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Proteasome Inhibitors Induce Apoptosis in Glucocorticoid-Resistant Chronic
Lymphocytic Leukemic Lymphocytes
By Joya Chandra, Irina Niemer, Joyce Gilbreath, Kay-Oliver Kliche, Michael Andreeff, Emil J. Freireich,
Michael Keating, and David J. McConkey
Our previous work showed that the nuclear scaffold (NS)
protease is required for apoptosis of both thymocytes and
chronic lymphocytic leukemic (CLL) lymphocytes. Because
partial sequencing of one of the subunits of the NS protease
revealed homology to the proteasome, we tested the effects
of classical proteasome inhibitors on apoptosis in CLL cells.
Here we report that proteasome inhibition caused high
levels of DNA fragmentation in all patients analyzed, including those resistant to glucocorticoids or nucleoside analogs,
in vitro. Proteasome inhibitor-induced DNA fragmentation
was associated with activation of caspase/ICE family cysteine protease(s) and was blocked by the caspase antagonist,
zVADfmk. Analysis of the biochemical mechanisms involved
showed that proteasome inhibition resulted in mitochondrial dysregulation leading to the release of cytochrome c
and a drop in mitochondrial transmembrane potential (DC).
These changes were associated with inhibition of NFkB, a
proteasome-regulated transcription factor that has been
implicated in the suppression of apoptosis in other systems.
Together, our results suggest that drugs that target the
proteasome might be capable of bypassing resistance to
conventional chemotherapy in CLL.
r 1998 by The American Society of Hematology.
C
cytochrome c.7 Precisely how caspases promote the downstream features of apoptosis is not clear, but studies with
specific peptide-based active site inhibitors indicate that they
are required for all of the major biochemical events observed in
apoptotic cells, including changes in cellular morphology, loss
of plasma membrane asymmetry (exposure of phosphatidylserine on the outer leaflet), and DNA fragmentation.8
We and others have obtained evidence that certain noncaspase proteases are also required for DNA fragmentation and
apoptosis. Specifically, we have shown that peptide-based
active site inhibitors of a Ca21-dependent nuclear protease,
termed the nuclear scaffold (NS) protease, block glucocorticoid- and nucleoside analog–induced DNA fragmentation in
CLL lymphocytes.9 Although the molecular characteristics of
the NS protease are at present unclear, preliminary evidence
obtained by another laboratory10 suggests that it is structurally
and functionally related to the 26S multicatalytic protease
complex (MPC), otherwise known as the proteasome. This
possible similarity may explain why NS protease inhibitors
block DNA fragmentation, because previous studies have
implicated the proteasome in the programmed cell death of
intersegmental muscles in the moth, Manduca sexta,11 and more
recent work in isolated mouse thymocytes12 and neuronal cells13
has shown that proteasome inhibitors block caspase activation and
other downstream events associated with apoptosis in these cells.
The results presented above suggested to us that the effects of
NS protease inhibitors in CLL cells might be due to proteasome
inhibition. To directly address this possibility, we tested the
effects of several specific proteasome inhibitors on caspase
activation and DNA fragmentation in isolated CLL lymphocytes, expecting that they would suppress apoptotic cell death.
On the contrary, here we report that proteasome inhibition
resulted in extraordinarily high levels of DNA fragmentation in
all patient isolates analyzed, including those found to be
completely resistant to glucocorticoid-induced apoptosis. Analysis of the biochemical mechanisms involved showed that the
effects are linked to inhibition of NFkB, a transcription factor
implicated in the maintenance of cell survival in other model
systems.14-17
HRONIC LYMPHOCYTIC leukemia (CLL) is an illness
characterized by an accumulation of monoclonal mature
B cells in the peripheral blood. Although CLL is the most
common leukemia in the Western world, little is known about
the biology of the disease. Treatment schemes rely heavily on
glucocorticoids, chlorambucil, and nucleoside analogs, and we
and others have shown that all of these agents trigger apoptosis
in CLL cells in vitro, suggesting that induction of apoptosis may
account for their therapeutic efficacy. Furthermore, recent work
has shown that apoptosis in vitro correlates with Rai stage,1,2
and rates of apoptosis detected following fludarabine treatment
correlate with clinical response in vivo.3 However, despite the
initial effectiveness of these drugs in patients with low-grade
disease, resistant cells ultimately emerge, leaving no effective
treatment options available. It is possible that drug-resistant
CLL cells possess intrinsic defect(s) in their ability to undergo
apoptosis.
Protease activation is required for completion of the apoptotic program in all cellular and cell-free systems interrogated to
date.4,5 Of central importance are members of the ICE/caspase
family of aspartate-specific cysteine proteases, which appear to
function at the core of the ‘‘effector’’ machinery for cell death.
Caspase activation can either be directly promoted by oligomerization of certain caspase-associated cell surface ‘‘death’’
receptors (Fas, TNF-RI)6 or by intramitochondrial events that
lead to the release of the electron transport chain intermediate,
From the Departments of Cell Biology and Hematology, The
University of Texas, M.D. Anderson Cancer Center, Houston, TX.
Submitted February 6, 1998; accepted July 22, 1998.
Supported by grants from the National Institutes of Health (CA16672,
CA55164, CA49639) (to M.A.), Physicians’ Referral Service, MDACC,
the American Cancer Society (RPG-97-169-01-CDD) (to D.J.M.), and
an American Legion Auxiliary Fellowship (to J.C.).
Address reprint requests to David J. McConkey, PhD, Department of
Cell Biology - 173, U.T. M.D. Anderson Cancer Center, 1515 Holcombe
Blvd, Houston, TX 77030; email: [email protected].
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.
r 1998 by The American Society of Hematology.
0006-4971/98/9211-0023$3.00/0
4220
MATERIALS AND METHODS
Materials. The esterified peptide caspase inhibitor, Z-VAD
(OMe)fmk, the fluorigenic caspase substrate, DEVD-AMC, and the
Blood, Vol 92, No 11 (December 1), 1998: pp 4220-4229
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APOPTOSIS IN CLL BY PROTEASOME INHIBITION
mouse anti-PARP monoclonal antibody C2-10 were purchased from
Enzyme Systems Products, Inc (Dublin, CA). A peptide inhibitor of the
NS protease, Z-APFcmk, and the caspase antagonist, Boc-Aspchloromethylketone (BDcmk) were purchased from Bachem Bioscience (King of Prussia, PA). Monoclonal antibodies for caspase-3, p53,
p27, and c-Jun were purchased from Transduction Laboratories (Lexington, KY). A monoclonal antibody to c-Fos and the proteasome inhibitors
lactacystin and MG-132 were obtained from Calbiochem (San Diego,
CA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit
antibodies were from Amersham Corp (Arlington Heights, IL).
Patients, cell isolation, and incubation criteria. All patients fulfilled the National Cancer Institute’s (NCI) criteria for the diagnosis of
CLL. Some of the patients had received prior therapy, although none
within the last 6 months before experimentation. Immunophenotyping
by dual-parameter flow cytometry showed coexpression of CD5 with
B-cell antigen and isotypic light chain expression. Clinical staging was
based on the system described by Rai.18 Freshly isolated peripheral
blood was fractionated by Ficoll-Hypaque (Winthrop Pharmaceuticals,
New York, NY) sedimentation at 4°C. Nonadherent mononuclear cells
were then immediately suspended in complete RPMI 1640 medium
supplemented with 10% fetal calf serum (FCS), 10 mmol/L HEPES (pH
7.5), and antibiotics at a cellular concentration of 1 to 2 3 106 cells/mL.
Cell viability was assessed by Trypan blue exclusion and exceeded 95%
following the isolation procedure.
Granulocyte-colony stimulating factor (G-CSF)–mobilized progenitor cells were obtained from pheresis samples by magnetic cell sorting
(MACS). The pheresis samples were resuspended in 50 mL of cold
RPMI medium, and two ‘‘soft-spins’’ (200g, 10 minutes) were performed to remove platelets. Cells were labeled with anti-CD34 antibody
and isolated with a commercial CD34 isolation kit (Miltenyi Biotec,
Auburn, CA) according to the manufacturer’s instructions. A MACS
buffer consisting of Ca21/Mg21-free Hanks’ buffered salt solution
(HBSS) containing 0.6% ACD-A (Baxter, Deerfield, IL), 0.5% bovine
serum albumin (BSA; Sigma, St Louis, MO), pH 6.5, was used
throughout staining and separation to prevent cell clumping while
maintaining optimal progenitor viability. Cells were separated on
VS-positive selection columns using a VarioMACS according to the
manufacturer’s instructions. Cell purity was assessed by flow cytometry
using CD34-phycoerythrin (PE) and CD45-fluorescein isothiocyanate
(FITC) as described previously.19
DNA fragmentation analysis. Quantification of apoptosis by propidium iodide (PI) staining and fluorescence-activated cell sorting
(FACS) analysis was performed as described previously.20 Following
incubation with various agents in vitro, cells were pelleted by centrifugation and resuspended in phosphate-buffered saline (PBS) containing
50 µg/mL PI, 0.1% Triton X-100, and 0.1% sodium citrate. Samples
were stored at 4°C for 16 hours and vortexed before FACS analysis
(FL-3 channel).
Cytochrome c release measurements. Release of cytochrome c
from mitochondria was measured by immunoblotting essentially as
described previously.21 Cells were incubated in the absence or presence
of 10 µmol/L APFcmk, 10 µmol/L MG-132, or 10 µmol/L methylprednisolone for 4 hours, obtained by centrifugation, and gently lysed for 30
seconds in an ice-cold buffer containing 250 mmol/L sucrose, 1 mmol/L
EDTA, 0.1% digitonin, and 25 mmol/L Tris, pH 6.8. Lysates were
centrifuged for 2 minutes at 12,000g, supernatants were mixed with 23
Laemmli’s reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and extracts from equal numbers
of cells (10 to 20 3 106) were resolved by 15% SDS-PAGE.
Polypeptides were transferred to nitrocellulose membranes (0.2 µm;
Schleicher & Scheull, Keene, NH), and cytochrome c was detected by
immunoblotting with a monoclonal antibody (clone 7H8.2C12; purchased from Pharmingen, San Diego, CA).
4221
Caspase activity assay. Protease activity measurements were conducted as described previously.9 Cells were lysed in 1 mL of a buffer
containing 25 mmol/L HEPES (pH 7.4), 5 mmol/L EDTA, 2 mmol/L
dithiothreitol, and 10 µmol/L digitonin for 15 minutes on ice. The
lysates were clarified by centrifugation (12,000g), and supernatants
were incubated with 50 µmol/L Asp-Glu-Val-Asp-aminomethylcoumarin (DEVD-AMC; Enzyme Systems Products, Inc at 37°C in the dark.
Relative activities were then measured in a spectrofluorimeter (400 nm
excitation, 505 nm emission); blanks included supernatants processed
as outlined above without dye and supernatants preincubated with
BDcmk (25 µmol/L).
Mitochondrial membrane potential measurements. The potentialsensitive fluorochrome JC-1 (Molecular Probes, Eugene, OR) was used
to measure DCmito. Cells were obtained by centrifugation and incubated
with 10 µmol/L JC-1 for 15 minutes at 37°C in the dark. Cells were
washed in PBS and analyzed by FACS on the FL-2 channel (FACScan;
Becton Dickinson, Mountain View, CA).
Annexin V binding. Exposure of surface phosphatidylserine was
quantified by surface annexin V staining as described previously.22 This
assay was used as a DNA fragmentation-independent endpoint to
confirm the involvement of apoptosis in the mechanism of cell death.
Cells were resuspended in binding buffer containing 1 µg/mL FITCconjugated annexin V (Nexins Research B.V., Hoeven, The Netherlands) and incubated for 30 minutes at 4°C, and cells were analyzed by
flow cytometry (FACScan, Becton Dickinson).
Immunoblotting. For detection of caspase-3, p53, p27, Fos, and
Jun, cells were lysed for 1 hour at 4°C in a buffer containing 150
mmol/L NaCl, 1% Triton X-100, a cocktail of protease inhibitors
(Complete Mini tablets; Boehringer-Mannheim, Indianapolis, IN), and
25 mmol/L Tris (pH 7.5). Debris was sedimented by centrifugation for 5
minutes at 12,000g, and the supernatants were solubilized for 5 minutes
at 100°C in Laemmli’s SDS-PAGE sample buffer containing 100
mmol/L dithiothreitol.
For analysis of PARP cleavage samples were denatured in a urea/SDS
buffer as follows: cells were incubated with various agents for 16 hours,
obtained by centrifugation, and resuspended in 25 µL ice-cold PBS.
Cells were subsequently disrupted by addition of 100 µL of a buffer
containing 6 mol/L urea, 2% SDS, 10% glycerol, 5 mmol/L EDTA, 5%
2-mercaptoethanol, and 100 mmol/L Tris (pH 6.8) followed by pipeting
through a 1-mL Pipetman tip, and samples were sonicated for 20
seconds at high power to sheer DNA. Samples were then incubated for
15 minutes at 65°C and centrifuged for 2 minutes at 12,000g before they
were loaded onto 8% SDS-PAGE gels.
Polypeptides were resolved at 100 V on 8% to 12% gels and
electrophoretically transferred to 0.2-µm nitrocellulose membranes
(Schleicher & Schuell Inc) for 1 hour at 100 V. Membranes were
blocked for 1 hour a TBS-T buffer (25 mmol/L Tris, pH 8.0, 150
mmol/L NaCl, and 0.05% Tween-20) containing 3% (wt/vol) nonfat
dried milk. Blots were then probed overnight with primary antibody and
developed using a horseradish peroxidase-coupled anti-mouse secondary antibody by enhanced chemiluminescence (Supersignal; Pierce
Chemical Co, Rockford, IL) according to the manufacturer’s instructions.
Electrophoretic mobility shift (EMSA) assays. Isolated nuclei were
prepared by lysis with Triton X-100 and centrifugation through a
glycerol cushion as described previously.23 Nuclear protein was extracted using a high salt, detergent-free buffer containing 20 mmol/L
HEPES (pH 7.9), 400 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L
EGTA, 1 mmol/L dithiothreitol, and 1 mmol/L phenylmethylsulfonyl
fluoride, for at least 20 minutes on ice. Extracts were centrifuged at 4°C
for 5 minutes at 12,000g, and protein content in supernatants was
measured by the Bradford method. A consensus double-stranded NFkB
probe (obtained from Promega Inc, Madison, WI) was end-labeled
using T4 polynucleotide kinase and g-32P-ATP. Five to 20 µg of nuclear
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4222
CHANDRA ET AL
Table 1. Effects of Proteasome Inhibitor on DNA Fragmentation
in CLL Patient Isolates
Patient
Number
Control*
MPS
Table 1. Effects of Proteasome Inhibitor on DNA Fragmentation
in CLL Patient Isolates (Cont’d)
MG132
Spontaneously
apoptotic isolates
Patient
Number
Control*
MPS
MG132
128
129
130
136
138
141
143
164
165
187
215
223
240
241
242
243
244
245
248
249
253
Mean(SD)
3.7
4.3
4.1
4.8
19.3
5.2
0.9
0.7
0.7
1.8
12.8
6.8
8
3.2
1.3
0.9
1.9
3.1
2.3
0.9
7.2
4 (4)
11
3.3
10.8
16.8
16.3
13
0.7
7
1.8
4.3
22.2
9.9
6.9
19.4
3.1
7.8
15.9
8.6
3.4
3.7
17.3
10 (6)
73.6
33.3
89
89.4
59.8
96.9
36.3
74.7
64.2
55
90.6
87.7
83
94.3
82
75.2
94.5
45.6
75
67.8
90.5
74 (19)
Glucocorticoidresistant isolates
156
160
180
213
227
228
230
232
233
235
Mean(SD)
46.1
27.4
89.8
24.9
37.9
51.2
32.4
25.6
29.5
38.5
40 (19)
60.5
34.1
90.1
48.5
49.8
71.2
57.7
37.8
31.8
57.6
54 (18)
87.3
82.7
96.1
70.1
93.8
97.3
90.4
91.2
72.8
89.9
87 (9)
131
132
134
135
137
140
144
146
147
148
149
154
155
157
188
209
210
211
212
214
218
220
222
229
238
247
250
251
Mean(SD)
0.7
3.7
8.9
15.7
11.2
2.4
4.6
13.4
1.4
10
1.6
4.1
16.7
8.7
3.3
8
3.7
8.7
13.8
3.1
2.3
7.6
2.5
10.7
5.2
3
18.5
16.1
7 (5)
66.3
51
23.4
90.6
37.9
24.3
34.1
31
19.6
49.4
30.1
99.4
36.9
33.3
33.3
34.8
37.1
48.9
41.8
20.4
23.5
30.3
30.5
44.9
31.7
44.8
33.2
61.6
41 (19)
99.5
95.4
99.6
88.8
88.6
90.4
50.9
78.5
92.5
95.2
99
98.3
99.8
55.3
36.8
95.7
88.9
98.6
89.3
92.9
96.7
95
80.1
77.2
92.2
95.7
82.7
82.9
87 (15)
Glucocorticoidsensitive isolates
extract was then incubated in binding buffer (supplied by the manufacturer) containing 1 µg/mL poly dI:dC (Promega), ensuring that the final
salt concentration was between 50 and 100 mmol/L. Reactions were
incubated for 20 minutes at room temperature, and 1 µL of end-labeled
probe was added. Samples were incubated for 30 minutes before
addition of loading buffer (Promega) and electrophoresis on 4%
nondenaturing polyacrylamide gels that were prerun in 0.53Tris-borateEDTA (TBE) buffer for 30 minutes at 100 V before use. Gels were run at
100 V in 0.53 TBE and dried, and DNA-protein complexes were
detected by autoradiography.
Statistical analyses. Mean values and standard deviations were
calculated with Microsoft Excel (Microsoft Inc, Redmond, WA).
Significance was evaluated using two-tailed paired Student’s t-tests
with SPSS software (SPSS Inc, Chicago, IL).
Apoptosis was quantified by PI staining and FACS analysis after 16
hours of culture as described in Materials and Methods. Spontaneously apoptotic isolates were arbitrarily defined as those exhibiting
greater than 20% DNA fragmentation in control (untreated) cultures.
Glucocorticoid-sensitive isolates were arbitrarily defined as those
exhibiting greater than 20% increase in DNA fragmentation relative to
controls following exposure to steroid.
Abbreviation: MPS, methylprednisolone.
*For spontaneously apoptotic isolates, 16-hour control was used.
RESULTS
Effects of proteasome inhibitor on DNA fragmentation and
surface exposure of phosphatidylserine. In a previous study
we showed that a specific inhibitor of the NS protease completely suppressed apoptosis-associated DNA fragmentation in
CLL cells treated with glucocorticoid or the nucleoside analog,
fludarabine.9 Because proteasome inhibitors block apoptosis in
thymocytes and neuronal cells,12,13 and another group has
reported that the NS protease is homologous to the proteasome,10 we tested the effects of proteasome inhibitors on DNA
fragmentation, measured by PI staining and FACS analysis, in
CLL cells to determine whether the effects of zAPFcmk might
be attributed to the proteasome. Levels were compared with
those observed in response to treatment with glucocorticoid
hormone. Our patient isolates fell into three general catetories:
(1) those exhibiting relatively high (mean 5 40%, n 5 10)
levels of apoptosis upon in vitro culture in the absence of
hormone (‘‘spontaneous’’); (2) those exhibiting low spontaneous apoptosis but strong (mean 5 40%, n 5 28) increases in
DNA fragmentation in response to glucocorticoid treatment
(‘‘sensitive’’); and (3) those exhibiting low spontaneous apoptosis and low levels of glucocorticoid-induced DNA fragmentation (mean 5 10%, n 5 21) (‘‘resistant’’; Table 1). Strikingly,
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APOPTOSIS IN CLL BY PROTEASOME INHIBITION
4223
Fig 1. Induction of apoptosis by proteasome inhibition in CLL patient isolates. (A) DNA fragmentation analysis. Cells from a representative
glucocorticoid-sensitive patient (see Table 1) were preincubated in the presence of 25 mmol/L zAPFcmk or 200 mmol/L zVADfmk for 1 hour and
then treated with 10 mmol/L MG132, and DNA fragmentation was measured at 16 hours by PI staining and FACS analysis. (B) Surface
phosphatidylserine exposure. Cells were incubated in absence or presence of 10 mmol/L methylprednisolone or 10 mmol/L MG132 with or
without 200 mmol/L zVADfmk, and surface PS exposure was quantitated by staining with annexin-FITC and measured by FACS analysis. Results
characteristic of three independent experiments with different patient isolates.
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4224
and contrary to our expectations, treatment with MG-132, a
peptide-based proteasome antagonist, promoted high levels of
DNA fragmentation in all three categories of cells (Table 1).
Proteasome inhibitors were effective in all patient isolates
analyzed (n 5 59). Similar results were obtained with another,
structurally distinct proteasome inhibitor, lactacystin (data not
shown). Proteasome inhibitors also induced surface phosphatidylserine exposure, another downstream event in apoptosis that
is thought to be independent of endonuclease activation (Fig
1B). Importantly, preincubation with zAPFcmk blocked MG132–induced DNA fragmentation (Fig 1A), indicating that these
inhibitors exert their effects on different biochemical activities.
Effects of MG-132 on DNA fragmentation in normal hematopoietic cells. In a preliminary attempt to determine whether
MG-132’s proapoptotic effects were selective for CLL cells, we
analyzed the effects of the proteasome antagonist on DNA
fragmentation in normal peripheral blood lymphocytes and in
G-CSF–mobilized CD341CD451 hematopoietic progenitor cells.
Normal lymphocytes were killed by the compound, but the
kinetics of the response were markedly delayed and the
maximal response shifted from 16 hours to 48 hours (Fig 2A).
Surface staining with specific B- and T-cell markers indicated
that MG-132 was substantially more toxic to normal T cells
than to B cells (data not shown). In contrast to the attenuated
responses of lymphocytes, mobilized stem cells were highly
sensitive to MG-132 (Fig 2B). Thus, proteasome inhibitors are
capable of inducing apoptosis in certain normal as well as
transformed hematopoietic cells.
Effects of proteasome inhibition on caspase activation.
Caspases are a family of cysteine proteases that are thought to
act at the core of the apoptotic pathway. Our previous work9 and
that of others24,25 has confirmed that caspases are required for
drug-induced apoptosis in CLL cells. We therefore investigated
whether or not caspases were also required for apoptosis
induced by proteasome inhibitors by four independent approaches. First, induction of apoptosis by proteasome inhibitors
was also associated with specific cleavage of the caspase
substrate, PARP, as detected by immunoblotting (Fig 3A). This
occurred in all patient samples analyzed (n 5 4), including one
that did not respond to glucocorticoid treatment (Fig 3A).
Second, proteasome inhibitors promoted hydrolysis of a specific caspase substrate (DEVD-AMC; Fig 3B). Third, proteasome inhibitors induced proteolytic processing of the inactive
form of caspase-3 (procaspase-3; Fig 3C), providing more
direct evidence for activation of caspase-3 and presumably
other caspases in the response. Finally, the caspase inhibitor
zVADfmk completely blocked MG-132–induced DNA fragmentation (Fig 1A) and surface exposure of phosphatidylserine (Fig
1B), confirming that caspase activation was required for
proteasome inhibitor–induced apoptosis.
Effects of proteasome inhibitor on mitochondrial function.
Disruption of mitochondria leading to the release of the electron
transport chain intermediate, cytochrome c, has recently been
implicated in caspase activation in other model systems.26
Although the mechanisms underlying cytochrome c release are
unclear, the event is associated with a drop in transmembrane
potential (DC), which may facilitate the opening of transmembrane pores in the mitochondrial membrane that would allow
CHANDRA ET AL
passage of cytochrome c and other proapoptotic factors from
the organelle.27 To determine whether this pathway of caspase
activation was induced by proteasome inhibitors in CLL cells,
we measured the effects of MG-132 on cytochrome c release in
digitonin-permeabilized cells. MG-132 promoted rapid release
of cytochrome c from mitochondria in intact CLL cells (Fig 4,
lane 3), effects that were also observed in cells treated with
zAPFcmk (Fig 4, lane 4) and to a lesser extent with glucocorticoid (Fig 4, lane 5). At this time point, control cells did not
exhibit either increased levels of cytosolic cytochrome c (Fig 4,
compare lanes 1 and 2) or significant caspase activation (Fig
3B). The effects of zAPFcmk on cytochrome c release are
consistent with earlier experments that showed that NS protease
inhibition results in caspase activation9 (Fig 3). Proteasome
inhibition also resulted in a drop in mitochondrial membrane
potential, measured with the potential-sensitive dye, JC-1 (Fig 5).
These results indicate that proteasome inhibitors promote
caspase activation via direct or indirect effects on mitochondria.
The proteasome is known to degrade many proteins implicated in the control of cell survival, including p53,28 Fos,29
Jun,30 Myc,31 p27,32 and IkBa33 (a protein inhibitor of the
transcription factor NFkB34). We did not observe any detectable
alteration in the levels of p53, Fos, Jun, Myc, or p27 in CLL
cells treated with MG-132 compared with cells incubated in
medium alone (data not shown), suggesting that these polypeptides may not participate in proteasome inhibitor-induced
apoptosis in this system. On the other hand, quantification of
NFkB activity by EMSA revealed that both zAPFcmk and
MG-132 drastically reduced the levels of active NFkB in
isolated nuclei from CLL cells (Fig 6). Thus, blockade of the
NFkB survival pathway may be responsible for triggering the
disruption of mitochondrial function and caspase activation in
these cells.
DISCUSSION
In spite of the development of nucleoside analogs (fludarabine
and cladribine) that have led to much better management of
disease burden in CLL patients, CLL cells ultimately develop
resistance to all currently available therapies, possibly because
of apoptosis suppression. Our data show that the proteasome
controls a central step in the maintenance of cell survival in
CLL cells, such that inhibitors are capable of inducing apoptosis
in all of them. The results support and extend independent work
recently published by another group, who reported that the
proteasome inhibitor lactacystin can promote radiation- and
tumor necrosis factor (TNF)-induced apoptosis in CLL cells.35
The mechanism underlying the responses involves mitochondrial alterations leading to the release of cytochrome c and loss
of mitochondrial membrane potential. The mitochondrial alterations are associated with caspase protease activation, as
measured by specific cleavage of hallmark endogenous (PARP)
and exogenous (DEVD-AMC) caspase substrates and proteolytic processing of procaspase-3. It is encouraging that we were
not able to identify a single patient isolate exhibiting de novo
resistance to proteasome inhibition among a fairly large (n 5
59) panel, some of which (n 5 21) showed marked resistance to
glucocorticoid-induced apoptosis. However, our work does not
address the issue of whether or not CLL cells can develop
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Fig 2. Effect of proteasome inhibitors on normal lymphocytes. (A) Effects on normal lymphocytes. Isolated peripheral blood lymphocytes
from normal donors were incubated in the presence of 10 mmol/L MG132 or 10 mmol/L methylprednisolone with or without 25 mmol/L zAPFcmk
for 16 hours, and apoptosis was assessed by PI staining and FACS analysis. Results of one experiment are representative of three independent
replicates. (B) Effects on hematopoietic progenitor cells. G-CSF–mobilized CD341CD451 progenitor cells were incubated in the absence or
presence of 25 mmol/L zAPFcmk or 10 mmol/L MG-132 and DNA fragmentation was measured after 16 hours by PI staining and FACS analysis.
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4226
CHANDRA ET AL
Fig 3. Caspase activation by proteasome inhibitors. (A) Cleavage of the caspase substrate, PARP, by treatment with proteasome inhibitor.
Cells were treated with either 10 mmol/L methylprednisolone, 25 mmol/L zAPFcmk, or various doses of MG132. PARP was detected by
immunoblotting. Intact (p116) and fragmented (p85) forms of PARP are indicated by arrows. (B) Effect of MG132 on DEVDase activity. Cells were
treated in the absence or presence of 10 mmol/L MG132 for 8 hours, and hydrolysis of the caspase substrate DEVD-AMC was measured in a
spectrofluorimeter. Results are from two experiments with independent patient isolates. Cells treated with 10 mmol/L BDcmk, a caspase
inhibitor, did not show DEVDase activity more than 50 U above baseline levels. DNA fragmentation from these patients was measured in parallel.
These patients are not included in Table 1. Patient no. 1: control 5 18.0, MG132 5 96.9; patient no. 2: control 5 1.4, MG132 5 32.5. (C) Activation of
caspase-3 by proteasome inhibitor. Cells were treated with either 25 mmol/L zAPFcmk, 10 mmol/L MG132, or 10 mmol/L methylprednisolone for
16 hours and procaspase-3 was detected by immunoblotting. Results of one experiment representative of three replicates with independent
patient isolates.
resistance to these agents under other conditions. Elimination of
proteasome function in yeast results in lethality,36 but recent
work suggests that mammalian cells contain another protease
complex that can compensate for loss of proteasome function in
cells chronically exposed to proteasome inhibitors.37
Although the precise mechanism(s) precipitating the mitochondrial changes await further investigation, our preliminary
efforts indicate that the effects of MG-132 are tightly linked to
suppression of NFkB activity and not to stabilization of several
other proteasome-regulated factors (p53, Fos, Jun, p27) that
have been implicated in the control of apoptosis in other
systems. Independent results obtained recently by another
group support the idea that suppression of NFkB leads to
apoptosis in CLL.35 The principal mechanism regulating NFkB
activation involves an inhibitor protein, IkBa, that binds to
NFkB and prevents its translocation to the nucleus.38 Stimulation of cells with NFkB-activating signals results in phosphorylation of IkBa and its coupling to ubiquitin,33 a small (8 kD)
polypeptide that forms polymers that serve to target proteins for
destruction by the proteasome.39 In other B-cell model systems,
constitutive NFkB activity is essential for cell survival40 and
inhibition of NFkB using protease inhibitors or mutant forms of
IkBa that cannot be degraded by the proteasome facilitates
apoptosis in a number of different cell types.15-17 Interestingly,
the immunosuppressive effects of glucocorticoid hormones are
linked to an inhibition of NFkB activity,41-44 suggesting that
suppression of NFkB may also be required for glucocorticoidinduced apoptosis. Our ongoing efforts are focused on further
Fig 4. Proteasome inhibition leads to release of cytochrome c
from mitochondria. Cells were incubated in the absence (control) or
presence of 10 mmol/L MG132, 25 mmol/L zAPFcmk, or 10 mmol/L
methylprednisolone for 6 hours, and cytosolic cytochrome c was
measured in digitonin-permeabilized cells by immunoblotting. Lane
1, 0 hours control; lane 2, 6 hours control; lane 3, MG132; lane 4,
zAPFcmk; lane 5, methylprednisolone. Results are typical of three
independent experiments with different CLL isolates.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
APOPTOSIS IN CLL BY PROTEASOME INHIBITION
4227
Fig 5. Effects of proteasome inhibition on mitochondrial membrane potential. Cells were incubated in the absence or presence of 10 mmol/L
methylprednisolone with or without 25 mmol/L zAPFcmk or 10 mmol/L MG132. Mitochondrial membrane potential was assessed by the
potential sensitive fluorochrome JC-1 and quantitated by FACS analysis.
characterizing the role of NFkB in the maintenance of survival
in CLL.
Even though some studies had shown that proteasome
inhibitors trigger apoptosis in tumor cell lines,45,46 the observation that proteasome inhibition results in apoptosis in CLL was
surprising to us. Proteasome inhibitors block glucocorticoidinduced apoptosis in immature thymocytes12 and the death of
neuronal cells deprived of neurotrophins,13 and ubiquitindependent pathways appear involved in developmentally regulated cell death in the hawkmoth Manduca sexta and in
radiation-induced apoptosis in tumor cells.47 Furthermore, we
had shown that inhibitors of the NS protease, a putative
proteasome homolog, completely block DNA fragmentation in
CLL isolates.9 However, NS protease inhibitors did promote
several other features of apoptosis, including caspase activation,
mitochondrial dysregulation, and PS exposure, suggesting that
their effects did overlap.9 Interestingly, a peptide inhibitor of the
protease complex that can compensate for loss of proteasome
function (AAFcmk)37 is very similar in sequence to our NS
protease inhibitor (APFcmk), both of which contain a phenylalanine (F) residue at the critical P1 position. In addition, although
MG132 failed to promote substantial accumulation of p53 in
CLL cells, zAPFcmk consistently did (D.J. McConkey, unpublished observations, April 1998), and the effects of the inhibitors
on NFkB activity are similar (Fig 6). We are currently isolating
the NS protease complex, and a detailed analysis of its structure
and biochemical regulation will reveal how it is related (if at all)
to the proteasome.
The proteasome is central to normal cell physiology and
hence complete inhibition of its activity is ultimately cytotoxic.
However, appropriate titration of proteasome activity can elicit
significant efficacy with limited side effects (Peter Elliot, Julian
Adams, Proscript Inc, personal communication, April 1998).
Indeed, the present report shows that proteasome inhibitors
induce marked apoptosis in mobilized hematopoietic progenitor
cells with more modest effects on normal lymphocytes. Moreover, extensive preclinical profiling of such compounds has
clearly shown that maximum tolerated doses have only modest
myelosuppressive activity (P. Elliot, J. Adams, personal communication). It is likely that progenitor cell mobilization and
probably other manipulations that induce cell cycling will in
general sensitize cells to proteasome inhibitor-induced apoptosis, as previous work suggests that proliferating cells are more
sensitive to their effects than are postmitotic cells.48 Additionally, the proteasome inhibitor, PS-341, has been shown to
possess antitumor activity of its own and this effect is enhanced
when combined with other chemotherapeutics (P. Elliot, J.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
4228
CHANDRA ET AL
ACKNOWLEDGMENT
The authors thank Virginia Snell for providing the purified hematopoietic stem cells, Yuko Miyamoto for purified peripheral lymphocytes,
and Julian Adams and Peter Elliot (Proscript Inc, Cambridge, MA) for
sharing preliminary data on PS-341.
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Fig 6. Inhibition of NFkB activity by NS protease and proteasome
inhibitors. Cells were incubated for 6 hours in the absence (control) or
presence of 25 mmol/L zAPFcmk or 10 mmol/L MG132, and NFkB
activity was measured in isolated nuclear extracts by EMSA using an
NFkB consensus element DNA probe. Lane 1, control extracts with
excess unlabeled probe (specificity control); lane 2, 6 hours control;
lane 3, zAPFcmk; lane 4, MG-132. Results of one experiment typical of
over 20 independent replicates.
Adams, personal communication). With the advent of acceptable phase I safety data, the present results would strongly argue
that PS-341 should be evaluated in patients with refractory
CLL.
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1998 92: 4220-4229
Proteasome Inhibitors Induce Apoptosis in Glucocorticoid-Resistant
Chronic Lymphocytic Leukemic Lymphocytes
Joya Chandra, Irina Niemer, Joyce Gilbreath, Kay-Oliver Kliche, Michael Andreeff, Emil J. Freireich,
Michael Keating and David J. McConkey
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