From The Jerome Lipper® Multiple Myeloma Center,
Department of Adult Oncology, Dana Farber Cancer Institute, Harvard
Medical School, Boston, Massachusetts 02115, the
§ Northwestern University Medical School, Robert H. Lurie
Cancer Center, Chicago, Illinois 60611, and ¶ The Burnham
Institute, La Jolla, California 92037
Received for publication, February 6, 2001, and in revised form, May 11, 2001
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ABSTRACT |
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Smac, a second mitochondria-derived activator of
caspases, promotes caspase activation in the cytochrome c
(cyto-c)/Apaf-1/caspase-9 pathway. Here, we show that
treatment of multiple myeloma (MM) cells with dexamethasone (Dex)
triggers the release of Smac from mitochondria to cytosol and activates
caspase-9 without concurrent release of cyto-c and Apaf-1
oligomerization. Smac binds to XIAP (an inhibitor of apoptosis protein)
and thereby, at least in part, eliminates its inhibitory effect on
caspase-9. Interleukin-6, a growth factor for MM, blocks
Dex-induced apoptosis and prevents release of Smac. Taken together,
these findings demonstrate that Smac plays a functional role in
mediating Dex-induced caspase-9 activation and apoptosis in MM cells.
The cellular response to diverse classes of stress inducers
includes growth arrest and activation of apoptosis. Apoptosis is
triggered through a controlled program that is associated with distinctive morphological changes, including membrane blebbing, cytoplasmic and nuclear condensation, chromatin aggregation, and formation of apoptotic bodies (1). The induction of apoptosis involves
a cascade of initiator and effector caspases that are activated
sequentially (2, 3). Caspases, a family of cysteine proteases with
aspartate substrate specificity, are present in cells as catalytically
inactive zymogens (2). Effector caspases, such as caspase-3, are
activated by initiator caspases, such as caspase-9. Once activated, the
effector caspases induce proteolytic cleavage of various cellular
targets, inducing poly(ADP-ribose) polymerase (4, 5),
DNA-dependent protein kinase, protein kinase C- Recent studies have shown that the inhibitor of apoptosis
(IAP)1 family of proteins
suppresses apoptosis by directly binding to and inhibiting caspases (7,
8). For example, XIAP, c-IAP-1, and c-IAP-2 bind to procaspase-9 and
prevent its activation (9), thereby blocking the downstream
apoptosis-related events such as proteolytic cleavage of caspase-3, -6, and -7 (10).
One of the major caspase cascades is triggered by the release of
mitochondrial apoptogenic protein, cytochrome c
(cyto-c) (11-13). Cytosolic cyto-c binds to the
CED-4 homolog Apaf-1 and induces caspase-9-dependent
activation of caspase-3 (14-17). Recent studies have identified
another important regulator of apoptosis, Smac (second
mitochondria-derived activator of caspase) or DIABLO, which is released
from mitochondria into the cytosol during apoptosis (18-20) and
functions by eliminating inhibitory effects of IAPs on caspases (20,
21).
Our prior study demonstrated that dexamethasone (Dex)-induced apoptosis
is independent of cyto-c release and associated with caspase-3 activation (22, 23). In the present study, we examined the
upstream signaling leading to caspase-3 activation. The results demonstrate that Dex-induced apoptosis in MM cells is mediated by Smac,
which activates caspase-9 by binding to and inhibiting XIAP.
Interleukin-6 (IL-6), a growth factor for MM, blocks Dex-induced release of Smac and apoptosis. Taken together, this study provides evidence for an Apaf-1-cyto-c-independent pathway mediating
caspase-9 activation via Smac. Moreover, these findings also
demonstrate a functional role of Smac in IL-6-mediated block during
Dex-induced apoptosis.
Cell Culture and Reagents--
Human MM.1S (Dex-sensitive) and
MM.1R (Dex-resistant) multiple myeloma cells (22, 24) were grown in
RPMI 1640 media supplemented with 10% heat-inactivated fetal
bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Mononuclear cells were isolated
from a patient with MM (PCL cells) by Ficoll-Hypaque density gradient
centrifugation and incubated with HB-7 (anti-CD38) mAb-biotin-streptavidin and 2H4 (anti-CD45RA)
mAb-fluorescein isothiocyanate on ice. Tumor cells (96 + 2%
CD38+45RA-) were isolated using an Epics C cell sorter (Coulter
Electronics, Hialeah, FL), washed, and resuspended in regular
growth media. Cells were treated with 10 µM Dex
(Sigma) in the presence or absence of 100 ng/ml of IL-6.
Preparation of Cytosolic and Mitochondrial Extracts from MM.1S,
MM.1R, and MM Patient Cells--
MM.1S or PCL cells were washed twice
with PBS, and the pellet was suspended in 3 volumes of ice-cold buffer
A (20 mM HEPES, pH 7.5, 1.5 mM
MgCl2, 10 mM KCl, 1 mM EDTA,
1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin and aprotinin and pepstatin A) containing 250 nM
sucrose. The cells were homogenized using a Dounce homogenizer, and
cytosolic or mitochondrial extracts were isolated as described
previously (12, 25).
Western Blot Analysis--
Proteins were separated from cell
lysates by SDS-PAGE, transferred to nitrocellulose, and probed with
anti-cyto-c (22), anti-Smac (kindly provided by Dr. Xiaodong
Wang), anti-tubulin (Sigma), anti-Hsp60 (Stressgen, Victoria,
British Columbia, Canada), anti-XIAP (Transduction Laboratories), as
well as anti-caspase-9, anti-casapase-8, and anti-caspase-3
(PharMingen) Abs. The blots were developed by enhanced
chemiluminescence (ECL) using the manufacturer's protocol (Amersham
Pharmacia Biotech).
Transient Transfections--
MM.1S cells were transiently
cotransfected with FLAG-Apaf-1 and T7-Apaf-1 using
SuperfectTM (Qiagen, Santa Clarita, CA) and treated with 10 µM Dex for 24 h. Lysates from transfectants were
then incubated with dATP and subjected to immunoprecipitation with
anti-FLAG M2 (Eastman Kodak Co.). The immunoprecipitates were
then analyzed by immunoblotting with anti-T7 (Novegen, Madison, WI) or
anti-FLAG. MM.1S cells were also transiently transfected with
pcDNA3-Myc-XIAP vector (26). Lysates were subjected to
immunoprecipitation with anti-Myc (Santa Cruz Biotechnology, Santa
Cruz, CA), and the immunoprecipitates were then analyzed by
immunoblotting with anti-caspase-9, anti-Smac, or anti-XAIP antibodies.
Caspase Activity Assays--
Caspase-9 activation was performed
using LEHD-pNA as a substrate, as per the manufacturer's instructions
(colorimetric assay kit, Biovision, Palo Alto, CA). MM.1S MM cells were
also treated with Dex (10 µM) in the presence or absence
of caspase-9 inhibitor LEHD-FMK (5 µM) for 24 h and
then analyzed for apoptosis.
Quantification of Apoptosis--
Flow cytometric analyses: dual
fluorescence staining with DNA-binding fluorochromes Hoechst 33342 (HO)
and propidium iodide (PI) was used to quantitate the percentage of
apoptotic (HO+PI To determine whether Dex-induced apoptosis in MM cells is
associated with the release of Smac, MM.1S MM cells were treated with
Dex for various times, and cytosolic and mitochondrial extracts were
analyzed for the levels of Smac. The results demonstrate that Dex
treatment is associated with an increase in Smac levels in the cytosol
at 24 and 48 h, with a concomitant decrease in mitochondrial Smac
levels (Fig. 1A). Dex-induced
increase in cytosolic Smac and a corresponding decrease in
mitochondrial Smac levels were specific, because no change was observed
in the levels of tubulin protein and mitochondrial matrix protein,
Hsp60, respectively (Fig. 1A). Similar results were obtained
when patient MM cells were exposed to Dex (Fig. 1B). These
results suggest that Dex-induced apoptosis is accompanied by
accumulation of Smac in the cytosol.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
,
and other substrates (6), ultimately leading to cell death.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-Radiation (IR) was performed as described previously (22). Cells
were also treated with anti-Fas as described previously (22).
) cells using flow cytometry (The Vantage,
Becton Dickinson), as described previously (24). DNA fragmentation
assays were also performed as described previously (24)
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Dex induces the release of Smac in the
cytosol. MM.1S (A) or MM patient cells (PCL)
(B) were treated with 10 µM Dex and harvested
at the indicated times. Cytosolic and mitochondrial extracts were
separated by 12.5% SDS-PAGE and analyzed by immunoblotting with
anti-Smac (cytosolic Smac, first panels; mitochondrial Smac,
third panels). As a control, filters were also analyzed by
immunoblotting (IB) with anti-tubulin (second
panels) or with anti-Hsp-60 (fourth panels).
C, MM.1S cells were treated with Dex or IR and harvested at
the indicated times. Cytosolic extracts were separated by 12.5%
SDS-PAGE and analyzed by IB with anti-cyto-c (upper
panel), anti-Smac (middle panel), or anti-tubulin
(lower panel). D, effects of Dex or IR on Apaf-1
oligomerization. MM.1S cells were transiently transfected with
FLAG-Apaf-1 (lanes 2 and 3) and T7-Apaf-1
(lanes 1-3) and treated with Dex (lane 2) or IR
(lane 3). Cytosolic extracts were subjected to
immunoprecipitation with anti-FLAG. The immunoprecipitates were then
analyzed by IB with anti-T7 (upper panel) or anti-FLAG
(lower panel).
Smac is known to promote caspase activation in the
cyto-c/Apaf-1/caspase-9 pathway; therefore, we next examined
the release of cyto-c triggered by Dex or IR in MM.1S MM
cells. The cytosolic extracts from Dex- or IR-treated cells were
subjected to immunoblot analyses with anti-cyto-c and
anti-Smac. As in our previous findings (22), treatment of MM.1S MM
cells with Dex did not induce release of cyto-c in the
cytosol (Fig. 1C, upper panel); in contrast, -radiation (IR) stimulated the release of cyto-c (Fig.
1C, upper panel), demonstrating that the release
cyto-c is functional in these cells. To assay for Smac
release these immunoblots were then stripped and reprobed with
anti-Smac. As seen in Fig. 1C, both Dex and IR induced
release of Smac in the cytosol. Furthermore, low to undetectable
cytosolic Smac or cyto-c levels were observed in the
untreated cells. Reprobing the immunoblots with anti-tubulin confirms
equal protein loading (Fig. 1C).
Since Dex-induced apoptosis is associated with Smac release, but not cyto-c release, we next determined whether Apaf-1 oligomerization is required for Smac-related signaling. For these experiments, we utilized the same MM.1S MM cells model (22) to determine whether IR or Dex induce Apaf-1 oligomerization. Cells were transiently cotransfected with FLAG-Apaf-1 and T7-Apaf-1 or empty vector and treated with Dex or IR. Cell lysates were incubated with dATP. As shown in Fig. 1D, IR (lane 3), but not Dex (lane 2), induces Apaf-1 oligomerization in MM.1S MM cells (Fig. 1D, lane 3). The finding that IR induces Apaf-1 oligomerization in MM.1S MM cells indicates that the Apaf-1 oligomerization system is functionally intact and served as a positive control. Taken together, these findings suggest that Dex-induced apoptosis in MM cells is mediated by Smac and is independent of cyto-c/Apaf-1 mechanism.
To examine whether Dex-induced Smac release and apoptosis are
associated with processing of caspase-9, the cytosolic extracts from
Dex-treated cells were subjected to immunoblot analysis with anti-caspase-9. The results demonstrate that treatment of MM.1S cells
with Dex induces proteolytic cleavage of procaspase-9 into 37- and
35-kDa fragments (Fig. 2A,
upper panel). Reprobing the immunoblot with anti-tubulin
confirms equal protein loading (Fig. 2A, lower
panel). We next assayed for catalytic activity of caspase-9 using
LEHD-pNA conjugated substrate in a colorimetric protease assays (26).
Incubation of cytosolic extracts from Dex-treated MM.1S cells with
LEHD-pNA was associated with efficient cleavage of LEHD-pNA (Fig.
2B). Although LEHD-pNA may be cleaved by caspases other than
caspase-9, our study indicates that activated caspase-8 in MM.1S MM
cells does not cleave this substrate (Fig. 2B), further supporting its specificity for caspase-9. Taken together, these findings demonstrate that treatment of MM.1S cells with Dex is associated with activation of caspase-9.
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We next asked whether caspase-9 activation is an obligatory event
during Dex-induced apoptosis. MM.1S MM cells were cultured with Dex in
the presence or absence of caspase-9 tetrapeptide inhibitor LEHD-FMK
for 24 h and then assayed for proteolytic cleavage of caspase-9
and caspase-3. LEHD-FMK abrogates Dex-induced cleavage of both
caspase-9 and caspase-3 (Fig. 2C, left and
right panel). In contrast, LEHD-FMK did not inhibit
anti-Fas-induced caspase-8 or caspase-8-mediated caspase-3 cleavage in
MM.1S MM cells (data not shown), further indicating the selectivity of
LEHD-FMK for caspase-9. We next determined whether blocking
caspase-9 activation affects Dex-induced apoptosis, MM.1S MM cells were
cultured with Dex in the presence or absence of caspase-9 inhibitor
LEHD-FMK for 24 h and then assayed for apoptosis using flow
cytometric analysis with PI and HO dual staining to determine the
percentage for PI and HO+ apoptotic cells. Dex-induced
apoptosis (51 + 3% apoptotic cells (n = 3)) was
significantly inhibited in cells pretreated with caspase-9 inhibitor
(27 + 2% apoptotic cells (n = 3)) (Fig.
2D). Other studies have demonstrated that caspase-9 proteolytically cleaves and activates procaspase-3 (14). In that
context, our previous studies have shown that Dex triggers caspase-3
activation in MM.1S MM cells (22, 23). Taken together, these results
suggest that Dex induces sequential activation of Smac
caspase-9
caspase-3 and is independent of cyto-c·Apaf-1 apoptosome complex formation.
We next determined the mechanism of Smac-mediated Dex-induced caspase-9 activation. Two potential mechanisms for capsase-9 activation have been suggested (18). First, the release of cyto-c leads to Apaf-1 oligomerization, which then activates caspase-9; second, Smac interacts with IAPs (inhibitors of apoptosis protein), such as X-chromosome-linked IAP (XIAP), and eliminates the inhibitory effects of IAPs on caspase-9 (27). Since Dex-induced apoptosis is not associated with cyto-c release or Apaf-1 multimerization, we asked whether XIAP interacts with Smac during Dex-induced apoptosis. MM.1S MM cells were transiently transfected with Myc-XIAP and treated with Dex for 24 h. Cytosolic extracts were subjected to immunoprecipitation with anti-Myc and immunoblotting with anti-caspase-9, anti-Smac, or anti-XIAP. As shown in Fig. 2E, Dex treatment induces an interaction between XIAP and Smac. Importantly, Dex treatment also leads to dissociation of XIAP from caspase-9 (Fig. 2E). Equivalent levels of transfected XIAP protein were confirmed by reprobing the filters with anti-XIAP (Fig. 2E). These findings are in concert with other studies demonstrating that Smac promotes caspase activity of initiator caspase-9 by binding to and inhibiting IAPs (7, 20, 21).
To explore the functional significance of Smac release during
Dex-induced apoptosis, we utilized IL-6, a known inhibitor of Dex-triggered apoptosis in MM cells. IL-6 abrogates Dex-induced apoptosis in MM.1S MM cells (Fig.
3A, Refs. 23 and 28), as evidenced by DNA fragmentation assay. To determine whether IL-6 affects
Dex-induced release of Smac, MM.1S MM cells were treated with Dex in
the presence or absence of IL-6 (100 ng), and cytosolic extracts were
analyzed for Smac levels. The Dex-induced increase in cytosolic Smac
was significantly abrogated in the cells pretreated with IL-6 (Fig.
3B). This IL-6-mediated block in the Dex-induced Smac levels
was specific, because no change was observed in the levels of tubulin
(Fig. 3B). Additional evidence for supporting the role of
Smac during Dex-induced signaling was obtained by utilizing
Dex-resistant (MM.1R) MM cells. MM.1R cells were treated with Dex or IR
for 48 h, and cytosolic extracts were analyzed for Smac levels. As
shown in Fig. 3C, IR, but not Dex, induces Smac release. The
finding that IR induces release of Smac from mitochondria to cytosol in
MM.1R MM cells indicates that the Smac release system is functionally
intact and served as a positive control. The observation that IL-6
prevents Dex-induced Smac release and apoptosis, coupled with the lack
of Smac release and apoptosis by Dex in MM.1R (Dex-resistant) cells,
supports a role for Smac in mediating Dex-induced apoptosis and is
consistent with the known resistance to Dex treatment in MM patients
with advanced disease and high serum levels of IL-6 (28, 29).
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Collectively, the present study demonstrates that Dex-induced apoptosis
in MM cells is associated with Smac release and caspase-9 activation,
without concurrent release of cyto-c and Apaf-1
oligomerization. In contrast, treatment of Dex-resistant cells with Dex
fails to induce release of Smac and apoptosis. Furthermore, IL-6 blocks Dex-induced apoptosis in MM cells and inhibits Smac release, thereby conferring Dex-resistance. Taken together, these findings provide evidence for a Smac-dependent activation of caspase-9 and
apoptosis, independent of Apaf-1/cyto-c (Fig.
3D), and suggest therapeutic strategies based upon targeting
both Smac and XIAP.
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ACKNOWLEDGEMENTS |
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We thank Dr. Xiaodong Wang for providing Smac-related reagents and helpful suggestions. We also thank Dr. Emad S. Alnemri for providing FLAG- and T7-Apaf-1 cDNAs. We appreciate the technical assistance of Kamal Chauhan and Guilan Li.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA 78373 awarded by the NCI, by a Multiple Myeloma Senior Research Scientist Award (to D. C.), and by a Doris Duke Distinguished Clinical Scientist Award (to K. C. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Adult Oncology,
Dana Farber Cancer Institute, Mayer 557, 44 Binney St., Boston, MA 02115. Tel.: 617-632-2144; Fax: 617-632-2569; E-mail:
kenneth_anderson@dfci.harvard.edu.
Published, JBC Papers in Press, May 16, 2001, DOI 10.1074/jbc.C100074200
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ABBREVIATIONS |
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The abbreviations used are: IAP, inhibitor of apoptosis; Smac, second mitochondria-derived activator of caspases; Apaf-1, apoptotic protease-activating factor-1; IR, ionizing radiation; cyto-c, cytochrome c; MM, multiple myeloma; Dex, dexamethasone; IL, interleukin; PAGE, polyacrylamide gel electrophoresis; Ab, antibody; mAb, monoclonal antibody; HO, Hoechst 33342; PI, propidium iodide; IB, immunoblotting; FMK, fluoromethylketone; pNA, p-nitroanilide.
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