Download BCL-2 Expression and Mitochondrial Activity in

From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
BCL-2 Expression and Mitochondrial Activity in Leukemic Cells With
Different Sensitivity to Glucocorticoid-Induced Apoptosis
By Lou A. Smets, Joop Van den Berg, Dennis Acton, Bert Top, Henny Van Rooij, and Manon Verwijs-Janssen
The present study investigates
the relationship between mitochondrial activity and the expression of the BCL-2 genein
a panel of six human and murine leukemia/lymphoma cell
lines. The cell lines all contained normal glucocorticoid receptors but differed widely in sensitivity to dexamethasone,
ranging from very sensitive S49 lymphoma to completely
resistant HL-60 acuteleukemiacells. In this panel,10- to
15-folddifferences in basaladenosinetriphosphate(ATP)
content and adenosine diphosphate (ADP)/ATP ratio were
correlated with up to fivefold differencesin bcl-2 protein (in
human cells) and approximately 25-fold difference in bel-2
mRNA content (all cell lines). Moreover, ATP content and
BCL-2 gene expression
were inversely correlated
with gluco-
corticoid sensitivity and cell cycle length. In resistant cell
lines, sensitivityto dexamethasonewas restored by the mitochondrial inhibitors rotenone andmeta-iodobenzylguanidine. This sensitization was not accompanied by detectable
reductions
in bcl-2 mRNA or protein content,
suggesting that the inhibitors were capable of overriding
BCL-2-mediated inhibition ofapoptosis.Increased
mitochondrial
activity
and
(overexpressed)
BCL-2
appeared
closely related properties of glucocorticoid-resistantcells,
sharing
common
cellular
targets in hormone-induced
apoptosis.
0 1994 by The American Society of Hematology.
T
murine and human leukemic cell lines, selected for widely
different susceptibility to the growth inhibitory and lytic
effects of dexamethasone. In this report, we explain that the
expression levels of the BCL-2 gene and cellular energy
status are highly correlated phenomena and that both parameters are inversely related to sensitivity for GC-induced
apoptosis and cell cycle length.
HE TRANSCRIPTIONALLY deregulated BCL-2 gene
increases the life-span of lymphoid cells'.' and confers
resistance to various inducers of programmed cell death
(apopt~sis).~.~
According to recent reports the bcl-2 protein
complexes with death-accelerating homologs7 and functions
in antioxidant pathways in preventing apoptosis.8The precise
biochemical activities of bcl-2 protein and its homologs are
not known, however. Because of its localization to mitochondrial membranes,' protection by BCL-2 has been previously
associated with mitochondrial functions." An exclusive role
of bcl-2 protein in mitochondria has been questioned, however, by the observation that in several cells the pattern of
immunofluorescent staining is consistent with its localization
to the endoplasmatic reticulum (ER) and the nuclear envelope as well.9"' In fact, neither mitochondrial localization
nor an integral membrane position are absolute requirements
for bcl-2 function according to Hockenberry et al.' Mitochondrial involvement has been specifically challenged by
the observation that fibroblasts lacking mitochondrial DNA
and thus deficient in oxidative phosphorylation, remain susceptible to protection by the transfected human BCL-2
gene."
On the other hand, cells deficient in mitochondrial DNA
still contain all nuclear encoded mitochondrial enzymes and
retain important mitochondrial functions that include succinate dehydrogenase activity" and the generation of an electrochemical gradient.13There is circumstantial evidence for
a critical role of mitochondrial activity in the susceptibility
of leukemic cells to glucocorticoid (GC)-mediated lysis.
Stru~tural'~
and functional" damage to mitochondria has
been observed early in GC-induced lysis of lymphoid cells,
although a causal sequence has not been established. However, several leukemic cell lines, including fully resistant
variants, can be sensitized to dexamethasone by inhibitors of
mitochondrial respiration.'"18 The observation that leukemic
cells are protected from GC-induced apoptosis by a relative
abundance of bcl-2 p r ~ t e i n but
~ . ~sensitized by respiration
inhibitors adds to a notion of functional relationships between bcl-2 protein and mitochondrial activity in the lysis
of leukemic cells.
To investigate the possibility relationship between BCL2 and mitochondria, we have compared mitochondrial activity and BCL-2 expression in a panel of GC receptor-positive
Blood, Vol 84, No 5 (September l), 1994: pp 1613-1619
MATERIALSANDMETHODS
Cell lines and cytological assays. Cell lines HL-60, L1210 (subline 56.3), and S49 and culture conditions were as described in
previous rep"t~.'~.'','~
Cell line JANEL was established by infection
of normal human B cells with Epstein-Barr virus. The human nonHodgkin's follicular lymphoma cell line DoHH2;' carrying translocation t(14; 18), was kindly donated by H. Kluin-Nelemans (Leiden
University, The Netherlands). Human T-cell leukemia CEM-C7 line
was obtained by courtesy of T. Schmidt (University of Iowa). Dexamethasone (DEX) was added from 1,000-fold ethanol concentrates
toafinal concentration of 10" m o w throughout and lysis was
scored microscopically. DNA per cell recordings were made by flow
cytometry on ethanol-fixed and ethidium bromide stained cells as
described previously.2'
Metabolic studies. DNAand protein synthesis was assessed
from the incorporation of'H-thymidineandI4C-leucine,
respectively, into acid-precipitable cell material and related to cellular
protein according to routine procedures. Adenosine triphosphate
(ATP) and adenosine diphosphate (ADP) content were determined
in cold 0.5 N perchloric acid extracts" and the relative ADP content
was expressed by the molar ratio ADP/ATP X 100%.
3H-dexamethasonebinding. Specific 3H-dexamethasonebinding
From the Divisions of Experimental Therapy and Molecular Genetics, The Netherlands Cancer Institute, Amsterdam.
Submitted January 20, 1994; accepted May 4, 1994.
Supported in part by the Dutch Cancer Society, Grant No. NKI89-2.
Address reprint requests to Lou A. Smets, PhD, The Division
of Experimental Therapy, The Netherlands Cancer Institute, 121
Plesmanlaan, NL-1066 CX, Amsterdam, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8405-03$3.00/0
1613
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1614
SMETS ET AL
Table 1. Characterization of the Cell Lines With Different
sites were measured in a whole-cell assay as described elsewhere."
Briefly, cells were incubated with 2 pCi of 'H-dexamethasone (5 X
Sensitivities t o Dexamethasone
10"' moliL) with or without a I ,000-fold excess of radioinert steroid
DEX
toassess nonspecific binding.After 20 minutespostincubationin
Cell Cycle
Binding
Time
Sites
Sensitivity
agonist-free growth medium the amount of cell-associated radioacCell Line
Description
(hours)
(per cell)
to DEX
tivity was determined.
Chemicals, antibodies, and buffers. Anti-receptor
antibody
HL-60 Human myeloid leukemia
48
18,000
GR49.1 was kindly donated by Dr H.M. Westphal (Marburg, GerDoHH2 Human N H lymphoma
36
15,000
+
many): monoclonal MAI-510 (clone BuGR2)
was purchased from
Jane1
Human B-cell leukemia
36
ND
2
Affinity Bioreagents (Neshanic Station, NJ). Anti bcl-2 monoclonal
CEM-C7 Human T-cell leukemia
18
20,000
+
antibody (clone 124)'? was a kind gift of Drs Mason and Pezella
L1210 Mouse leukemia
12
9,000
++
(Oxford, U,K). Anti-actin antibody (clone C4) was purchased from
S49
Mouse lymphoma
10-12
20,000
+++
Boehringer Mannheim Biochemica (Mannheim, Germany). HorseCell
cycle
time
was
estimated
from
the
doubling
time
during
exporadish peroxidase conjugated goat-anti-mouse (GAM-HRP) IgG was
nential
growth.
Specific
W
D
E
X
binding
sites
were
determined
as
from Pierce (Rockford, IL), fluorescein isothiocyanate (FITC) coudescribed in Materials and Methods. Sensitivity to IO" mol/L of dexapled sheep-anti-mouse IgG was from Sigma (St Louis, MO), nitromethasone was rated as strong growth inhibition and massive cell
cellulose filters werefromBio-Rad(Richmond,VA)andPVDF
lysis after 24 hours (+++), growth inhibition with marginal cell lysis
lmmobilonmembraneswerefromMillipore(Bedford,MA).Enhanced chemiluminescence (ECL) detection kit for Western blotting, after 48 hours (+) or no effect ( 6 ) .
Abbreviation: ND. not done.
'H-thymidine (25 Ci/mmol), I4C-leucine (300 mCi/mmol), 'H-dexamethasone(37Ciimmol),and
'251-IgG sheep-anti-mouse ("'ISAM, 16 pCi/pg) were from Amersham International (Buckinghamshire, UK).
rescenceafter fixation withglutaraldehyde (0.2% inPBSfor10
Buffers used included phosphate buffered saline (PBS); blocking
minutes at 4 T ) , permeabilization with 0.2% Triton X-l00 (30 minbuffer no. I : Carnation (Los Angeles, CA) nonfat dry milk (5% (wt/
utes at room temperature) and reduction with NaBH4(3 X 5 minutes
vol)) and 0.1% (vol/vol) Tween-20 in PBS; blocking buffer no. 2:
at room temperature). The slides were
then incubated in blocking
0.15% (wt/voi) bovine serum albumin (BSA), 0.5 mmol/L EDTA,
buffer no. 2for 1 houratroomtemperatureandincubatedwith
0.5% (vol/vol) Tween-20 in PBS; SSC (20X): 88.2 g sodinmcitrate,
undiluted anti bcl-2 antibody in the cold for 6 hours, followed by
175.3 g NaCI, pH 7; lysis buffer: 250 mmol/L sucrose, 20 mmol/L
three washes with cold PBS and incubation in FITC-coupled RAM
KH2P04 pH 7, 1 rnmol/L EDTA, CaCl, and MgCl,, both at 0.1 5
IgG for 10 hours in the cold (1:5000
in blocking buffer no. 2). DNA
mmol/L.
was stained by propidium iodide in a concentration of 0.02 pg/mL
Western blotting. The intracellular distribution of glucocorticoid
for 30 minutes at room temperature. A BioRad MRC 600 confocal
receptors was determined by Western blotting. After disruption
of
laser microscope was used to visualize the protein (excitation at488
the cells in lysis buffer, cytosolic and nuclear fractions were preparednm) and nuclei (at 350 nm).
by centrifugation at 14,000.g and cell equivalent amounts of protein
were separated by sodium dodecyl sulfate-polyacrylamide gel elecRESULTS
trophoresis(SDS-PAGE)(7.5%)andtransferredtonitrocellulose
Characterization of cell lines. The presence of GC-bindfilters. The filters were blocked with buffer no. I and probed with
anti-receptor antibody GR49.1or BuGR2 (1:5,000 in blocking buffer ing sites, ranging from 9,000 in L1210 to 20,000 sitedcell
in S49 and CEM-C7, wasconfirmed in conventional wholeno. 1) using GAM-HRP(1 :1000 in blocking buffer no.1) as secondcell binding assays. Nuclear translocation of DEX-liganded
ary antibody. The immunoreactive proteins were visualized by the
ECL technique according to instructions of the manufacturer.
cytosolic receptors was also confirmed by immunoblotting
Relative levels of bcl-2 protein were determined
in samples of
of the GC receptor protein in cytosolic and nuclear fractions
IO" cells boiled in sample buffer, separated by SDS-PAGE (15%)
before and after 2 hours of incubation with the steroid. In
and transferred to Immobilon-P, PVDF membranes. The membranes spite of the presenceofnormal
GC receptors and of the
were incubated in blocking buffer no. 2 and probed with anti bcl-2
capability of nucleartranslocation, thecelllines differed
antibody ( 1 :20 in blocking buffer no. 2). Detection was performed
widelyinsusceptibility
to the growthinhibitory and lytic
by GAM-HRP (1:3,000 in blocking buffer no. 2) as a second antieffects
of
m
o
l
L
DEX.
(Table 1). In S49 cells, 80%
body and staining with 4-chloro-l-naphthol. Actin was detected on
was
lysed
within
24
hours,
whereas
lysis of L1210 cells was
the same membrane with anti-actin (1 :10,000 in blocking buffer no.
observed only after 2 days.19 In CEM-C7 cells inhibition of
2) as primary antibody. For quantification, pre-stained bands were
relabeled with "'l-SAM (2 pCiin blocking buffer no. 2), cut out, and growth started after 72 hours and was accompanied by the
the associated radioactivity was measured by scintillation counting.
appearance of 20% to 40%lysed cells after 92 hours. DoHH2
Northern blotting. TotalRNAwasisolatedfrom
=50 X 10'
and JANEL cellsresponded to DEXby a transient inhibition
cells.24 For Northern blot analysis 20 pg of RNA was separated on
of growth during the first 24 hours, accompanied by a mara 1 % agarose gel and transferred to nitrocellulose filters. Filters were ginal degree of cell death of 10% to 20%. Subsequently,
hybridized with a mouse cDNA probe, derived from a mouse cDNA these cells resumed normal growth in the continuous preslibrary and comprising the bcl-2 codon region as described by Neence of DEX. HL-60 cells were completely insensitive to
grini et al? and labeled by the random priming method. Filters were
Overall, the cell
DEX for 72 hours as reported previou~ly.'~
washed in 0.1 X standard sodium citrate (SSC), 1% SDS at 6VC,
dried and exposedto x-ray film. The radioautograms were quantified lines with shorter cell cycle time were better responders to
the steroid,but there was no relationwith the number of
by densitometry. Total RNA and actin mRNA were used as loading
specific GC binding sites.
controls.
Mitochondrial activity. The cell lines differed considerImmunocytochemistry of hcl-2. Intracellular distribution of bcl2 was investigated in cytospin preparationsby indirect immunofluoably in basal ATP content and energy status as reflected by
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1615
bcl-2 AND GLUCOCORTICOIDSENSITIVITY
80
tein was not available, BCL-2 gene expression in all cell
lines was assayed on Northern blots using a mouse cDNA
probe. This probe detects a 7.5-and2.4-kb transcript as
previously observed by Negrini et al. Because of the absence
of cross-hybridization with the 28s rRNA, an additional 5kb message is also detected. The 7.5-kb bcl-2 mRNA levels
in L12 IO and S49 cells were compared by densitometry, and
normalized against the 2-kb actin mRNA signal. The results
(Fig 3C) indicated at least three-fold higher levels in L1210
cells. Mouse L1210 cells werecomparedwithhuman
HL-60DoHA2 Janel C E M L1210 S49
DoHH2 cells, using the mouse cDNA probe. At comparable
total mRNA loading levels (Fig 3A), expression of the major
Fig 1. ATPlevels (W) and relative ADP content (0)in celllines
with (from left to right)increasing sensitivity to dexamethasone Id,
7.5-kb transcript in DoHH2 cells was about eight-fold higher
Table 1). ADP content is expressed aspercentage of molar ATP conthan in L1 210 cells (Fig 3B). Considering the differences in
tent and isdefined as the reciproque of cellular energy charge. Values
the nucleotide sequences between mouse and human BCL2,
are mean ? SEM of duplicates of six to eight independent experithis eight-fold higher level may be an underestimate. Howments.
ever, no obvious differences in mRNA levels were observed
between DoHH2and the other human cell lines HL-60.
JANEL,
and CEM-C7 (data not shown).
ADP/ATP ratio (Fig l). ATP levels were expressed relative
Reversal of GC-resistance by mitochondrial inhibitors.
to protein content to compensate for differences among the
To test if increased ATP levels wereinstrumental in GC
various cell lines. The observed values were well above (HLresistance, the cells were assayed for dexamethasone-medi60) or below (S49; L1210) the average ATP content reported
ated
lysis in the presence of the inhibitors rotenone or metafor various leukemic and non-leukemic cell lines of -4 to
iodobenzylguanidine (MIBG). Inhibition of mitochondrial
6 nmol/lO' cells,''~*' corresponding with -20 mg/g protein.
respiration with these complex I inhibitors" or with doxycyLikewise, the relative ADP content ranged significantly becIineI8allows for a compensatory increase in glycolytic flux,
low and above the average value of = 10% in actively growmaintaining ATP at lower but vital levels. MIBGcaused
ing cells in vitro. Overall, sensitivity to dexamethasone was
a dose-dependent cytolytic response to dexamethasone in
inversely correlated with ATP content and proportional with
refractory DoHH2 cells. This became already apparent as
decreasing energy status, ie, increasing ADP/ATP ratio.
early as 12 hours after combined treatment (Fig 4). Rotenone
BCL-2 expression. The bcl-2/actin ratio was highest in
in the non-toxic concentration range of 0.5 to 2.0 X IO-'
HL-60 cells, exceeding by two-fold that of DoHH2 cells.
molL also potentiated lysis (Fig 5). An excess of IO-' mol/
EBV-transformed JANEL cells contained lower levels of
L of the GC-antagonist RU486 completely blocked rotenonebcl-2 protein but these were still higher thanin CEM-C7
induced sensitization to dexamethasone, indicating that lysis
cells (Fig 2). The bcl-2/actin ratio of reference human leukowas initiated by the hormone and not caused by toxic side
cytes was 0.05, indicating the relative abundance of the proeffects of the inhibitor. The effects of rotenone and MIBG
tein in the human cell lines. As expected, mouse L1210 cells
in JANEL cells were similar to those observed in DoHH2
were negative for the human-specific antibody.
Because an antiserum directed against murine bcl-2 procells. Sensitization of CEM-C7 and L1210 cells was as re-
"1 T
1 .oo
r
I
actin
]
0.80
Fig2.Levels of bcl-2 protein
in human leukemic cell
lines
with increasing
sensitivity
to
DEX es described in the legend
to Fig 1. Bel-2 protein content is
expressed relative to actin content in an arbitrary scale and values are mean ? SE of three to
four independent experiments
with mouse L1210 cells
as a negative control (background levels). A representative immunoblot is shown in the insert and
cell linesare indicatedby initials.
The dashed line represents the
bcl9lactin ratio in normal human peripheral lymphocytes.
H J D C L
0.60
0.40
0.20
0.00
HL-60
DoHH2
Janel
CEM
L1210
S49
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
SMETS ET AL
1616
A
B
1 2 3
1 2 3
C
1 2
S
100
.
80
kb
kb
- 7.6
-5
60
-7.6
40
-4.8
-2.1
20
- 2.4
.’
-.S
-
0
0.1
1
1
10
rotenone (10-6M)
Fig 5. Sensitization of DoHH2 cells by rotenone. Cells were incubated in graded concentrations of rotenonewith (VIor without (01
lo” mol/L DEX and lysis was scored after 24 hours of incubation.
Mean values 2 SE of four independent experiments.
Fig 3. Bcl-2 mRNA expression in leukemic cell lines. (A) TotalRNA
loading control ofB. (B1 Northern blot of
bcl-2 mRNA levels in mouse
L1210 cells (lane l),
control human DoHH2 cells (lane 2). and DoHH2
mol/L rotenone (lane 3). (Cl
cells incubated for 21 hours with
Northern blot of bcl-2 and actin mRNA levels in L1210 (lane 1) and
S49 (lane 21 cells.
ported previously” but observations in S49 cells were inconclusive because of toxicity of both inhibitors in the concentrations used, probably because of critically low basal ATP
levels. However, HL-60 cells remained completely refractory to DEX in the presence of rotenone. With MIBG a weak
and delayed (ie, after 4 days) response to the steroid was
induced as reported earlier.“ ATP levels were reduced in
8o
6o
t
t
0 ’
0
10
20
30
40
I
50
incubation time(hour)
Fig 4. Sensitization of DoHH2 cells t o DEX (lo” mol/L) by MIBG;
control: W 5 p g / m L (01, 10 p g / m L ( W , 20 p g / m L (01.Exponentially growing cells (4 x 1O5/mL1were incubatedwith DEX and MIBG
and scored for thepercentage of apoptotic cells at regularintervals.
Mean values f SE of four t o five independent experiments.
cells grown with rotenone ( IOv6 mol/L) or MIBG ( I O pg/
mL). After 21 hours, the reductions variedbetween 70%
(CEM-C7, L1210) and 40% (HL-60) and were all accompanied by a proportional decrease in energy charge. In HL-60
cells, refractory to sensitization by the inhibitors, the ATP
levels (37 mg/g protein) and the relative ADP content (6%)
of treated cells remained well above (ATP) or below (ADP/
ATP) the basal values in the sensitive L1210 and S49 cells
(cf, Fig l ) .
Efects on BCL-2 expression and cell proliferation. The
effect of mitochondrial inhibitors was studied in more detail
in DoHH2 cells. There was no reduction in the level of bcl2 protein during incubation in rotenone for 4 or 16 hours
(Fig 6). In Northern blots, the major 7.5-kb messenger signal
in DoHH2 wasnot detectably altered by incubation with
MIBG or rotenone for 21 hours (Fig 3A,B). Immunocytochemistry with confocal laser scanning microscopy of bcl-2
protein distribution showed predominant perinuclear localization with the typical patchy staining as described for several other cells?” At this level of resolution, no gross effects
of the inhibitors alone or in combination with dexamethasone
on bcl-2 content nor its intracellular distribution could be
observed (data not shown). Consistent with the finding on
bcl-2 protein levels (Fig 6), the incorporation of I4C-leucine
was not significantly affected during 21 hours of incubation
with the inhibitors. The number of specific ‘H-DEX binding
sites was only marginally reducedfrom 15,000 to 11,000
sites per cell, probably because of cell cycle
Cell
multiplication in 24 hours was reduced from 1.8 in controls
to 1.2 in cells grown the presence of rotenone or MIBG.
Flow-cytometric analysis of DNA per cell content indicated
that this inhibition of growth was accompanied byan increase in the fraction of cells in G, phase from 0.35 to 0.75.
The biochemical and cell kinetic findings in DoHH2 cells
were similar to the reported effects of doxycyclineIx and
MIBGIh.17 ’In leukemic cells, showing that inhibitionof mitochondrial respiration with simultaneous glycolytic compen-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
bcl-2 AND GLUCOCORTICOIDSENSITIVITY
control 4 hrs 16 hrs
1 2 3 4 5 6
1617
kDa
-55
-
26
-1 4
Fig 6. Effect of rotenone on bcl-2 protein levels in DoHH2 cells.
lmmunoblot of bcl-2 protein in DoHH2 cells incubatedwithout (lanes
l , 2) and with 10.' mol/L rotenone for 4 hours (lanes3,4) or 16 hours
(lanes 5, 6).
sation allows completion of the ongoing cell cycle but blocks
cells in G, phase of the next division cycle.
DISCUSSION
The protective effect of the overexpressed RCL-2 gene
against several apoptotic stimuli has been well documented:.'
but the biochemical mechanisms involved are still a matter
of intensive research. In view of its preferential association
with mitochondrial membranes,' protection from apoptosis
by bcl-2 has been previously associated with mitochondrial
functions. According to recent reports, bcl-2 may act in the
control of intracellular Ca" repartitioning',''
and functions
in antioxidant pathways.'.''
The present observations in a panel of leukemic cell lines
revealed a correlation between BCL-2 expression (Figs 2 and
3) and mitochondrial activity as reflected by ATP levels and
energy status (Fig l ) . Moreover, BCL-2 expression level and
ATP content were inversely related to the susceptibility to
dexamethasone and the rate of cell proliferation (Table l ) .
Variation in sensitivity to dexamethasone was not associated
with the amount of 'H-DEX binding sites nor with the nuclear translocation capability of the liganded GC-receptor.
The notion thatincreased mitochondrial activity can interrupt
the lytic signal, once initiated by binding of GC's to their
cognate receptors, finds additional support in the present and
previous observations'"'X that inhibitors of mitochondrial
respiration can potentiate GC-action and even induce sensitivity to steroid-mediated lysis in resistant cell lines. A comparison of the basal ATP levels (Fig 1) and the sensitizing
effect of inhibitors suggests that a level of about 20 mg ATP/
g protein (4 nmol/lO" cells) or the corresponding ADP/ATP
ratio of about 0.20 are critical discriminators between sensitivity and insensitivity to GCs, irrespective of leukemic cell
lineage. These observations agree with similar alterations in
ATP levels and relative ADP content in Molt-4 leukemia
cells sensitized to DEX by doxycycline.IxIn this view. failing sensitization of HL-60 cells by rotenone and MIBG can
be ascribed to the inability of the drugs to lower high basal
ATP levels below this critical level. Conversely, the sensitivity of morerapidly growing cell lines (CEM-C7, L1210.
S49) is plausibly explained by an intrinsically lower energy
status as a consequence of enhanced energy requirements
for rapid protein and nucleic acid synthesis.
Unlike in the human cell lines, the relative bcl-2 protein
levels in themurine L1210 and S49 cells couldonly be
estimated frommRNA
levels. Althoughcomparison
of
mRNA levels can only show a trend in bcl-2 protein content,
there was a marked, ie, 25-fold difference in BCL-2 gene
expression between fully resistant HL-60 and moresensitive
S49 cells detected with a mouse cDNA probe. Because of
sequence differences betweenhumanandmurinemessengers, this difference is probably an underestimate. Despite
this, the relationship between BCL-2 gene expression on the
one hand and the correlated variations in ATP content, cell
cycle time and DEX sensitivity (Fig 1: Table I ) on the other,
is not an obvious one. However. abrogation of GC-resistance
by the inhibitors in DoHH2 cells without detectable effects
on bcl-2 protein (Fig 6) or mRNA (Fig 3) content may
exclude a trivial explanation that a low energy status on its
own reduces RCL-2 expression. Therefore, overexpression
of RCL-2 andelevated energy status may each afford protection by totally different mechanisms and the observed correlations could be coincidental. ie, the result of co-selection
for two unrelated mechanisms of GC-resistance.
On the other hand, there are some grounds for the hypothesis that mitochondrial activity and bcl-2 protein may cooperate in modulating GC sensitivity. In cultured hematopoietic
cells and in the B-cell compartment of transgenic mice, overexpression of the bcl-2 proteindoes not stimulate cell proliferation per se, but promotes survival by preventing irreversible cell cycle exit leading to cell death."' In fact. the inverse
correlation in our cell panel between RCL-2 expression and
cell cycle length suggests that the proto-oncogene can reduce
the rate of cell proliferation, allowing the recovery of energy
charge required for the acquisition of resistance toDEX.
The notion that the BCL-2 gene can suppress proliferation
finds support in the observations that elevated levels of bcl2 protein are associated with slowly growing, indolent tumors3" and can afford protectionagainst the lethal effects of
excessive or inappropriate mitogenic signals.."
Finally, the observation that mitochondrial inhibitors were
capable of overriding RCL-2-mediated protection in DoHH2
cells could indicate a more direct relationship between bcl2 proteinandmitochondrial activity. Current explanations
on bcl-2action"
concentrate on twoequally
attractive
hypotheses. namely the regulation of intracellular Ca'' repartioning and scavenging of reactive oxygen species. It is
conceivable that any role of bcl-2 in antioxidant pathways
will be dependent on the cellular redox state, and thus, sensitive to complex I inhibitors in cells with a functional respiratory chain. Likewise, mitochondria are directly and indirectly
implicated in Ca" homeostasis. It is of note that incubation
of hepatocytes with MIBG results in an increased size of the
mitochondrial Ca2+pool,33a process that is typically blocked
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1618
SMETS ET AL
by bcl-2 overproduction in a hematopoietic cell line resistant
to apoptosis induced by growth factor ~ithdrawal.~’
In spite of suggestive correlations, the present experiments
cannot provide for direct evidence that bcl-2 protein level
is a metabolic checkpoint of GC-sensitivity, acting through
mitochondrial functions. However, it is obvious that the
physiologic role of bcl-2 in the response of leukemic cells
to GC treatment can be fully appreciated only in the context
of cell cycle control and cellular energy status. Irrespective
of the precise mechanisms involved, the apparent possibility
to override resistance to GC hormones in bcl-2 expressing
human leukemicflymphoma cells may be of clinical relevance. Increased bcl-2 protein levels are found inan expanding spectrum of hematologic malignancies, often without involvement of translocation t( 14;18).23,34In tissue
culture models, the proto-oncogene appears a potential arbiter of response to GC hormones and various antileukemic
drug^^.^ and BCL-2 expression has been recently associated
with poor outcome of chemotherapy in acute myeloid leukemia.35Accordingly, antagonism of bcl-2 action by pharmacologic interventions of the type described in this report would
appear a feasible strategy in the chemotherapy of leukemid
lymphoma with high expression of bcl-2 protein.
ACKNOWLEDGMENT
The authors gratefully acknowledge Dr H. Westphal for providing
the anti-GR antibody, Drs F. Pezella andD. Mason for the antibcl-2 Moab, Dr H. Kluin-Nelemans for the DoHH2 cells, Nienke
Oldenburg for assistance in the ATP assays, and Dr C. van den
Bogert for valuable suggestions.
REFERENCES
1 . Hockenberry D, Nuiiez G, Milliman C, Schreiber R,Korsmeyer SJ: bcl-2 is an inner mitochondrial membrane protein that
blocks programmed cell death. Nature 348:334, 1990
2. McDonnel TJ, Deane N, Platt FM, Nuiiez G, Jaeger U,
McKearn JP, Korsmeyer SJ: bcl-2-immunoglobulin transgenic mice
demonstrate extended B cell survival and follicular lymphoproliferation. Cell 57:79, 1989
3. Tsujimoto Y: Stress-resistance conferred by high level of bcl2 alpha protein in human B lymphoblastoid cell. Oncogene 4: 1331,
1989
4. Alnemri ES, Femandes TF, Haldar S, Croce CM, Litwack G:
Involvement of BCL-2 in glucocorticoid-induced apoptosis of human pre-B-leukemias. Cancer Res 52:491, 1992
5. Miyashita T, Reed JC: Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 8 1:151,
1993
6. Miyashita T, Reed JC: bcl-2 gene transfer increases relative
resistance of ,549.1 and WEH17.2 lymphoid cells to cell death and
DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs. Cancer Res 52:5407, 1992
7. Oltvai Z, Milliman C, Korsmeyer SJ: bcl-2 heterodimerizes in
vivo with a conserved homolog, Bax, that accelerates programmed
cell death. Cell 74509, 1993
8. Hockenberry DM, Oltvai Z, Xiao-Ming Y, Milliman C, Korsmeyer SJ: bcl-2 functions in an antioxidant pathwayto prevent
apoptosis. Cell 75:241, 1993
9. Chen-Levy Z, Nourse J, Cleary M: The bcl-2 candidate protooncogene product is a 24-kilodalton integral-membrane protein
highly expressed in lymphoid cell lines and lymphomas carrying the
t(14;18) translocation. Mol Cell Biol 9:701, 1988
IO. De Jong D, Prins A, Mason D, Reed J, Van Ommen GJ,
Kluin P: Subcellular localization of bcl-2 protein in malignant and
normal lymphoid cells. Cancer Res 54:256, 1994
11. Jacobson M, Burne J, King M, Myashita T, Reed J, Raff M:
bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature
361:365, 1993
12. Loveland B, Johns T, Mackay I, Vaillant F, Wang Z-X, Hertzog
P: Validation of theMTT dye assay for enumeration of cells in proliferative and antiproliferative assays. Biochem Intern27501, 1992
13. Van den Bogert C, Herzberg N, Spelbrink J, Dekker H, Groen
B, Bolhuis P: Mitochondrial function and biogenesis in cultured
mammalian cells without functional respiratory chains. In S Papa,
A Azzi, J Tager (eds): Adenine nucleotides in cellular energy transfer
and signal transduction. Basel, Switzerland, Birkhauser Verlag,
1992, p 92
14. Gallili U, Leizorowitz R, Moreb J, Gamliel H, Gurfel D,
Palliack A: Metabolic and ultrastructural aspects of the in vitro lysis
of chronic lymphocytic leukemia cells by glucocorticoids. Cancer
Res 42: 1433, 1982
15. Dowd D, Miesfield R: Evidence that glucocorticoid- and cyclic AMP-induced apoptotic pathways in lymphocytes share distal
events. Mol Cell Biol 12:3600, 1992
16. Smets LA, Metwally EA, Knol E, Martens M: Potentiation
of glucocorticoid-induced lysis in refractory and resistant leukemia
cells by inhibitors of ADP-ribosylation. Leuk Res 12:737, 1988
17. Smets LA, Loesberg C, Janssen M, Van Rooij H: Intracellular
inhibition of mono(ADP-ribosylation) by metu-iodobenzylguanidine: Specificity, intracellular concentration and effects on glucocorticoid-mediated cell lysis. Biochim Biophys Acta 1054:49, 1990
18.Van den Bogert C, Dontje B, Melis T, Van der Veen C,
Kroon A: Inhibition of mitochondrial protein synthesis influences
the glucocorticoid sensitivity of lymphoid cell. Biochim Biophys
Acta 972:302, 1988
19. Van den Berg J, Smets LA, Van den Elshout M, VanGee1
I, Janssen M: Temperature dependence of glucocorticoid binding in
sensitive and refractory murine leukaemia cells. Leuk Res 17:263,
1993
20. Kluin-Nelemans HC, Limpens J, Meerabux J, Beverstock GC,
Jansen JH, de Jong D,Kluin PM: A newnon-Hodgkin’s B-cell
line (DoHH2) with a chromosomal translocation t(14; 18)(q32;q21).
Leukemia 5:221, 1991
21. Smets LA, Bout B, Brouwer M, Tulp A: Cytotoxic effects of
dexamethasone restricted to noncycling, early GI-phase cells of
L1210 leukemia. J Cell Physiol 116:397, 1983
22. Lundin A, Hasenson M, Persson J, Pouselle A: Estimation of
biomass in growing cell lines by adenosine triphosphate assay. Methods Enzymol 133:141, 1986
23. Pezzella F, Tse AG, Cordell JG, Pulford U, Gatter KC,
Mason DY: Expression of the bcl-2 oncogene protein is not specific
for the 14; 18 chromosomal translocation. Am J Pathol 137:225,
1990
24. Chomczynski P, Sacchi M: Single step method of RNA isolation by acid guanidinium thiocyanate phenol cloroform extraction.
Anal Biochem 162:156, 1987
25. Negrini M, Silini E, Kozak C, Tsujimoto Y, Croce CM: MOlecular analysis of mbcl-2: Structure and expression of the murine
gene homologous to
the
human gene involved in follicular
lymphoma. Cell 49:455, 1987
26. Loesberg C, Van Rooij H, Nooijen W, Meijer AJ, Smets LA:
Impaired mitochondrial respiration and stimulated glycolysis by mIodobenzylguanidine (MIBG). Int J Cancer 46:276, 1990
27. Baffy G, Miyashita T, Williamson JR, Reed JC: Apoptosis
induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intra-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
bcl-2 AND GLUCOCORTICOIDSENSITIVITY
cellular calcium and is blocked by enforced bcl-2 oncoprotein production. J Biol Chem 268:6511, 1993
28. Kane DC, Sarafian TA, Anton R, Hahn H, Gralla EB, Valentine JS, &d T, Bredesen DE: bcl-2 inhibition of neural death: Decreased generation of reactive oxygen species. Science 262:1274,
1993
29. Korsmeyer S: bcl-2 initiates a new category of oncogenes:
Regulators of cell death. Blood 80:879, 1992
30. Pezzella F, Turley H, Kuzu I, Tungekar MF, Dunnill MS,
Pierce CB, Harris A, Gatter KC, Mason DY: bcl-2 protein in nonsmall-cell lung carcinoma. New Engl J Med 329:690, 1993
31. Fanidi A, Harrington E, Evan G: Cooperative interaction between c-myc and bcl-2 proto-oncogenes. Nature 359:554, 1992
1619
32. Reed JC: bcl-2 and the regulation of programmed cell death.
J Cell Biol 124:1, 1994
33. Juedes MJ, Kass GE, Orrenius S : m-Iodobenzylguanidine increases the mitochondrial Ca2+pool in isolated hepatocytes. FEBS
Lett 313:39, 1992
34. Delia D, Aiello A, Soligo D, Fontanella E, Melani C, Pezzella
F, Pierotti MA, Della Porta G: bcl-2 proto-oncogene expression
in normal and neoplastic human myeloid cells. Blood 79:1291,
1992
35. Campos L, Rouault J-P, Sabido 0,Oriol P, Roubi N, Vasselon
C, Archimbaud E, Magaud J-P, Guyotat D: High expression of bcl2 protein in acute myeloid leukemia cells is associated with poor
prognosis to chemotherapy. Blood 81:3091, 1993
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1994 84: 1613-1619
BCL-2 expression and mitochondrial activity in leukemic cells with
different sensitivity to glucocorticoid-induced apoptosis
LA Smets, J Van den Berg, D Acton, B Top, H Van Rooij and M Verwijs-Janssen
Updated information and services can be found at:
http://www.bloodjournal.org/content/84/5/1613.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.