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2007;67:782-791. Cancer Res
Shuang Chen, Yun Dai, Hisashi Harada, et al.
Translocation
Cooperatively Inducing Bak Activation and Bax
Mcl-1 Down-regulation Potentiates ABT-737 Lethality by
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Mcl-1 Down-regulation Potentiates ABT-737 Lethality by
Cooperatively Inducing Bak Activation and
Bax Translocation
Shuang Chen,
1
Yun Dai,
1
Hisashi Harada,
1
Paul Dent,
2
and Steven Grant
1,2,3
Departments of
1
Medicine,
2
Biochemistry, and
3
Pharmacology, Virginia Commonwealth University and Massey Cancer Center,
Richmond, Virginia
Abstract
The Bcl-2 antagonist ABT-737 targets Bcl-2/Bcl-xL but not
Mcl-1, which may confer resistance to this novel agent. Here,
we show that Mcl-1 down-regulation by the cyclin-dependent
kinase (CDK) inhibitor roscovitine or Mcl-1-shRNA dramati-
cally increases ABT-737 lethality in human leukemia cells.
ABT-737 induces Bax conformational change but fails to
activate Bak or trigger Bax translocation. Coadministration
of roscovitine and ABT-737 untethers Bak from Mcl-1 and
Bcl-xL, respectively, triggering Bak activation and Bax trans-
location. Studies employing Bax and/or Bak knockout mouse
embryonic fibroblasts (MEFs) confirm that Bax is required
for ABT-737 F roscovitine lethality, whereas Bak is primarily
involved in potentiation of ABT-737–induced apoptosis by
Mcl-1 down-regulation. Ectopic Mcl-1 expression attenuates
Bak activation and apoptosis by ABT-737 + roscovitine,
whereas cells overexpressing Bcl-2 or Bcl-xL remain fully
sensitive. Finally, Mcl-1 knockout MEFs are extremely sensitive
to Bak conformational change and apoptosis induced by
ABT-737, effects that are not potentiated by roscovitine.
Collectively, these findings suggest down-regulation of Mcl-1
by either CDK inhibitors or genetic approaches dramatically
potentiate ABT-737 lethality through cooperative interactions
at two distinct levels: unleashing of Bak from both Bcl-xL and
Mcl-1 and simultaneous induction of Bak activation and Bax
translocation. These findings provide a mechanistic basis for
simultaneously targeting Mcl-1 and Bcl-2/Bcl-xL in leukemia.
[Cancer Res 2007;67(2):782–91]
Introduction
Cell death decisions are regulated by the complex interplay
between two groups of Bcl-2 family members: proapoptotic
proteins (e.g., multidomain: Bax and Bak; BH3-only: Bad, Bim,
Bid, and Noxa) and antiapoptotic proteins (e.g., Bcl-2, Bcl-xL,
Bcl-w, Mcl-1, and Bfl-1/A1; refs. 1, 2). In disorders such as
leukemia, increased expression of antiapoptotic proteins, such as
Bcl-2, is required for disease maintenance (3), confers drug
resistance (4), and is associated with poor clinical outcome (5).
These observations have prompted the development of small-
molecule Bcl-2 inhibitors (refs. 6, 7; e.g., HA14-1) that disable Bcl-2,
resulting in induction of apoptosis in leukemia cell lines (8).
Alternative strategies include the use of antisense oligodeoxynu-
cleotides (e.g., G3139; ref. 6) or stabilized forms of BH3 peptides (9),
among others. Recently, a novel inhibitor (ABT-737) of Bcl-2,
Bcl-xL, and Bcl-w, which is significantly more potent than previous
compounds of this type, has been developed. This compound acts
by mimicking the capacity of the BH3-only protein Bad to dock to
the hydrophobic groove of antiapoptotic Bcl-2 family proteins,
thereby diminishing their ability to antagonize apoptosis (10).
ABT-737 lowers the apoptotic threshold for chemotherapeutic
agents or ionizing radiation and has shown impressive precli-
nical activity against hematopoietic malignancies as well as solid
tumors in vitro and in vivo (10). Recently, ABT-737 was shown to
overcome drug resistance (e.g., toward imatinib) in Bcr/Abl
+
leukemic cells (11). However, ABT-737 has a low affinity for other
antiapoptotic Bcl-2 family proteins (e.g., Mcl-1 and A1; ref. 10)
and thus may exhibit limited cytotoxic effects in cells with high
endogenous levels of Mcl-1 (12). Moreover, ABT-737 efficiently kills
interleukin-3 (IL-3)–dependent cells (e.g., FL5.12) only after IL-3
withdrawal (13), suggesting that additional death signals may be
required for lethality. Notably, Mcl-1 is a highly expressed anti-
apoptotic protein (14) and a critical survival factor for various
malignant hematopoietic cells (14, 15). Recent evidence suggests
that more than one antiapoptotic protein (e.g., Mcl-1 and Bcl-xL)
cooperate to sequester multidomain proapoptotic proteins, such
as Bak, thereby preventing its activation (16). Thus, the impaired
capacity of ABT-737 to induce apoptosis in tumor cells expressing
high Mcl-1 levels may stem from a requirement for inhibition
of multiple antiapoptotic proteins. A corollary is that down-
regulation/inhibition of ABT-737–nontargeted proteins (e.g., Mcl-1
or A1) may enhance the lethality of this compound (12).
One candidate strategy to down-regulate/inhibit Mcl-1 involves
the use of cyclin-dependent kinase (CDK) inhibitors. In preclinical
studies, CDK inhibitors, including flavopiridol and the roscovitine
derivative CYC202 (seliciclib), are potent inducers of apoptosis in
malignant hematopoietic cells, including leukemia cells (17, 18).
Notably, results from several laboratories have established that
CDK inhibitors (e.g., flavopiridol and CYC202) act, at least in part,
by inhibiting CDK9, a kinase intimately involved in transcription
initiation and elongation through activation of the positive
transcription elongation factor-b, resulting in down-regulation of
several short-lived proteins, including Mcl-1 (17, 19).
Here, we report that Mcl-1 down-regulation by either CDK
inhibitors or a small hairpin RNA (shRNA) approach leads to a
dramatic increase in ABT-737–mediated apoptosis in human
leukemia cells. Our results also indicate that this phenomenon
stems from a mechanism involving two levels of cooperation
between antiapoptotic and multidomain proapoptotic proteins of
the Bcl-2 family: (a) simultaneous untethering of Bak from Bcl-xL
(by ABT-737) and Mcl-1 (e.g., by roscovitine) and (b) the resulting
Note: S. Chen and Y. Dai contributed equally to this work.
Requests for reprints: Steven Grant, Division of Hematology/Oncology, Virginia
Commonwealth University/Massey Cancer Center, MCV Station Box 230, Richmond,
VA 23298. Phone: 804-828-5211; Fax: 804-828-2178; E-mail: stgrant@vcu.edu.
I2007 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-3964
Cancer Res 2007; 67: (2). January 15, 2007
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Research Article
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activation of both Bak and Bax, culminating in Bax mitochondrial
translocation and engagement of the apoptotic cascade. These
findings may provide a theoretical framework for combinatorial
approaches that target diverse antiapoptotic proteins that
cooperate in the efficient induction of apoptosis in malignant cells.
Materials and Methods
Cells and reagents. Human leukemia U937, HL-60, and Jurkat cells were
provided by the American Type Culture Collection (Rockville, MD) and
maintained in RPMI 1640 containing 10% FCS as previously reported (20).
U937/Bcl-2 and U937/Bcl-xL were obtained by stable transfection of cells
with full-length Bcl-2 and Bcl-xL cDNA, respectively (21). U937 cells stably
overexpressing Mcl-1 were kindly provided by Dr. Ruth Craig (Dartmouth
Medical School, Hanover, NH; ref. 22). All experiments used logarithmically
growing cells (3–5
10
5
cells/mL). Peripheral blood samples were obtained
with informed consent according to the Declaration of Helsinki from the
peripheral blood of three patients with acute myeloblastic leukemia (AML;
FAB subtype M2) undergoing routine diagnostic aspirations with approval
from the Virginia Commonwealth University Institutional Review Board.
Leukemic blasts were isolated as previously described (20). Wild-type (wt),
Bax
/
, Bak
/
, and Bax
/
/Bak
/
(double knockout) mouse embryonic
fibroblast (MEF) were kindly provided by the laboratory of Dr. Stanley
Korsmeyer (Dana-Farber Cancer Institute, Boston, MA; ref. 23). Mcl-1
/
MEFs were kindly provided by Dr. Joseph Opferman (St. Jude Children’s
Research Hospital, Memphis, TN). All experiments were initiated in cells
cultured at f60% confluence.
ABT-737 was kindly provided by Dr. Gary Gordon (Abbott Laboratories,
Abbott Park, IL; ref. 10). It was dissolved in DMSO, aliquoted, and stored at
80jC. Roscovitine and R-roscovitine were purchased from Calbiochem
(San Diego, CA), dissolved in sterile DMSO, aliquoted, and stored at 20jC.
In all experiments, the final concentration of DMSO did not exceed 0.1%.
Assessment of apoptosis. The extent of apoptosis was evaluated by flow
cytometric analysis using Annexin V-FITC staining as described previously
(20). To analyze the extent of cell death in MEFs, cells were trypsinized and
harvested together with those in the culture supernatant and then stained
with 5 Ag/mL 7-amino-actinomycin D (7AAD; Sigma, St. Louis, MO) in PBS
for 20 min at 37jC. The percentage of dead cells (7AAD
+
) was assessed by
flow cytometry using a Becton Dickinson FACScan (Becton Dickinson, San
Jose, CA).
Immunoblot. For subcellular fractionation, cells were lysed in digitonin
lysis buffer (20). After centrifugation, the supernatant (S-100, cytosolic
fraction) and pellets (organelle/membrane fractions) were collected and
subjected to immunoblot. Samples (30 Ag protein for each condition) from
whole-cell pellets or subcellular fractions were subjected to immunoblot
following procedures previously described (20). Where indicated, the blots
were reprobed with antibodies against h-actin (Sigma) or a-tubulin
(Oncogene, La Jolla, CA) to ensure equal loading and transfer of proteins.
The following antibodies were used as primary antibodies: anti-Mcl-1, anti-
caspase-9, and anti-caspase-3 (BD PharMingen, San Diego, CA); anti-Mcl-1,
anti-Bcl-2, anti-Bcl-xS/L, anti–cytochrome c, anti-AIF, anti-Bak, and anti-
Bax (Santa Cruz Biotechnology, Santa Cruz, CA); anti–cleaved caspase-3
(Asp
175
), anti–cleaved poly(ADP-ribose) polymerase (PARP; Asp
214
), and
anti-Bcl-xL (Cell Signaling, Beverly, MA); anti–RNA polymerase II (pol II)
and anti–phospho-pol II (Upstate, Lake Placid, NY); anti-human Bcl-2 onco-
protein (DAKO, Carpinteria, CA); anti-caspase-8 (Alexis, San Diego, CA);
anti-PARP (Biomol, Plymouth Meeting, PA).
Immunoprecipitation. The interaction between Bak and Mcl-1 or Bcl-
xL was evaluated by coimmunoprecipitation analysis by using ExactaCruz
kits (Santa Cruz Biotechnology) as per manufacturer’s instructions. For
these studies, CHAPS buffer was employed to avoid artifactual associations
reported with other buffers (24). Briefly, cells were lysed by syringing with a
23-gauge needle in lysis buffer [20 mmol/L Tris (pH 7.4), 135 mmol/L NaCl,
1.5 mmol/L MgCl
2
, 1 mmol/L EDTA, 10% glycerol] containing 1% CHAPS
(Pierce, Rockford, IL); 800 Ag of protein per condition was used for
immunoprecipitation with anti-Bak antibody (Santa Cruz Biotechnology),
and the immunoprecipitated protein was subjected to immunoblot analysis
using antibodies to either Mcl-1 (BD PharMingen) or Bcl-xL (Cell Signaling).
Analysis of Bak and Bax conformational change. Cells were fixed and
permeabilized using FIX & PERM Cell Permeabilization Reagents (Caltag
Lab, Burlingame, CA) as per manufacturer’s instructions. Fixed cells were
incubated with either anti-Bak (Ab-1 for U937 cells and Ab-2 for MEFs,
Calbiochem) or anti-Bax (clone 3 for U937 cells, BD Transduction Lab,
Lexington, KY; YTH-6A7 for MEFs, Trevigen, Gaithersburg, MD) on ice for
30 min and then with FITC-conjugated goat-anti-mouse IgG (Southern
Biotech, Birmingham, AL) for 30 min in the dark. After washing, the
samples were analyzed by flow cytometry. The results for each condition
were calibrated by values for cells stained with mouse IgG (Southern
Biotech) as the primary antibody. Values for untreated controls were
arbitrarily set to 100%. In parallel, cells for each condition were stained with
antibodies to total Bak (Santa Cruz Biotechnology) for comparison.
RNA interference. The pSUPER.retro.puro vector containing the human
H1 RNA promoter for expressing shRNA was obtained from Oligoengine
(Seattle, WA). DdRNAi oligonucleotides (5¶-GATCCCCGCGGGACTGGC-
TAGTTAAACTTCAAGAGAGTTTAACTAGCCAGTCCCGTTTTTA-3¶; ref. 25)
were cloned into pSUPER.retro.puro vector (pSUPER/shMcl-1). U937 cells
were transiently transfected with the pSUPER/shMcl-1 construct and its
empty vector by using the Amaxa Nucleofector device (program V-001) with
Kit V (Amaxa GmbH, Cologne, Germany) as per manufacturer’s instructions.
Reverse transcription-PCR. Total RNA was isolated using RNeasy Mini
kit with QIAshredder spin column (Qiagen, Valencia, CA) as per
manufacturer’s instructions; 1 Ag per condition of total RNA was subjected
to reverse transcription-PCR (RT-PCR) reaction using One-Step RT-PCR kit
(Qiagen) and Thermal Cycler (MJ Research, Inc., Reno, NV). The primers
forward, 5¶-ATCTCTCGGTACCTTCGGGAGC-3¶ and reverse, 5¶-CCTGATGC-
CACCTTCTAGGTCC-3¶ (26) were used for Mcl-1. PCR products of Mcl-1
(442 bp) were analyzed in 2% agarose gel with ethidium bromide staining.
Statistical analysis. The values represent the means F SD for at least
three independent experiments done in triplicate. The significance of
differences between experimental variables was determined using the
Student’s t test. Analysis of synergism was done according to median dose-
effect analysis using Calcusyn software (Biosoft, Ferguson, MO; ref. 20).
Results
CDK inhibitors transcriptionally down-regulate the expres-
sion of Mcl-1 and synergistically interact with ABT-737 to
induce apoptosis in U937 cells. Earlier reports indicated that
various CDK inhibitors, including roscovitine (27), transcriptionally
down-regulate expression of short-lived proteins, such as Mcl-1.
As shown in Fig. 1A, treatment with either CDK inhibitor at
concentrations >10 Amol/L resulted in significant declines in Mcl-1
protein levels, but no change in expression of Bcl-2 and Bcl-xL was
observed. This phenomenon was associated with inhibition of
phosphorylation of pol II CTD and a pronounced reduction in
Mcl-1 mRNA levels (Fig. 1B).
Attempts were then made to determine what effect CDK
inhibitors would have on the response of cells to ABT-737. Whereas
exposure to 12 Amol/L roscovitine alone modestly increased
apoptosis, ABT-737 at a concentration range of 150 to 750 nmol/L
had very little effect (Fig. 1C). However, combined treatment
resulted in a dramatic increase in the loss of mitochondrial
membrane potential (DW
m
; data not shown) and apoptosis.
Identical results were obtained with R-roscovitine. Moreover, lower
concentrations (V10 Amol/L) of either roscovitine or R-roscovitine,
which failed to down-regulate Mcl-1 (Fig. 1A), did not enhance
ABT-737 lethality (data not shown). Median dose-effect analysis,
employing apoptosis determined by Annexin V-FITC as an end
point, yielded combination index values <1.0 (Fig. 1C, inset),
denoting synergistic interactions.
Mcl-1 Down-regulation Enhances ABT-737 Lethality
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Exposure of U937 cells to roscovitine F ABT-737 resulted in
marked down-regulation of Mcl-1 protein, whereas ABT-737 by itself
failed to modify Mcl-1 expression (Fig. 1D). In contrast, no change in
Bcl-2 or Bcl-xL expression was observed (data not shown). Lastly,
effects of cotreatment with ABT-737 and roscovitine were examined
in relation to mitochondrial events. Cotreatment with roscovitine
and ABT-737 triggered a pronounced increase in cytochrome c and
AIF release into the cytosolic fraction (Fig. 1D). Combined treat-
ment also induced a modest but discernible increase in caspase-9
and caspase-8 cleavage, and a marked increase in cleavage of
caspase-3, accompanied by cleavage of PARP (Fig. 1D ). However,
transfection with a dominant-negative caspase-8 construct (21)
failed to protect U937 cells from mitochondrial damage and
apoptosis induced by ABT-737/roscovitine (data not shown),
arguing against involvement of the extrinsic apoptotic pathway.
Together, these findings indicate that CDK inhibitors dramatically
increase ABT-737–mediated apoptosis in association with Mcl-1
down-regulation.
Roscovitine markedly down-regulates Mcl-1 and increases
ABT-737 lethality in various human leukemia cells, including
primary AML cells. Parallel studies were done in human HL-60
promyelocytic and Jurkat lymphoblastic leukemia cells. First, these
leukemia cells exhibited differing susceptibilities to ABT-737–
mediated lethality (IC
50
:1.3Amol/L for U937, 420 nmol/L for
Jurkat, 190 nmol/L for HL-60). Immunoblot showed that Bcl-2 and
Mcl-1 protein levels varied between the cell lines, whereas Bcl-xL
expression was equivalent. Interestingly, HL-60 cells, which were
the most sensitive of the three lines, exhibited very low Mcl-1
expression but higher levels of Bcl-2 (Fig. 2A, inset). These results
suggest that levels of Mcl-1 and/or the ratio between Mcl-1 and
Figure 1. CDK inhibitors interact with ABT-737 to induce mitochondria-related apoptotic signaling events in association with down-regulation of Mcl-1. A, U937 cells
were exposed to 7.5 to 15 Amol/L of either roscovitine (Rosc)orR-roscovitine (R-rosc ), after which immunoblot analysis was done to monitor expression of Bcl-2,
Bcl-xL, and Mcl-1. B, immunoblot analysis was conducted to assess phosphorylation status of pol II (P-pol II) CTD after 24 h of treatment with roscovitine (12 A mol/L).
Alternatively, total RNA was extracted, and RT-PCR was done to monitor Mcl-1 mRNA levels. C, U937 cells were treated with 150 to 750 nmol/L ABT-737
(ABT) F 12 Amol/L roscovitine or 300 nmol/L ABT-737 F 12 Amol/L R-roscovitine for 24 h, after which flow cytometry was conducted to determine the percentage of
apoptotic cells (Annexin V
+
). Moreover, U937 cells were exposed (24 h) to varying concentrations of ABT-737 and roscovitine alone and in combination at fixed ratio
1:100. At the end of this period, the percentage of Annexin V
+
cells was determined for each condition, and median dose-effect analysis was then employed to
characterize the nature of the interaction between these agents (inset). Combination Index (C.I.) values <1.0 denote a synergistic interaction. Representative for three
separate experiments. D, U937 cells were exposed (24 h) to 150 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine, and immunoblot was then done to evaluate
expression of Mcl-1 as well as cleavage of caspases (casp ) and PARP. Alternatively, S-100 cytosolic fractions were prepared and subjected to immunoblot analysis.
Primary antibodies were employed as indicated. CF, cleavage fragment; Cyt c, cytochrome c. A, B, and D, representative results from one experiment, and two
additional studies yielded equivalent results.
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Bcl-2 expression may represent as determinants of ABT-737
sensitivity in leukemia cells.
Cotreatment with marginally toxic concentrations of ABT-737
and roscovitine markedly induced mitochondrial damage (data not
shown) and apoptosis in Jurkat and HL-60 cells (Fig. 2A). Roughly
equivalent results were obtained when R -roscovitine was coad-
ministered (data not shown). Moreover, roscovitine, administrated
alone F ABT-737 dramatically down-regulated Mcl-1 in Jurkat cells,
and combined treatment marked increased PARP cleavage (Fig. 2B).
Effects of this regimen on primary leukemia blasts isolated from
three patients with AML (Fig. 2C) were similar to those obtained in
leukemia cell lines. Furthermore, roscovitine F ABT-737 almost
completely abrogated Mcl-1 expression in AML blasts and induced
pronounced PARP (Fig. 2D), indicating that ABT-737/roscovitine
interactions occur in association with Mcl-1 down-regulation in
both continuously cultured human leukemia cell lines differing in
their sensitivity to ABT-737 as well as in primary AML blasts.
Roscovitine enhances ABT-737–mediated Bax conforma-
tional change, whereas Bak activation is induced only by
combined ABT-737 and roscovitine administration. Bax and
Bak are essential for mitochondrial outer membrane permeabiliza-
tion (28, 29), a critical cell death determinant (30). Whereas Bak is
normally localized at its site of action (i.e., organellar membranes),
Bax is monomeric and found in the cytosol of healthy cells.
Following death stimuli, Bax undergoes conformational change
and translocates to organellar membranes. Activation of both Bak
and Bax is associated with a conformational change, which can be
detected by antibodies recognizing only the active protein
conformers (16). The effect of ABT-737 F roscovitine on Bak/Bax
conformational change was then examined. When U937 cells were
exposed to ABT-737 (150–500 nmol/L) alone, a clear dose-
dependent increase in Bax conformational change was observed
(Fig. 3A). Interestingly, Bax conformational change was unaccom-
panied by Bax translocation (see below; Fig. 3C) nor did it induce
Figure 2. Roscovitine down-regulates Mcl-1 and promotes ABT-737 lethality in multiple human leukemia cell lines and primary AML cells. A, untreated U937, HL-60,
and Jurkat cells were lysed and subjected to immunoblot analysis to detect protein levels of Bcl-2, Bcl-xL, and Mcl-1 (inset ). Jurkat and HL-60 cells were exposed for
24 h to ABT-737 (Jurkat, 100–200 nmol/L; HL-60, 30–50 nmol/L) F 12 Amol/L roscovitine, after which the percentage of Annexin V
+
cells was determined by flow
cytometry. B, Jurkat cells were treated as described in (A), after which immunoblot analysis was done to monitor Mcl-1 expression and PARP cleavage. C, blasts were
isolated from the peripheral blood of three AML patients (designed as #1–3; FAB subtype M2) and then incubated (24 h) with 150 nmol/L ABT-737 F roscovitine
(#1 and #2, 10 Amol/L; #3, 12 Amol/L). At the end of this period, the percentage of Annexin V
+
cells was determined by flow cytometry. Columns, means for an
experiment done in triplicate; bars, SD. Representative experiment (patient #3). D, the blasts (patient #3 ) were incubated (24 h) with 150 and 300 nmol/L
ABT-737 F 12 Amol/L roscovitine and then lysed for immunoblot using indicated primary antibodies. C-PARP, cleaved PARP. A (inset), B , and D, representative for
three separate experiments.
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apoptosis (Fig. 1C). On the other hand, roscovitine (12 Amol/L)
alone minimally induced Bax conformational change. Notably, cells
coexposed to roscovitine and ABT-737 displayed a further increase
in Bax conformational change compared with cells treated ABT-737
alone (Fig. 3A), an effect accompanied by marked increases in Bax
translocation (see below; Fig. 3C ) and lethality. These findings
suggest that ABT-737–induced Bax conformational change by itself
may not be sufficient to trigger Bax translocation and apoptosis.
Parallel studies were then done in U937 cells to assess Bak
conformational change. In sharp contrast to results involving
Bax, ABT-737 by itself had little or no effect on Bak activation
(Fig. 3A), whereas roscovitine also failed to induce Bak activation.
However, combined treatment with ABT-737 and roscovitine
induced pronounced Bak activation. No change in total Bak was
noted for any condition (data not shown). Together, these results
indicate that (a) roscovitine enhances ABT-737–mediated Bax
conformational change and cooperates to trigger Bax transloca-
tion, and that (b) ABT-737 alone is ineffective in triggering Bak
conformational change; instead, roscovitine coadministration is
required for ABT-737–mediated Bak activation.
Coadministration of ABT-737 and roscovitine disrupts the
association of Bak with both Bcl-xL and Mcl-1 and induces Bax
translocation. One of the mechanisms by which Mcl-1 opposes
apoptosis is by binding/sequestering Bak and preventing its
activation (31). Furthermore, there is evidence that Bcl-xL, but
not Bcl-2, Bcl-w, or A1, acts analogously to block Bak activation
(16), and that both Mcl-1 and Bcl-xL must be neutralized (e.g., by
Noxa and Bad, respectively) to displace Bak in order to trigger cell
death (16, 32). Consequently, the effects of ABT-737 and roscovitine
on interactions between Bak and Bcl-xL or Mcl-1 were assessed.
No change in total Bak levels was observed with any treatment
(see below; Fig. 3C). U937 cells exposed to ABT-737 F roscovitine
were lysed in CHAPS buffer, and associations between Bak and
Mcl-1 or Bcl-xL were assessed (Fig. 3B). Treatment with ABT-737
Figure 3. Bax is necessary for induction of cell death by both ABT-737 alone and in combination with roscovitine, whereas Bak activation is only required for synergistic
interactions between these agents. A, U937 cells were treated (24 h) with 150 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine, after which cells were stained with
anti–conformationally changed Bax (clone 3)/FITC-conjugated goat-anti-mouse IgG and subjected to flow cytometry. In parallel, Bak activation was monitored by
flow cytometry after staining with anti–conformationally changed Bak (Ab-1)/FITC-conjugated goat-anti-mouse IgG. Representative histograms (dotted, untreated
controls). Values, mean FSD for three separate experiments done in triplicate. B, U937 cells were exposed for 24 h to 150 to 300 nmol/L ABT-737 F 12 Amol/L
roscovitine, after which cells were lysed in CHAPS buffer and subjected to immunoprecipitation (IP) using anti-Bak and then immunoblotted (IB) with either anti-Mcl-1 or
anti-Bcl-xL antibody. For comparison, the right lanes (designed as C) were loaded with whole-cell lysates. C, U937 cells were treated as described in (A ), after
which S-100 cytosolic and pelleted organellar membrane fractions were prepared and subjected to immunoblot analysis using an anti-Bax antibody. Alternatively, total
levels of Bax and Bak were monitored in whole-cell lysates. D, untreated wt, Bak
/
, Bax
/
, and Bax
/
/Bak
/
[double knockout (DKO)] MEFs were lysed and
subjected to immunoblot to detect expression of both proapoptotic (Bak and Bax) and antiapoptotic (Mcl-1, Bcl-2, and Bcl-xL) proteins (inset). Various MEFs were
exposed (24 h) to 300 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine, after which the cells, including those in the culture supernatant, were collected for each condition.
Cells were then stained with 7AAD and subjected to flow cytometry to determine the percentage of 7AAD
+
cells. *, P < 0.01, significantly greater than values for
treatment with each concentration of ABT-737 alone. B, C, and D (inset), representative results from one experiment, and two additional studies yielded equivalent
results.
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alone modestly but discernibly increased the amount of Mcl-1
associating with Bak. Notably, this effect was largely abrogated by
roscovitine treatment, presumably due to Mcl-1 down-regulation
(Fig. 1). Reciprocal effects were noted when the amount of Bcl-xL
coimmunoprecipitating with Bak was monitored (i.e., roscovitine
substantially increased the amount of Bcl-xL associating with Bak,
whereas ABT-737 essentially reversed this phenomenon). Together,
these findings suggest that cotreatment with roscovitine and ABT-
737 antagonizes interactions of Bak with both Mcl-1 and Bcl-xL.
Lastly, the effect of coexposure to roscovitine and ABT-737 on
intracellular Bax localization was examined. Treatment with either
ABT-737 or roscovitine alone had little or no effect on the
intracellular disposition of Bax (Fig. 3C). However, ABT-737/
roscovitine coadministration induced a major translocation of
Bax from the cytosolic compartment to the pelleted organellar
membrane fraction. This suggests that roscovitine and ABT-737
cooperate to untether Bak from Mcl-1 and Bcl-xL, leading to Bak
activation and accompanying Bax translocation to organellar
membranes. These findings also suggest that concomitant
activation of Bax and Bak may be responsible for the dramatic
induction of apoptosis in cells coexposed to roscovitine and
ABT-737.
Bax knockout in MEFs substantially diminishes the lethality
of ABT-737 F roscovitine, whereas Bak knockout primarily
blocks synergistic interactions between these agents. To
evaluate further the functional roles of Bax and Bak in the lethality
of ABT-737 and its interactions with roscovitine, Bax
/
, Bak
/
,
and Bax
/
/Bak
/
(double knockout) MEFs were employed
(Fig. 3D, inset). Immunoblot analysis revealed that antiapoptotic
Bcl-2 family protein levels (e.g., Mcl-1, Bcl-2, and Bcl-xL) were
roughly equivalent in each of the cell types. Coadministration of
roscovitine clearly increased the lethality of ABT-737 in wt MEFs
(P < 0.01, compared with cells exposed to ABT-737 alone; Fig. 3D).
However, the lethality of ABT-737 F roscovitine was substantially
blunted in Bax
/
MEFs, indicating that Bax is critical for this
phenomenon. In marked contrast, ABT-737 was still able to induce
cell death in Bak
/
MEFs; in fact, these cells were slightly more
sensitive than wt cells. Significantly, coadministration of roscovi-
tine failed to increase the lethality of ABT-737 in Bak
/
MEFs.
Finally, Bax/Bak double knockout MEFs displayed essentially no
response to any of these treatments. Together, these findings sug-
gest that Bax is required for induction of cell death by both ABT-
737 F roscovitine, whereas Bak activation, although dispensable
for ABT-737 lethality, is nevertheless required for ABT-737/rosco-
vitine synergism. They also support the notion that cooperation
between Bak and Bax is critical for ABT-737/roscovitine lethality.
Roscovitine down-regulates Mcl-1 expression and attenu-
ates ABT-737 resistance in leukemic cells ectopically express-
ing Bcl-2 or Bcl-xL. Because ABT-737 targets Bcl-2 and Bcl-xL (10),
it might be predicted that the relative abundance of antiapoptotic
proteins, such as Bcl-2, would be related to ABT-737 sensitivity.
Consequently, the effect of Bcl-2 or Bcl-xL expression on the
susceptibility of cells to ABT-737 F roscovitine was examined using
U937 cells ectopically expressing Bcl-2 or Bcl-xL. Ectopic expres-
sion of Bcl-2 or Bcl-xL provided significant protection from the
lethal effects of etoposide (VP-16), a potent inhibitor of DNA
topoisomerase (Fig. 4A and B). Notably, Bcl-2 or Bcl-xL over-
expression attenuated ABT-737–mediated lethality but did not
affect roscovitine cytotoxicity. However, Bcl-2 or Bcl-xL over-
expression failed to protect cells from mitochondrial damage
(i.e., loss of DW
m
; data not shown) and apoptosis induced by
roscovitine and ABT-737 coadministration (P > 0.05, for each ABT-
737 concentration, compared with empty vector controls U937/
pCEP or U937/3.1). Cotreatment with roscovitine/ABT-737 induced
an equivalent decline in Mcl-1 expression and enhanced PARP
cleavage in U937/Bcl-2, U937/Bcl-xL, and controls but did not
modify Bcl-2 or Bcl-xL expression (Fig. 4C and D). Collectively,
these findings indicate that although ectopic expression of either
Bcl-2 or Bcl-xL reduces human leukemia cell sensitivity to ABT-737,
they are unable to prevent the ABT-737/roscovitine regimen from
diminishing Mcl-1 levels and inducing apoptosis.
Ectopic expression of Mcl-1 but not Bcl-2 blocks Bak
activation and apoptosis triggered by combined exposure of
leukemia cells to ABT-737 and roscovitine. Because Bcl-2/Bcl-xL
and Mcl-1 may play disparate functional roles in blocking apoptosis
(32), studies were done employing U937 cells ectopically expressing
Mcl-1 (22). As in cells overexpressing Bcl-2 or Bcl-xL, ectopic
expression of Mcl-1 substantially protected cells from VP-16– and
ABT-737–mediated lethality (Fig. 5A). However, in striking contrast
to the former cells, enforced Mcl-1 expression significantly
diminished mitochondrial damage (loss of DW
m
; data not shown)
and apoptosis (Fig. 5A) induced by the ABT-737/roscovitine
regimen (P < 0.001, versus U937/pCEP).
Immunoblot revealed that combined treatment with ABT-737/
roscovitine clearly diminished Mcl-1 expression in U937/pCEP cells
but failed to do so in their U937/Mcl-1 counterparts (Fig. 5B ).
Moreover, PARP cleavage induced by ABT-737/roscovitine was
almost completely abrogated in U937/Mcl-1 cells. Basal Bcl-2
expression was somewhat lower in U937/Mcl-1 cells compared
with controls but was not modified appreciably in either line
with any treatment. Together, these findings argue strongly that
down-regulation of Mcl-1 plays a functional role in potentiation
of ABT-737–mediated lethality by roscovitine.
Finally, the effects of ectopic expression of Bcl-2 or Mcl-1 were
compared with respect to conformational change of Bax and Bak
induced by the ABT-737/roscovitine regimen. Consistent with their
inability to block ABT-737/roscovitine–mediated lethality, Bcl-2
overexpression failed to attenuate conformational change of Bax or
Bak in cells exposed to ABT-737 and roscovitine in combination
(Fig. 5C), although it did reduce Bax conformational change
induced by ABT-737 alone (data not shown). Similar results were
obtained in cells ectopically expressing Bcl-xL (data not shown).
However, in sharp contrast, ectopic expression of Mcl-1, which also
attenuated Bax conformational change mediated by ABT-737 alone
(data not shown), essentially abrogated Bak activation triggered by
the ABT-737/roscovitine regimen and also partially reduced Bax
conformational change after exposure to the combination (Fig. 5D).
These findings provide further evidence that disruption of Mcl-1
function plays a critical role in ABT-737/ roscovitine interactions
associated with Bax/Bak activation and apoptosis.
RNA interference or gene knockout of Mcl-1 dramatically
sensitizes cells to ABT-737 but abrogates the capacity of
roscovitine to potentiate Bak activation and lethality. To
evaluate further the functional role of Mcl-1 in Bax/ Bak activation
as well as apoptosis mediated by ABT-737 F roscovitine, a shRNA
strategy and Mcl-1
/
MEFs were employed. First, U937 cells were
transiently transfected with a construct encoding shRNA against
Mcl-1 mRNA, and immunoblot analysis documented Mcl-1 down-
regulation (Fig. 6A, inset). Mcl-1 down-regulation by this approach
dramatically sensitized human leukemia cells to ABT-737 lethality
(P < 0.02–0.001, for each ABT-737 concentration, compared with
those transfected with empty vector; Fig. 6A ).
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Further studies were done in wt and Mcl-1
/
MEFs. Immuno-
blots revealed that levels of Bcl-2, Bcl-xL, Bak, and Bax in Mcl-1
/
cells were roughly equivalent to those in wt MEFs, whereas Mcl-1
was absent (Fig. 6B, inset ). Mcl-1
/
cells exhibited a dramatic
increase in sensitivity to ABT-737 (50–100 nmol/L) compared with
wt cells (P < 0.001; Fig. 6B). Significantly, roscovitine was unable
to enhance cell killing mediated by ABT-737 in Mcl-1
/
cells
(P > 0.05). Moreover, Bak conformational change was observed only
after coexposure of wt MEFs to ABT-737 and roscovitine, consistent
with preceding findings in human leukemia cells. In striking
contrast, ABT-737 alone dramatically induced Bak activation in
Mcl-1
/
cells, a phenomenon that was not further enhanced by
roscovitine (Fig. 6C). Analogously, Mcl-1
/
cells displayed greater
Bax conformational change following ABT-737 exposure compared
with their wt counterparts (Fig. 6C). However, as in the case of Bak,
roscovitine failed to increase ABT-737–mediated Bax conforma-
tional change in Mcl-1
/
cells. Together, these results provide clear
evidence that Mcl-1 is a critical determinant of both ABT-737
actions as well as the capacity of roscovitine to potentiate ABT-737
lethality through cooperative activation of Bak and Bax.
Discussion
ABT-737, a novel small-molecule Bcl-2/Bcl-xL/Bcl-w inhibitor
currently in development as an anticancer agent, has a relatively
low affinity for the more divergent antiapoptotic Bcl-2 family
proteins (e.g., Mcl-1 and A1; ref. 10). ABT-737 is less efficient in
killing tumor cells exhibiting relatively high levels of Mcl-1 (12).
Because Mcl-1 has a short half-life (i.e., <2 h; ref. 33), it is
particularly susceptible to down-regulation by agents that disrupt
its de novo synthesis. Consequently, attention has recently focused
on the capacity of several clinical relevant CDK inhibitors (e.g.,
flavopiridol and the roscovitine derivative CYC202) to transcrip-
tionally down-regulate Mcl-1 through CDK9 inhibition (17, 19).
Moreover, we previously reported that flavopiridol enhanced the
lethality of HA14-1, although the mechanism underlying this
interaction was not determined (34). Consistent with these
findings, roscovitine and its R enantiomer R-roscovitine inhibited
phosphorylation of RNA polymerase II CTD, diminished Mcl-1
mRNA levels, and markedly down-regulated protein levels.
Significantly, such actions correlated closely with synergistic
interactions with ABT-737. Although the possibility that other
Figure 4. Overexpression of Bcl-2 or Bcl-xL fails to protect cells from roscovitine/ABT-737–mediated lethality. A and B, U937/Bcl-2 (A) and U937/Bcl-xL (B ), as well
as their empty-vector controls (U937/pCEP and U937/pcDNA3.1), were treated (24 h) with 150 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine or 25 Amol/L VP-16 (6 h)
for comparison, after which the percentage of Annexin V
+
cells was assessed by flow cytometry. **, P < 0.001, significantly less than values for empty vector controls.
C, U937/Bcl-2 and U937/pCEP cells were treated as described in (A), after which cells were lysed and subjected to immunoblot for expression of Bcl-2 and Mcl-1 as
well as PARP cleavage. D, U937/Bcl-xL and U937/pcDNA3.1 cells were exposed for 24 h to ABT-737 (top, 300 nmol/L; bottom, 150–500 nmol/L) F 12 Amol/L
roscovitine, after which immunoblot analysis was done to monitor protein levels of Bcl-xL and Mcl-1 as well as PARP cleavage. B and D, representative for three
separate experiments.
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activities (e.g., CDK disruption) contribute to the lethality of
combination regimens involving CDK inhibitors cannot be com-
pletely excluded (35), it is noteworthy that Mcl-1 down-regulation
plays an important role in apoptosis in malignant hematopoietic
cells (17, 19, 36). On the other hand, in certain transformed cells,
Mcl-1 down-regulation, although required, seems to be incapable
by itself of initiating apoptosis (31, 37), indicating that other
cooperating factors may be necessary for cell death. These findings
suggest that interventions targeting more than one antiapoptotic
protein may be required for optimal cell killing.
Whereas resistance to ABT-737 may reflect the compensatory
actions of Mcl-1 (12), the present investigation was prompted by
recent evidence that that Bak activation requires simultaneous
disruption of its associations with Mcl-1 (e.g., by Noxa) and Bcl-xL
(e.g., by Bad; ref. 16). Moreover, increased production of Noxa can
oppose Mcl-1 antiapoptotic functions, leading to simultaneous
activation of Bax and Bak (38). The present results suggest that
coadministration of ABT-737 and roscovitine recapitulate the
actions of more physiologic proapoptotic BH3-only proteins.
Specifically, ABT-737, by binding to hydrophobic groove within
the Bcl-xL BH3 domain (10), untethers Bak from Bcl-xL, analogous
to the actions of BH3-only proteins such as Bad (39). On the other
hand, Mcl-1 down-regulation by roscovitine, mimicking the actions
of Noxa in displacing Bak from Mcl-1 (16), reciprocally released Bak
from Mcl-1 sequestration. Thus, coadministration of ABT-737 and
roscovitine markedly diminished the association of Bak with both
Bcl-xL and Mcl-1, inducing Bak activation. In support of this
hypothesis, Bak activation was observed only when roscovitine
and ABT-737 were administrated concomitantly, but not after
ABT-737 alone. The notion of cooperativity in the regulation of
ABT-737 lethality is further supported by the results obtained in
Mcl-1 knockout MEFs, in which ABT-737 alone, in contrast to its
actions in wt cells, markedly induced Bak activation. Significantly,
roscovitine was unable to enhance ABT-737 lethality further in
these cells presumably because Mcl-1 was absent and levels could
not be reduced further. These findings provide strong support for
the concept that disruption of more than one antiapoptotic protein
of the Bcl-2 family (i.e., Mcl-1 and Bcl-2/Bc-xL) represents a highly
potent apoptotic stimulus (12).
The finding that ABT-737 induced Bax conformational change but
did not trigger apoptosis by itself suggests that the lethality of the
ABT-737/roscovitine regimen involves not simply activation of
either Bax or Bak, but cooperativity between these proteins.
Untethering of Bak from both Mcl-1 and Bcl-xL allows Bak confor-
mational change, homo-oligomerization (16), as well as possible
associations with Bax (40). Nevertheless, there is evidence that
Bax and Bak may interact to promote apoptosis (41). Results
obtained with Bax and Bak knockout MEFs are fully compatible with
a model in which Bax and Bak cooperate to trigger cell death. For
example, Bax knockout cells displayed marked resistance to ABT-
737 given alone or in combination with roscovitine. This suggests
that the presence of Bax is essential for both ABT-737 lethality and
synergistic interactions with roscovitine. In striking contrast, Bak
knockout cells remained fully sensitive to ABT-737–mediated cell
Figure 5. Ectopic expression of Mcl-1, but not Bcl-2, diminishes Bak activation and potentiation of apoptosis in cells coexposed to ABT-737 and roscovitine. A, U937/
Mcl-1 and its empty-vector control (U937/pCEP) cells were exposed to 150 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine (24 h) or 25 Amol/L VP-16 (6 h) for
comparison, after which the percentage of Annexin V
+
cells was determined by flow cytometry. **, P < 0.001, significantly less than values for U937/pCEP cells.
B, U937/Mcl-1 and U937/pCEP cells were treated (24 h) with 150 to 500 nmol/L ABT-737 F 12 Amol/L roscovitine, and then immunoblot analysis was done to monitor
expression of Mcl-1 and Bcl-2 as well as PARP cleavage. Representative for three separate experiments. C and D, U937/Bcl-2 (C ) and U937/Mcl-1 (D) cells,
as well as their empty-vector controls (U937/pCEP), were treated for 24 h with 300 nmol/L ABT-737 + 12 Amol/L roscovitine, after which conformational change of Bax
or Bak was monitored by flow cytometry using anti-Bax (clone 3) or anti-Bak (Ab-1) antibody, respectively.
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killing, indicating that Bak is not absolutely required for the lethality
of an agent targeting Bcl-2/Bcl-xL. However, it is significant that
roscovitine failed to potentiate ABT-737 lethality in these cells,
arguing strongly that Bak is required for full engagement of the
apoptotic program following disruption of the Bcl-2/Bcl-xL axis.
Moreover, Bax translocation and cell death occurred only in human
leukemia cells coexposed to ABT-737 and roscovitine (Fig. 3C ).
Although the mechanisms responsible for potentiation of ABT-737–
mediated Bax translocation by roscovitine are presently unclear,
there are several plausible explanations. These include the
possibilities that (a) Mcl-1 may be involved in Bax regulation, either
through a process involving ‘‘activator’’ BH3-only proteins such as
Bim and/or tBid (13, 42–45), or directly by itself (46); or that (b)
activated Bak may promote Bax translocation in an as yet to be
defined way. Collectively, the present results support the notion
that simultaneous interruption of Mcl-1 and Bcl-xL function frees
and activates Bak, which, in the setting of Bax conformational
change, results in Bax translocation, leading in turn to full engage-
ment of the apoptotic machinery.
It is noteworthy that overexpression of Bcl-2 or Bcl-xL failed
to protect leukemia cells from the ABT-737/roscovitine regimen,
reflecting the important contribution of Mcl-1 down-regulation
to the lethality of this regimen. The finding that ectopic expression
of Mcl-1 diminished potentiation of ABT-737 lethality by rosco-
vitine highlights the central role of Mcl-1 down-regulation in
synergism between these agents. This interpretation is further
supported by results showing that roscovitine was unable to
enhance ABT-737–mediated apoptosis in Mcl-1 knockout
MEFs. Moreover, overexpression of Mcl-1, but not Bcl-2/Bcl-xL,
essentially abrogated Bak activation following exposure to ABT-
737/roscovitine, strongly arguing that Mcl-1 plays a major role in
regulating Bak. This concept is consistent with previous findings
indicating that Mcl-1 binds with considerably greater affinity
to Bak compared with Bcl-xL (IC
50
< 10 versus < 100 nmol/L;
ref. 16).
Whereas recent studies suggest that ABT-737 and the more
specific Bcl-xL inhibitor A-385358 increase the antitumor activity
of conventional cytotoxic drugs (10, 47), this phenomenon may
reflect a generic lowering of the apoptotic threshold. On the other
hand, the present results suggest that a mechanism-based
approach combining agents that target distinct antiapoptotic
molecules (e.g., CDK inhibitors that down-regulate Mcl-1 expres-
sion and small-molecule Bcl-2/Bcl-xL inhibitors like ABT-737)
deserve attention. These findings also highlight the importance of
Figure 6. Knockout or down-regulation of Mcl-1 sensitize cells to cell death induced by ABT-737. A, U937 cells were transiently transfected with either empty
vector (EV) or shMcl-1/pSUPER for 24 h, after which cells were lysed and subjected to immunoblot analysis (inset ). After transfection with empty vector or shMcl-1,
cells were incubated for 6 h and then exposed to the indicated concentrations of ABT-737 for an additional 24 h, after which the percentage of viable cells (7AAD
)
was determined by flow cytometry after stained with 7AAD. *, P < 0.02; **, P < 0.001, significantly less than values for cells transfected with empty vector.
B, immunoblot analysis was done to monitor levels of antiapoptotic (Mcl-1, Bcl-2, and Bcl-xL) and multidomain proapoptotic proteins (Bax and Bak) in untreated wt
and Mcl-1 knockout MEFs (inset ). MEFs were exposed to 50 to 100 nmol/L ABT-737 alone or in the presence of 12 Amol/L roscovitine for 24 h, after which cells,
including those in the culture supernatant, were harvested together for flow cytometry using 7AAD staining. **, P < 0.001, significantly greater than values for wt cells.
C, MEFs (wt and Mcl-1
/
) were incubated (24 h) with 100 nmol/L ABT-737 F 12 Amol/L roscovitine, after which cells were stained with anti–conformationally changed
Bak (Ab-2) or Bax (YTH-6A7)/FITC-conjugated goat-anti-mouse IgG and subjected to flow cytometry.
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cooperative interactions between such agents at two separate but
interrelated levels in cell death induction: (a) release of Bak from
both Bcl-xL and Mcl-1 and (b) simultaneous activation of both
Bax and Bak, which may be essential for Bax translocation and
ABT-737 lethality. Whether a strategy combining CDK inhibitors,
or other transcriptional repressors capable of down-regulating
Mcl-1, with Bcl-2/Bcl-xL antagonists will result in enhanced
therapeutic efficacy will depend upon multiple factors, including
the capacity of such agents to diminish Mcl-1 expression in vivo,
and whether the therapeutic index is enhanced. In this context, it
is noteworthy that ABT-737 displays in vivo antitumor selectivity
in preclinical studies (10). In any case, the present findings
suggest that in addition to combining Bcl-2/Bcl-xL antagonists
with conventional cytotoxic drugs, combination strategies involv-
ing targeted agents that down-regulate Mcl-1, a protein that can
compensate for the loss of Bcl-2/Bcl-xL function, represents a
potentially useful alternative approach.
Acknowledgments
Received 10/25/2006; accepted 11/28/2006.
Grant support: National Cancer Institute grants CA63753, CA 93738, and CA
100866; Leukemia and Lymphoma Society of America award 6045-03; V Foundation;
and Department of Defense.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Joseph T. Opferman for Mcl-1 knockout MEFs.
Mcl-1 Down-regulation Enhances ABT-737 Lethality
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Cancer Res 2007; 67: (2). January 15, 2007
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