensed chromatin (significantly more compact
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4482
LEE AND SHACTER
than normal nuclei). Samples were randomized and examined after
blinding. At least 200 cells for each treatment were counted. Experiments were repeated at least three different times.
Transfection of BL cells with bcl-2. A human bcl-2 expression
plasmid was prepared by subcloning a 910-bp EcoRI complementary
DNA from the plasmid pB4 (American Type Culture Collection,
Rockville, MD) into the pcDNA3.1 expression vector (Invitrogen,
San Diego, CA). The resulting recombinant plasmid pcDNA3.1–
bcl-2 and the parental vector were linearized with Sca I and transfected into JLP 119 cells (20 mg DNA/6 1 106 cells) by electroporation (200 V, 1,600 mF). Stably transfected cells were selected with
1 mg/mL G418 (GIBCO BRL, Gaithersburg, MD), expanded, and
screened for expression of the Bcl-2 protein by Western blotting.
RESULTS
Susceptibility of BL cell lines to killing by H2O2 . The
goal of this research was to identify tumor cells with differing degrees of resistance to oxidative stress so that the
cellular pathways responsible for conferring resistance could
be examined. To this end, nine different cell lines were
seeded at a density of 5 1 105 cells/mL and exposed to a
bolus addition of different concentrations of H2O2 (50 to 500
mmol/L). The panel of cell lines examined included Burkitt’s
lymphoma cell lines that are infected (SHO) or not infected
(BJAB, ST-486, JLP 119, EW-36, CA-46, and JD-38) with
EBV and cells that contain only wild-type (wt) p53 (JLP
119, EW-36, and SHO) or either one (BJAB and ST-486)
or two (CA-46 and JD-38) mutated (mut) alleles of p53.
EBV-immortalized lymphoblastoid cell lines (LCLs; Tory
and VDSO) were used as nontumor controls. Viabilities were
assessed by trypan blue staining after 24 hours of incubation.
As shown in Fig 1, a range of different susceptibilities to
H2O2 -induced killing was observed. LD50 values (concentration of H2O2 required to kill 50% of the cells) ranged from
approximately 35 to 500 mmol/L H2O2 . SHO (EBV-positive,
p53 wt/wt) and BJAB (EBV-negative, p53 wt/mut) were the
most resistant to H2O2 -induced killing (LD50 , Ç500 mmol/
L), followed by the LCLs (Tory and VDSO; LD50s, Ç350
mmol/L). CA-46 (EBV-negative, p53 mut/mut) showed an
intermediate level of resistance to H2O2 -induced killing
(LD50 , Ç100 mmol/L). The remaining lymphoma cell lines
were relatively sensitive to H2O2 treatment (LD50s, Ç35 to
75 mmol/L). In general, there was no correlation between
p53 mutation status and susceptibility to H2O2 toxicity.
To determine whether different levels of expression of
Bcl-2 might be responsible for the observed differences in
the H2O2 -sensitivity of Burkitt’s lymphoma cells, Bcl-2 protein was assayed in cell extracts by Western blot immunoassay using a monoclonal antibody to human Bcl-2 (Fig 2).
The EBV-immortalized LCLs (Tory and VDSO) were used
as positive controls because they are known to express elevated levels of Bcl-2.38,39 As shown in Fig 2, the Bcl-2 level
varied among the BL cell lines: EW-36 expressed an unusually high level of Bcl-2 protein, whereas JD-38 and SHO
expressed moderately elevated levels that were similar to
those found in the LCLs. All of the other cell lines showed
little or no Bcl-2 expression. There was no clear correlation
between the level of expression of Bcl-2 and either the EBV
infection status (eg, compare SHO with EW-36 and JD-38)
or the p53 mutation status.
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Fig 1. Relative susceptibility and resistance of BL cell lines and
LCLs to killing by H2O2 . Cells were seeded at a density of 5 Ì 105/
mL in growth medium. H2O2 was added as a bolus at the indicated
concentrations. After 24 hours of incubation at 377C, the number of
live and dead cells was determined using trypan blue. The percentage
of viability is defined as the number of live cells divided by the number of live plus dead cells. Also indicated are the p53 mutation (m
indicates a mutated allele) and EBV infection status (" indicates infected) for each cell line. Cell line (EBV, p53): ( ) SHO (", w/w); (m)
BJAB (Ï, w/m); (Ì) Tory (", w/w); (j) VDSO (", w/w); (n) CA-46
(Ï, m/m); (s) EW-36 (Ï, w/w); ( ) JD-38 (Ï, m/m); (●) JLP 119 (Ï,
w/w); (h) ST-486 (Ï, w/m).
As shown in Fig 3, the relative susceptibilities of the
different cell lines to H2O2 -induced killing (as defined by
LD50 values) did not correlate with the respective levels of
Bcl-2 expression. EW-36, which expresses Bcl-2 at unusually high levels, and JD-38, which expresses a moderately
elevated level of Bcl-2, were among the most sensitive cell
lines (LD50s, Ç75 mmol/L), whereas BJAB and CA-46,
which do not show detectible Bcl-2, were relatively resistant
to H2O2 treatment (LD50 , Ç500 and 150 mmol/L, respectively). Treatment with H2O2 did not alter the steady-state
Bcl-2 protein levels in the cells (data not shown). There was
no apparent correlation between the presence of a mutated
p53 allele and resistance to H2O2 -induced killing.
The different BL cell lines were tested for their ability
to degrade H2O2 to establish whether differences in their
antioxidant defense systems (eg, peroxidase activities) might
be affecting their susceptibility to H2O2 toxicity. The ability
of the cells to deplete H2O2 from the surrounding medium
was measured by the phenol red assay,46 as described in
Materials and Methods. As shown in Fig 4, there was very
little difference in the rate of removal of H2O2 by the BL
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BCL-2 AND OXIDANT-INDUCED CELL DEATH
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Fig 2. Western blot analysis of Bcl-2 levels in BL
cell lines and EBV-transformed LCLs. Total cell extracts from equal numbers of cells (Ò50,000) were
loaded in each lane. The figure shows immunoblot
results obtained using a monoclonal antibody to human Bcl-2 (see the Materials and Methods). Also indicated are the p53 mutation (m indicates presence of
1 or 2 mutated alleles) and EBV infection status ("
indicates infected) for each cell line.
cell lines. The cell line with the highest Bcl-2 level (EW36) was somewhat (Ç2-fold) slower in removing H2O2 than
the other cell lines, but there was no consistent correlation
between the level of expression of Bcl-2 and the rate of
removal of H2O2 . JD-38 has an intermediate level of Bcl-2
expression, but removed H2O2 at the same rate as cell lines
with little or no Bcl-2 protein expression. All six lines removed at least 80% of the H2O2 within 2 hours. The viability
Fig 3. Correlation between susceptibility to H2O2 toxicity and the
level of expression of Bcl-2 protein. The data are compiled from Figs
1 and 2. LD50 is defined as the concentration of H2O2 that kills 50%
of the cells after 24 hours of incubation. The level of Bcl-2 protein
expression was unaffected by H2O2 treatment (not shown).
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assays shown in Fig 1 were conducted 18 to 24 hours after
the addition of H2O2 .
Correlation between Bcl-2 expression and protection from
apoptosis induced by calcium ionophore, serum depletion,
and g-irradiation. One possible explanation for the lack
of protection of BL cells from H2O2 -induced killing could
be that the Bcl-2 expressed in these cells is not functional.
Because activity of Bcl-2 is defined by its ability to inhibit
apoptosis, we tested this possibility by subjecting cells to
treatments that have been shown to induce apoptosis in different cells, including BL cell lines.10,11,53 Among these are
the calcium ionophore A23187, serum depletion, and g-irradiation.
To eliminate possible confounding effects caused by EBV
infection and p53 mutations, two cell lines that express
widely different levels of Bcl-2 but are both EBV-negative
and p53 wild-type were compared. These are EW-36 (Bcl2hi) and JLP 119 (Bcl-2lo). The LD50 values for H2O2 -induced
killing in these two cell lines differed by less than twofold,
whereas the Bcl-2 levels differed by more than 20-fold.
There was no difference in the levels of expression of other
Bcl-2 family members, Bax54 and Bcl-x55 (Fig 5).
Apoptosis was assayed by induction of DNA ladders using
agarose gel electrophoresis49 and a TUNEL method coupled
with FACS analysis.50 Cell viability was determined by trypan blue exclusion. This latter assay is not ideal, because
cells at the early stages of apoptosis do not manifest membrane leakage and may still have the ability to exclude vital
dye.56,57 Thus, trypan blue exclusion underestimates cell
death when it occurs by an apoptotic mechanism and the
values obtained can only be viewed as estimates. As shown
in Fig 6A, A23187 (1 and 2 mmol/L), serum depletion (reduction from 10% to 1% or 0.2%), and g-irradiation (600
and 1,200 rads) killed both cell lines in a concentration/
dose-dependent manner. However, JLP 119 was significantly
more sensitive to all treatments than EW-36. The biggest
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Fig 4. Depletion of H2O2 by BL cell lines. The ability of BL cell lines
to degrade H2O2 (peroxidase-like activity) was assessed as described
in the Materials and Methods. A concentration of 100 mmol/L H2O2
was added to each cell suspension (5 Ì 105 cells/mL). After various
times of incubation at 377C, aliquots were removed and the medium
was assayed for the presence of residual H2O2 . The X’s show the
stability of H2O2 incubated in medium in the absence of cells. (n) ST486; (s) CA-46; (●) BJAB; (L) JD-38; (j) EW-36; (h) JLP 119; (Ì) H2O2
only.
poration of FITC-labeled dUTP and the cell population remained in the high FSC/low SSC region of the scattergram.
The percentage of apoptotic cells induced by treatment of
both cell lines with different concentrations of A23187 was
quantified from the TUNEL assay and the results are shown
in Fig 7B. Roughly 70% of JLP 119 cells underwent
apoptosis in response to 2 mmol/L A23187 compared with
only 5% of EW-36 cells.
The ability of the TUNEL apoptosis assay to reflect differences in cellular Bcl-2 levels was confirmed using two additional cell lines: JD-38, which expresses an intermediate
level of Bcl-2, and ST-486, in which little or no Bcl-2 is
detected. As shown in Fig 8, JD-38 was much more resistant
to A23187-induced apoptosis than ST-486 but was more
sensitive than EW-36, which expresses the highest bcl-2
levels.
Inefficient induction of apoptosis in BL cell lines by H2O2 .
The comparisons mentioned above show that the Bcl-2hi
expressor cell line EW-36 is more resistant to being killed
by A23187, serum depletion, and g-irradiation than JLP 119.
However, EW-36 was highly sensitive to toxicity from H2O2
(see Fig 1). Because Bcl-2 is believed to protect cells primarily from apoptosis, we investigated whether the reason for
the discrepancy might derive from the mechanisms of cell
killing, ie, that H2O2 kills by inducing necrosis and not
apoptosis. The following experiments show that this is partly
correct. When total DNA was extracted from cells treated
with H2O2 and checked for the presence of ladders by agarose
gel electrophoresis, we found that H2O2 induced little if any
of the DNA fragmentation that is characteristic of apoptosis
in either the low or high Bcl-2 expressor. Because it is
difference in viability between the two cell lines was seen
with A23187, which is also the agent that induced the most
profound degree of apoptosis. Agarose gel electrophoresis
of total DNA (Fig 6B) shows that cell killing by these agents
is mainly apoptotic in JLP 119. In contrast, EW-36 was
resistant to induction of apoptosis.
The results obtained by agarose gel electrophoresis were
confirmed by FACS analysis of cells treated with A21387.
Two types of measurements were performed; a TUNEL
assay was used to quantify apoptotic cells,50 whereas forward
and side light scatter were used to estimate the number of
total live and dead cells.52 Cells incorporating high levels
of fluorescein isothiocyanate (FITC)-labeled dUTP in the
presence of terminal deoxynucleotidyl transferase (TUNEL
assay) represent apoptotic cells containing fragmented DNA.
Cells exhibiting high forward scatter (FSC) and low side
scatter (SSC) are considered to be live cells, whereas those
exhibiting low FSC and high SSC are considered to be
dead.52 When these analyses were performed on JLP 119
and EW-36 treated with A23187, the results shown in Fig 7A
were obtained. A23187 induced a significant, concentrationdependent incorporation of FITC-conjugated dUTP into the
DNA of JLP 119 cells and caused a clear shift in the cell
population from one of high FSC/low SSC (live cells) to
one of low FSC/high SSC (dead cells). In contrast, identical
treatment of EW-36 had no such effect; there was little incor-
Fig 5. Western blot analysis of Bax and Bcl-x levels in JLP 119
and EW-36. Total cell extracts from 50,000 cells were loaded in each
lane. The figures show typical immunoblot results obtained using a
polyclonal antibody to human Bax or human Bcl-x (see the Materials
and Methods). The cell line BJAB was found to express high levels
of bcl-xL and is shown as a positive control.
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BCL-2 AND OXIDANT-INDUCED CELL DEATH
4485
Fig 6. Effect of A23187, serum depletion, and girradiation on cell viability or induction of DNA fragmentation in EW-36 and JLP 119. Cells (5 Ì 105/mL)
were treated with A23187 (1 or 2 mmol/L), serum
depletion (reduced from 10% to either 1% or 0.2%),
or g-irradiation (600 or 1,200 rads). After 48 hours of
incubation, cell viability was determined by trypan
blue exclusion (A). Fragmentation of total cellular
DNA (1 Ì 106 cells) was assessed by agarose gel
electrophoresis (B). The viability results (j, EW-36;
, JLP 119) represent the mean Ô SEM of quadruplicate (A23187) or duplicate (serum depletion and girradiation) experiments. The DNA fragmentation
data shown are from a representative study that was
repeated two to four times.
thought that apoptotic death occurs at low levels of cellular
damage, whereas necrotic death is induced under more extreme conditions,58 we tested concentrations of H2O2 (25 to
100 mmol/L) that show a range of degrees of cell killing
(see Fig 1): 25 mmol/L H2O2 , which kills less than 10% of
the cells; 50 mmol/L H2O2 , which induces moderate killing
(20% to 60%); and 75 and 100 mmol/L H2O2 , which kill the
majority (Ç80%) of the cells. As shown in Fig 9, there was
no detectible DNA fragmentation in EW-36 at any concentration of H2O2 tested. Fragmentation of a relatively small
portion of the DNA of JLP 119 cells treated with 50 or 75
mmol/L H2O2 was seen, but most of the DNA still remained
in the high molecular weight fraction. At 100 mmol/L H2O2 ,
more than 80% of cells from both lines were dead, but the
DNA still remained in the high molecular weight fraction,
suggesting that most of the cell killing by H2O2 was nonapoptotic. This poor induction of DNA ladders by H2O2 was
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seen additionally with four other lymphoma cell lines (CA46, BJAB, JD-38, and ST-486; data not shown). This phenomenon is in stark contrast to the effects of A23187 and
serum depletion, which both led to complete loss of high
molecular weight DNA in JLP 119 at the highest concentrations tested (see Fig 6B). The low level of induction of
nucleosomal DNA fragmentation by H2O2 was confirmed by
the TUNEL assay coupled with FACS analysis (Fig 10).
Increasing the concentration of H2O2 induced significant
shifts in the cell populations of both JLP 119 and EW-36
from high FSC/low SSC to low FSC/ high SSC populations,
consistent with the results obtained by trypan blue counting.
However, only a small number of cells (°20%) from either
cell line showed significantly increased incorporation of
FITC-dUTP, indicating that most of the cells did not exhibit
the DNA fragmentation characteristic of death by apoptosis.
The Bcl-2hi cell line showed the same amount of TUNEL
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Fig 7. Flow cytometric analysis of JLP 119 and EW-36 treated
with A23187. (A) Flow cytometer
plots. Cells (5 Ì 105/mL) were incubated in growth medium containing the indicated concentrations of A23187 (0, 0.5, 1.0, or 2.0
mmol/L) for 48 hours. FACS analysis of fixed cells was performed
as described in the Materials and
Methods. Rows 1 and 3, cytograms showing forward and side
light scatter (FSC and SSC, respectively) for 10,000 cellular
events. Rows 2 and 4, histograms of FITC-labeled DNA for
the cells shown in the cytograms. Rows 1 and 2, results for
JPL-119. Rows 3 and 4, results
for EW-36. The population with
high FITC intensity in each histogram (right-hand peak) represents apoptotic cells. The data
shown are from a representative
study that was repeated three
times. (B) Proportion of JLP 119
(m) and EW-36 (n) cells that are
undergoing apoptosis at each
concentration of A23187. The
percentage of apoptosis equals
the high FITC-labeled population
divided by the total cell population. Each point is derived from
10,000 cellular events and represents the mean Ô SEM of triplicate cultures.
activity as the Bcl-2lo cell line, indicating that Bcl-2 had no
effect on this activity.
Morphologic assessment of apoptosis. Both of the
assays described above measure apoptosis based on induction of nucleosomal DNA fragmentation. Because A23187
produced a positive response using both methods, we conclude that Burkitt’s lymphoma cells can be induced to undergo nucleosomal DNA fragmentation but that H2O2 is inefficient at accomplishing this. However, these results do not
rule out the possibility that H2O2 induces other features of
apoptosis that may be independent of endonuclease activity.59-61 This possibility was examined using nuclear staining
and fluorescence microscopy to look for the ability of H2O2
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to induce the morphologic changes that define apoptotic
death.62 Hoechst 33342 is a blue fluorescent dye that is permeable to both live and dead cells, whereas propidium iodide
(red fluorescence) only enters cells in which the membrane
integrity has been damaged (defined as dead). Based on this
assay, we found that A23187 (2 mmol/L) induced substantial
nuclear fragmentation in 87% of JLP 119 but only 3% of
EW-36 cells. Very little necrotic cell death (Ç5%) was observed in either cell line after treatment with A23187. In
contrast, H2O2 induced morphologic changes characteristic
of both apoptosis and necrosis in JLP 119, whereas the predominant form of death in EW-36 was necrotic (Fig 11). It
should be pointed out that, in general, the morphologic
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BCL-2 AND OXIDANT-INDUCED CELL DEATH
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cells compared with 12% to 14% of control cells). The overall viability of the cells was determined by trypan blue staining and it was found that transfected Bcl-2 was not protective
(average of 61% and 66% viability in control and bcl-2–
transfected cells, respectively). This result was confirmed
using Hoechst/propidium iodide staining to quantify cell viability and mechanism of cell death. In this experiment, the
topoisomerase II inhibitor etoposide was used as a positive
control for induction of apoptosis because it acts more
quickly than A23187 and can thus be assayed at the same
time as H2O2 (ie, after 24 hours). As shown in Fig 14, treatment with 100 mmol/L H2O2 killed 60% to 75% of cells
regardless of whether they expressed transfected Bcl-2. The
mechanism of cell killing was both necrotic and apoptotic.
In contrast, treatment with etoposide (5 mg/mL) killed Ç80%
of control cells but only Ç5% of bcl-2–transfected cells.
Almost all of the cell death induced by this agent was apoptotic.
DISCUSSION
Fig 8. Correlation between TUNEL activity and the level of Bcl-2
expressed in BL cells. The flow cytometric TUNEL assay was performed and analyzed on the cell lines indicated in the figure as described in the legend to Fig 7. The relative levels of Bcl-2 expression
(see Fig 2) were EW-36 ÛÛ JD-38 ÛÛST-486 Ò JLP-119. (●) ST-486;
(s) JLP 119; (m) JD-38; (n) EW-36.
There is a substantial body of literature that suggests that
Bcl-2 protects cells from death by protecting them from
oxidative stress.16,17,28-31,63 We intended to build on this phenomenon so that we could investigate specific oxidative processes from which Bcl-2 protects but found that Bcl-2 had
no effect on H2O2 -induced killing in our experimental system. Two BL cell lines, EW-36 and JLP 119, that contained
significantly different levels of Bcl-2 but were wild-type for
p53 protein, were EBV-negative, and showed similar levels
of expression of Bax and Bcl-x were compared. The cells
changes induced by H2O2 were atypical. Nuclei classified
as apoptotic were cleaved into 2 or 3 spheres that were
significantly larger than the small fragments induced by
A23187. Intact nuclei classified as necrotic were not necessarily larger than control (untreated) nuclei but were not
adequately condensed to be defined as apoptotic .
Effect of Bcl-2 overexpression induced by transfection.
The results of the present study show that endogenous Bcl2 is ineffective at protecting BL cells from H2O2 toxicity.
However, interpretation of the results is complicated by the
fact that the cell lines examined are not isogenic. Despite
our efforts to use matched cell lines, the possibility exists
that unknown pathways could be modulating the responses
of these cells to the various agents tested. To test directly
whether overexpression of Bcl-2 by itself can protect Burkitt’s lymphoma cells from H2O2 -induced cytotoxicity, the
human bcl-2 gene was transfected into JLP 119 cells. In Fig
12, the levels of expression of transfected Bcl-2 are shown
for two positive clones (H6 and H10) and two control clones
(G4 and G21). The results of TUNEL analyses performed
with these cell lines are shown in Fig 13. Control cells carrying vector only were highly susceptible to A23187-induced
DNA fragmentation, similar to the parental cell line, whereas
both bcl-2–transfected cell lines exhibited marked resistance. However, H2O2 (50 mmol/L) induced little TUNEL
activity in any of the four cell lines. Transfected Bcl-2 prevented some of this activity (positive TUNEL in Ç6% of
Fig 9. Effect of H2O2 concentration on induction of DNA fragmentation in EW-36 and JLP 119. Cells (5 Ì 105/mL) were incubated for
24 hours in growth medium containing the indicated concentrations
of H2O2 (0, 25, 50, 75, or 100 mmol/L). Fragmentation of total cellular
DNA (extract of 1 Ì 106 cells/lane) was assessed by agarose gel
electrophoresis. The data shown are from a representative study that
was repeated three times.
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Fig 10. Flow cytometric analysis of JLP 119 and EW-36
treated with different concentrations of H2O2 . (A) Flow cytometer data. Cells (5 Ì 105/mL)
were incubated in growth medium for 24 hours after the addition of the indicated concentrations of H2O2 (0, 25, 50, 75, or 100
mmol/L). FACS analysis of fixed
cells was performed as described in the Materials and
Methods. Rows 1 and 3, cytograms showing forward and side
light scatter (FSC and SSC, respectively) for 10,000 cellular
events. Rows 2 and 4, histograms of FITC-labeled DNA for
the cells shown in the cytograms. Rows 1 and 2, results for
JPL-119. Rows 3 and 4, results
for EW-36. Cells with a high FITClabeling intensity represent apoptotic cells. The data shown are
from a representative study that
was repeated three times. (B)
Proportion of JLP 119 (m) and
EW-36 (n) cells that are undergoing apoptosis at each concentration of H2O2 . The percentage
of apoptosis equals the high
FITC-labeled population divided
by the total cell population. Each
point is derived from 10,000 cellular events and represents the
mean Ô SEM of triplicate cultures.
showed markedly different susceptibilities to induction of
apoptosis by A23187 that correlated with their respective
levels of expression of Bcl-2, consistent with the conclusion that Bcl-2 prevents cell death by inhibiting
apoptosis.5,10,11,26,27,39,64 But these cell lines showed little difference in susceptibility to H2O2 -induced killing. The cell
line with low Bcl-2 (JLP 119) was slightly more susceptible
(°2-fold) to toxicity (loss of viability) from 50 mmol/L H2O2
compared with the high Bcl-2 expressor (EW-36; see Fig
1), but the differences were small relative to the substantial
(ú20-fold) differences in both the levels of expression of
Bcl-2 (see Fig 2) and the amount of protection afforded from
A23187-induced apoptosis (see Figs 6 and 7).
One experimental difference that might have explained
the disparity between our findings and those reported pre-
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viously17,29-31 is that most previous investigations were performed using murine or rat cell lines that were transfected
with the human bcl-2 gene,16,26,27,29,31,63,65 whereas we examined BL cells expressing different levels of endogenous Bcl2 protein. Transfection experiments offer the advantage that
the protein is expressed in an otherwise isogenic background.
However, transfected genes are under different regulatory
control from endogenous genes and functional differences
between transfected and endogenous Bcl-2 proteins can influence the experimental results.30,53 We tested this possibility directly by transfecting human bcl-2 into JLP 119 BL
cells and obtained results that were very similar to those
achieved with the endogenous protein, ie, that transfected Bcl-2 did not prevent H2O2 -induced killing in these
cells.
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BCL-2 AND OXIDANT-INDUCED CELL DEATH
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Fig 11. Quantitation of extent and type of H2O2 - and A23187-induced cell death induced in BL cell lines by Hoechst/propidium iodide
nuclear staining. EW-36 and JLP 119 cells were treated for 24 hours
with 100 mmol/L H2O2 or for 48 hours with 2 mmol/L A23187. Cell
death (the percentage of propidium iodide-stained cells) and morphology were quantified as described in the Materials and Methods.
The data represent the results from four separate experiments. (j)
Necrotic death; ( ) apoptotic death.
The absence of a protective effect of Bcl-2 on H2O2 induced cell death in BL cells may be explained in part
by the fact that H2O2 was relatively inefficient at inducing
apoptosis. Using nucleosomal DNA fragmentation to assay
for apoptosis (agarose gel electrophoresis and TUNEL
assays), none of the cell lines that we examined (a total of
6) showed a high degree of apoptotic death in response to
H2O2 treatment even though a range of concentrations was
tested. In contrast, A23187 and serum depletion killed the
cells primarily by apoptosis and cells expressing high levels
of Bcl-2 were protected from these treatments. Using a morphologic assay for apoptosis, a somewhat different result
was seen; by this measure, roughly half of the bcl-2lo (JLP
119) cells died by apoptosis and half by necrosis. However,
it should be noted that the morphologic apoptosis induced
by H2O2 was different from that seen with A23187. H2O2
Fig 12. Western blot immunoassay of JLP 119 cells transfected
with bcl-2. JLP 119 cells with a stable transfection of either vector
alone (G4 and G21) or plasmid expressing the human bcl-2 gene (H6
and H10) were tested for expression of the Bcl-2 protein as described
in the legend to Fig 2. The level of expression of endogenous Bcl-2
in EW-36 cells is shown for comparison.
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Fig 13. Flow cytometric TUNEL assay of JLP 119–bcl-2 transfectants treated with H2O2 or A23187. JLP 119 cells with a stable transfection of either control plasmid (G4 and G21) or plasmid expressing
the human bcl-2 gene (H6 and H10) were tested for induction of
TUNEL activity as described in the legends to Figs 7 and 10. ( ) H2O2
was added at 50 mmol/L and cells were assayed 24 hours later. ( )
A23187 was added 1 mmol/L and the cells were assayed 48 hours
later. The data are taken from one representative experiment. (j) No
treatment.
induced nuclear pyknosis and cleavage into 2 to 3 spheres,
but the fragmentation into the smaller particles that was
characteristic of A23187 treatment was uncommon. At the
same time, the necrosis induced by H2O2 was also somewhat
atypical in that the nuclei were intact but were not necessarily
larger than control nuclei. Additional experiments will be
required to characterize the mechanism of cell death induced
by H2O2 . The poor induction of chromatin fragmentation
at the microscopic level correlated with the low levels of
molecular DNA fragmentation assessed through biochemical
techniques. Taken together, the results obtained to date suggest that H2O2 induces only partial apoptosis, as has been
observed in some other experimental systems.59-61
Our studies suggest that Bcl-2 prevents cell death only
when the mechanism of cell death is primarily apoptotic.
The apoptotic death induced by H2O2 was inhibited by overexpression of Bcl-2. However, the cells still died (as determined by trypan blue and propidium iodide staining), apparently by a necrotic mechanism. This finding is inconsistent
with previous reports suggesting that Bcl-2 protects cells
from necrotic as well as apoptotic death. Kane et al16,28 concluded that, in neural cell lines, diminution of antioxidant
defenses (glutathione depletion) kills cells by a necrotic
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on performing and interpreting the Hoechst/propidium iodide morphology assays. Finally, we thank Giovanna Tosato, Wendy Shores,
Melanie Vacchio, and Joy Williams for careful reading of the manuscript and for many useful suggestions.
REFERENCES
Fig 14. Quantitation of extent and type of H2O2 -induced cell death
induced in bcl-2–transfected JLP 119 cells by Hoechst/propidium iodide nuclear staining. JLP 119 cells transfected with either control
plasmid (G4 and G21) or plasmid expressing the bcl-2 gene (H6 and
H10) were treated with 100 mmol/L H2O2 or 5 mg/mL etoposide. In
both cases, cells were collected after 24 hours and examined for
cell death (propidium iodide staining) and morphologic features of
apoptosis (chromatin condensation) as described in the Materials
and Methods. The data represent the average of four independent
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present investigation, these findings challenge the theory that
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ACKNOWLEDGMENT
The authors thank Kishor Bhatia, Ian McGrath, and Barry Cherney
for supplying the cell lines and their p53 mutation and EBV infection
statuses. We are grateful to Apurva Sarin for invaluable instructions
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1997 89: 4480-4492
Bcl-2 Does Not Protect Burkitt's Lymphoma Cells From Oxidant-Induced
Cell Death
Yang-ja Lee and Emily Shacter
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