Identification of Chelerythrine as an Inhibitor of BclXL Function*,
Shing-Leng Chan,
Mei Chin Lee,
Kuan Onn Tan,
Lay-Kien Yang
,
Alex S. Y. Lee
,
Horst Flotow
,
Nai Yang Fu,
Mark S. Butler
,
Doel D. Soejarto
,
Antony D. Buss
and
Victor C. Yu ¶
From the
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore
117609, Republic of Singapore,
MerLion Pharmaceuticals Pte. Ltd., 59A Science Park Drive, The Fleming,
Singapore Science Park, Singapore 118240, Republic of Singapore,
Faculty of Medicinal Chemistry and Pharmacognosy, University of Illinois at
Chicago, Chicago, Illinois 60612
Received for publication, March 31, 2003
, and in revised form, April 15, 2003.
 |
ABSTRACT
|
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The identification of small molecule inhibitors of antiapoptotic Bcl-2
family members has opened up new therapeutic opportunities, while the vast
diversity of chemical structures and biological activities of natural products
are yet to be systematically exploited. Here we report the identification of
chelerythrine as an inhibitor of BclXL-Bak Bcl-2 homology 3 (BH3) domain
binding through a high throughput screening of 107,423 extracts derived from
natural products. Chelerythrine inhibited the BclXL-Bak BH3 peptide binding
with IC50 of 1.5 µM and displaced Bax, a
BH3-containing protein, from BclXL. Mammalian cells treated with chelerythrine
underwent apoptosis with characteristic features that suggest involvement of
the mitochondrial pathway. While staurosporine, H7, etoposide, and
chelerythrine released cytochrome c from mitochondria in intact
cells, only chelerythrine released cytochrome c from isolated
mitochondria. Furthermore BclXL-overexpressing cells that were completely
resistant to apoptotic stimuli used in this study remained sensitive to
chelerythrine. Although chelerythrine is widely known as a protein kinase C
inhibitor, the mechanism by which it mediates apoptosis remain controversial.
Our data suggest that chelerythrine triggers apoptosis through a mechanism
that involves direct targeting of Bcl-2 family proteins.
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INTRODUCTION
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Proteins of the Bcl-2 family are central regulators of apoptosis. While the
precise molecular mechanisms by which these proteins confer their biological
activities remain to be determined, they are thought to act directly on the
mitochondria (1). Members of
the Bcl-2 family can be divided into three subfamilies based on several
conserved sequence motifs known as Bcl-2 homology
(BH)1 domains. The
antiapoptotic members (Bcl-2, BclXL, Mcl-1, Bcl-1-w, and Ced-9) share all four
BH domains, designated as BH14; the proapoptotic members Bax, Bak, Bok,
and BclXs contain BH13 domains, but other proapoptotic members (Bid,
Bad, Bik, and Egl1) only have a BH3 domain. Antiapoptotic Bcl-2 family members
appear to function, at least in part, by interacting with and antagonizing
proapoptotic family members
(2). The BH13 domains of
BclXL form an elongated hydrophobic groove, which is the docking site for the
BH3 domains of proapoptotic binding partners
(3). It has been demonstrated
that BH3 domains from Bak and Bad proteins are required for binding to BclXL
and for mediating their proapoptotic effect
(4). A synthetic peptide
derived from the Bak BH3 domain binds recombinant BclXL protein in
vitro with high affinity
(3). Furthermore the Bak BH3
peptide alone is able to induce apoptosis when introduced into various cell
lines (5), suggesting that the
interaction between the BH3 domains from proapoptotic proteins and BclXL is
important in mediating the proapoptotic signal. Therefore small molecular
weight compounds that inhibit the BclXL-BH3 domain interaction could
potentially act as apoptotic modulators. Indeed significant progress has been
made in isolating compounds of this nature
(6). BH3I-1 and BH3I-2 were
discovered by screening a library of 16,320 compounds for ones that disrupted
the interaction between BclXL and a Bak BH3 peptide
(7), whereas antimycin A was
discovered serendipitously (8).
A few other compounds have also been discovered in silico, and they
are of diverse structures (9,
10,
11).
Natural products cover a molecular diversity not available from synthetic
libraries with an unrivaled success rate as drug leads
(12). We have, therefore,
carried out a large scale high throughput screen of natural product extracts
to uncover compounds that would disrupt the interaction between BclXL and the
Bak BH3 peptide. Here we report the identification of chelerythrine
(1,2-dimethoxy-12-methyl[1,3]benzodioxolo[5,6-c]phenanthridinium),
which is a natural benzophenanthridine alkaloid and a known protein kinase C
inhibitor (13), as an
inhibitor of BclXL-Bak BH3 peptide binding. Chelerythrine released cytochrome
c (CytC) from isolated mitochondria and induced apoptosis in
BclXL-overexpressing cells that were completely resistant to staurosporine or
etoposide. Chelerythrine thus represents the first BH3 mimetic identified
through high throughput screening of natural products.
 |
EXPERIMENTAL PROCEDURES
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Reagents and Cell LinesHuman SH-SY5Y and MCF7 cells were
maintained as described previously
(14). HCT116 cells and FDC-P1
cells were gifts from Bert Vogelstein (Dana Farber Cancer Institute) and David
C. Huang (The Walter and Eliza Hall Institute), respectively. LipofectAMINE
(Invitrogen) was used for transfections according to the user's manual, and
BclXL stably transfected SH-SY5Y cells were selected with 400 µg/ml
hygromycin B (Calbiochem) and maintained in medium containing 100 µg/ml
hygromycin B after 23 weeks of selection. The peptide protease
inhibitor z-VAD-fmk was from Enzyme System Products, Livermore, CA.
Staurosporine, H7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine), and
etoposide were from Sigma, and BH3I-1 was from Calbiochem.
Fluorescence Polarization (FP) AssayThe Bak BH3 peptide
labeled with fluorescein at the N terminus was synthesized by Mimotopes
(Clayton, Victoria, Australia) and purified by HPLC. The peptide was dissolved
in Me2SO at 1 mM. The reaction was carried out in a
total volume of 50 µl/well containing 50 µg/ml glutathione
S-transferase (GST)-BclXL
C19 and a 60 nM
concentration of the labeled peptide in assay buffer (50 mM Tris,
pH 8, 150 mM NaCl, and 0.1% bovine serum albumin). 5 µl of
natural product extracts were added to the wells, the reaction was incubated
at room temperature for 1 h, and FP values were determined using a Tecan Ultra
plate reader.
Isolation of ChelerythrineA
MeOH/CH2Cl2 (1:1) extract from the stems of Bocconia
vulcanica (Papaveraceae) was separated using a modified Kupchan solvent
partition method (15) to give
an active CH2Cl2 fraction. The active fraction was
fractionated using gradient C18 HPLC (2030% ACN, H2O
buffered with 0.1% formic acid) and preparative TLC on silica gel (1% MeOH,
CHCl3) to yield chelerythrine (0.9 mg), identical in all respects
to that reported previously
(16). Experiments subsequent
to the identification of the compound were performed with chelerythrine
chloride purchased from Sigma.
AntibodiesAnti-BCL-2 (100), anti-Bcl-xS/L
(S-19), anti-Bax (N-20), and anti-Bid (C-20) antibodies were purchased from
Santa Cruz Biotechnology. Anti-Bak (Ab2) was from Oncogene Research Products.
Anti-CytC (7H8.2C12) antibody was from BD Biosciences Pharmingen, and
antibodies against actin and cytochrome oxidase IV were from Sigma.
Production of GST-BclXL The DNA sequence encoding
BclXL
C19 was inserted into the GST fusion protein vector pGEX-TK4E
(14). The plasmid was
transformed into the Escherichia coli strain BL 21, and the fusion
protein was isolated as described previously
(14). GSTBclXL
19 was
eluted with 100 mM glutathione, 50 mM Tris-HCl (pH 8.0).
Eluate was dialyzed against phosphate-buffered saline containing 15% glycerol
and concentrated to 1 µg/ml using Amicon centrifugal concentrating
devices.
In Vitro Binding AnalysisLabeled Bax was prepared by in
vitro transcription/translation of pXJHA-Bax
(17) using the TNT
T7-coupled reticulocyte lysate system from Promega. The GST binding assay was
performed as described previously
(14) except that increasing
concentrations of chelerythrine were incubated with GSTBclXL
19 30 min
prior to the addition [35S]Bax.
CytC Release from Isolated MitochondriaMitochondria were
isolated from SH-SY5Y cells. Cells were suspended in isolation buffer (320
mM sucrose, 1 mM EDTA, 50 mM HEPES (pH 7.5),
1 µM dithiothreitol) and disrupted by 10 expulsions through a
27-gauge needle. Disrupted cells were centrifuged two times at 1000 x
g for 5 min to remove cell debris and nucleus. The supernatants were
centrifuged at 3000 x g to pellet the mitochondria. The
mitochondria pellets were resuspended in assay buffer (250 mM
sucrose, 2 mM KH2PO4, 5 mM sodium
succinate, 2 mM EGTA, and 10 mM HEPES (pH 7.5)) at 0.5
mg/ml and treated at room temperature with the indicated compounds for 15 min
followed by centrifugation. CytC released into the supernatant was subjected
to fractionation by 10% SDS-PAGE followed by Western blotting analysis.
Flow CytometryFor detection of sub-G1 DNA, cells
were washed once, resuspended in 200 µl of phosphate-buffered saline, and
fixed in a 50-fold excess of ice-cold 70% ethanol. Cells were recovered by
centrifugation at 1000 x g for 5 min at 4 °C, washed,
stained with 50 mg/ml propidium iodide for 30 min at room temperature, and
analyzed in a FACScan flow cytometer (BD Biosciences). Mitochondrial potential
change as measured by JC-1 staining was performed in accordance with the
manufacturer's instructions (Molecular Probes). A minimum of 10,000
cells/sample were analyzed.
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RESULTS AND DISCUSSION
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Identification of Chelerythrine as an Inhibitor of BclXL and BH3
Peptide InteractionA high throughput screen based on FP
(7) was devised to identify
compounds that disrupt the interaction between BclXL and the BH3 domain of
Bak. A total of 107,423 extracts prepared from plants, actinomycetes, fungi,
marine invertebrates, and marine bacteria were screened. Twelve extracts were
chosen for isolation of active compounds, and the active principle of four
extracts from plants was found to be chelerythrine
(Fig. 1A).
Chelerythrine displaced the fluorescently labeled BH3 domain peptide from a
recombinant GST-BcLXL fusion protein with IC50 of 1.5
µM (Fig.
1B). Similar concentrations of chelerythrine with GST
protein and the labeled peptide produced no significant change in polarization
(Fig. 1B).

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FIG. 1. Chelerythrine is an inhibitor of BclXL-BH3 domain peptide
interaction. A, structure of chelerythrine. B,
displacement of fluorescein-labeled BH3 peptide from GST-BclXL by
chelerythrine (filled diamond). Displacement of the BH3 peptide is
indicated by a decrease in polarization. mP, millipolarization.
Polarization readings with BH3 peptide and GST protein in the presence of
increasing concentrations of chelerythrine are shown with open triangles.
C and D, dose-dependent displacement of [35S]Bax from
GST-BclXL by chelerythrine. C, GST-BclXL bound on
Sepharose-glutathione beads was preincubated with the indicated concentrations
of chelerythrine for 30 min followed by the addition of an equal amount
(500,000 cpm) of [35S]Bax. GST protein bound on beads was used as a
negative control. Autoradiographs of [35S]Bax and Coomassie Blue
images of GST-BclXL and GST are shown. D, the autoradiograph in
C was scanned using a densitometer to quantify the bands, and the
relative amount of Bax bound to the beads was determined. CTR,
control.
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Chelerythrine Disrupts the Interaction between BclXL and
BaxThe ability of chelerythrine to displace the Bak BH3 peptide in
the FP assay suggests that it may be able to displace BH3-containing proteins
from BclXL. In vitro translated [35S]Bax bound
specifically to GST-BclXL immobilized on glutathione beads, and the addition
of chelerythrine resulted in a dose-dependent decrease in Bax binding
(Fig. 1, C and
D). Chelerythrine was, however, unable to disrupt
interaction between the Caenorhabditis elegans sex determination
proteins [35S]FEM3 and GST-FEM2
(18) immobilized on
glutathione beads (data not shown), suggesting that the action of
chelerythrine on Bax and BclXL was specific. The solution structure of Bcl-2
has been solved (19), and the
data suggest that Bcl-2 and BclXL have highly similar three-dimensional
structures, including the hydrophobic groove. Interestingly we found that the
binding of Bax to Bcl-2 was disrupted by chelerythrine in a dose-dependent
manner (data not shown).
Chelerythrine-mediated Apoptosis Exhibits Characteristic Features
Similar to Cell Death Induced by Proapoptotic Members of the Bcl-2
FamilySince the mitochondria play a key role in the control of
apoptosis and it is the main site where BclXL and Bcl-2 exert their function,
we evaluated mitochondrial function in response to chelerythrine with the
fluorescent dye JC-1 that allows the analysis of mitochondrial potential
(
m). Treatment of human neuroblastoma SH-SY5Y
cells (20) with chelerythrine
at 2.5 and 5 µM for 16 h induced a substantial decrease in
mitochondrial potential as indicated by an increase in JC-1 green fluorescence
(Fig. 2, A and
B). Chelerythrine-induced mitochondrial potential changes
were partially inhibited by the broad spectrum caspase inhibitor ZVAD
(Fig. 2, A and
B), similar to reports showing that Bax-induced
mitochondrial potential change was partially sensitive to caspase inhibition
(21). Treatment of SH-SY5Y
cells with chelerythrine also induced the appearance of sub-G1 DNA
that is indicative of apoptosis (Fig. 2,
C and D). The appearance of sub-G1
DNA is totally blocked by the addition of ZVAD
(Fig. 2, C and
D), which is consistent with the notion that DNA
fragmentation is dependent on caspase activation
(22). The ZVAD-treated cells
without sub-G1 DNA, however, were not viable since they were unable
to grow upon replating on fresh tissue culture plates (data not shown).
Similar to apoptosis mediated by proapoptotic members of the Bcl-2 family,
inhibition of caspases only slows down but does not abrogate the cell death
process (23,
24). The change in
mitochondrial potential and the appearance sub-G1 DNA upon
chelerythrine treatments were observed in two other cell lines, HCT116, a
colon carcinoma cell line, and MCF7, a breast cancer cell line (data not
shown), suggesting that the effect is not limited to SH-SY5Y cells.

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FIG. 2. Chelerythrine mediates apoptosis through the mitochondrial pathway.
A, chelerythrine induces mitochondrial depolarization that is
partially blocked by ZVAD-fmk. SH-SY5Y human neuroblastoma cells were treated
with the indicated concentrations of chelerythrine in the presence or absence
of the broad spectrum caspase inhibitor ZVAD-fmk (20 µM). After
16 h, cells were harvested, stained with JC-1, and analyzed by flow cytometry.
The increases in JC-1 green fluorescence (FL1) indicate the degree of
depolarization in the mitochondria. B, chelerythrine-induced DNA
fragmentation in SH-SY5Y cells is efficiently blocked by ZVAD-fmk. SH-SY5Y
cells were treated with the indicated concentrations of chelerythrine in the
presence or absence of ZVAD-fmk (20 µM), and 48 h later they
were fixed and stained with propidium iodide. Samples were analyzed by flow
cytometry. Percentages of sub-G1 DNA are shown. Data are
representative of at least three experiments. DNA Con., DNA
content.
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Chelerythrine Triggers CytC Release from Isolated
MitochondriaMany death stimuli trigger apoptosis through the
release of CytC from the mitochondrial intermembrane space to activate Apaf-1,
thus coupling this organelle to caspase activation. Treatment of SH-SY5Y cells
with etoposide, staurosporine, and H7
(20) as well as chelerythrine
induced mitochondrial potential change, CytC release from the mitochondria
(Fig. 3, A and
B), and the appearance of sub-G1 DNA (data not
shown), which are hallmarks of apoptosis. However, if the action of
chelerythrine is on BclXL or Bcl-2 on the mitochondria, it should be able to
trigger CytC release directly from isolated mitochondria as observed with
proapoptotic Bcl-2 family members
(25,
26). To investigate this,
mitochondria were isolated from healthy SH-SY5Y cells and subjected to
treatment with various death stimuli. Chelerythrine released CytC from
isolated mitochondria in a dose-dependent manner
(Fig. 3, C and
D). Etoposide and other protein kinase C inhibitors such
as H7 and staurosporine were unable to do so
(Fig. 3, C and
D) even at concentrations exceeding the required amount
to induce apoptosis in intact cells (Fig.
3, BD, and data not shown). The interactions of
Bax or Bak with BclXL in the mitochondrial preparation, if any, appeared to be
very weak (data not shown). It was therefore technically difficult to
determine whether there was a reduction in heterodimerization between the
proteins upon chelerythrine treatment. It is possible that the CytC release
represents a direct antagonistic effect of chelerythrine on BclXL/Bcl-2
function.

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FIG. 3. Chelerythrine targets mitochondria directly to release CytC.
A, chelerythrine (CHE), staurosporine (Sts), H7,
and etoposide (Etop) are apoptotic stimuli that induce mitochondrial
potential change in SH-SY5Y cells. Cells were treated with either
chelerythrine (10 µM), staurosporine (0.5 µM), H7
(50 µM), or etoposide (50 µM). After 16 h, cells
were harvested, stained with JC-1, and analyzed by flow cytometry. B,
apoptosis stimuli induce CytC release in intact cells. Cells were treated with
the indicated compounds at their respective concentrations as in A
for 24 h and harvested for cytosol isolation. CytC released in the isolated
cytosol was analyzed by Western blotting analysis. The same blot was stripped
and probed for actin to indicate equal loading of cytosolic extracts.
C, chelerythrine, but not the other apoptotic stimuli, selectively
mediates release of CytC from isolated mitochondria. Mitochondria were
isolated as under "Experimental Procedures" and incubated at 30
°C with the indicated compounds for 15 min. CytC released into the
supernatant was determined by Western blotting analysis. Triton X-100
(TX100) was used to lyse the mitochondria to show the total pool of
CytC. The presence of the integral mitochondrial protein cytochrome oxidase IV
(COX) in the supernatant was only detected in the Triton
X-100-treated sample. D, dose-dependent release of CytC from isolated
mitochondria by chelerythrine. The autoradiograph in C was scanned
using a densitometer to quantify the bands, and the data were plotted as a
histogram to show the relative amount of CytC released. Data are
representative of at least three experiments. CtR, control.
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Chelerythrine Induces Apoptosis in BclXL-overexpressing SH-SY5Y
CellsOverexpression of Bcl-2 or BclXL is able to block cell death
induced by many forms of death stimuli, e.g. radiation and most
chemotherapeutic drugs (2,
27). The limited
concentrations of endogenous factors serving the apoptotic signaling pathway
preceding the mitochondria step enable BclXL overexpression to block these
signals. On the other hand, if a compound acts directly on BclXL, it should be
able to overcome the effect of overexpression of the protein easily since
cellular protein concentration, even in a state of overexpression, is limited
in comparison to concentrations achievable with small molecular weight
compounds. To test our hypothesis, we generated SH-SY5Y cells that overexpress
BclXL. In these cells the level of BclXL is greatly enhanced, while other
members of the Bcl-2 family such as Bax, Bak, and Bid stay relatively constant
with a moderate down-regulation of Bcl-2 level
(Fig. 4A). Treatment
of BclXL-overexpressing cells with staurosporine up to 1 µM did
not induce cell death as indicated by the lack of mitochondrial potential
change (Fig. 4B, data
not shown) as well as the absence of sub-G1 DNA
(Fig. 4C). In
contrast, the vector line was very sensitive to staurosporine-induced
apoptosis. Nearly 100% of the cells exhibited mitochondrial potential change,
and 80% of the cells contained sub-G1 DNA when only a 100
nM concentration of the drug was added
(Fig. 4, B and
C). Similarly the apoptotic effects of etoposide
(Fig. 4, B and C) and H7 (data
not shown) were abolished by BclXL overexpression. Interestingly, although the
staurosporine- and etoposide-treated BclXL-overexpressing cells did not
undergo apoptosis, they were arrested at the G2 and S phase of the
cell cycle, respectively (see the supplemental figure). These observations are
consistent with previous reports indicating that cell cycle arrests induced by
genotoxic drugs are not affected by BclXL overexpression
(28). Overexpression of BclXL
was able to confer resistance to the killing effect of chelerythrine at low
concentration of up to 2 µM. At higher concentrations,
chelerythrine overcame the protective effect of BclXL and induced apoptosis in
these cells effectively (Fig.
4D). Similar results were obtained with a
Bcl-2-overexpressing, mouse interleukin-3-dependent, promyelocytic cell line,
FDC-P1, in which chelerythrine at concentrations higher than 1.25
µM was able to overcome the protective effect of Bcl-2 (data not
shown). The data suggest that chelerythrine, unlike staurosporine, H7, and
etoposide, induces apoptosis by inhibiting BclXL/Bcl-2 directly.

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FIG. 4. Chelerythrine is an effective apoptosis inducer in BclXL-overexpressing
cells. A, expression profiles of Bcl-2 family proteins in
BclXL-overexpressing cells. Total cell extracts were prepared from SH-SY5Y
cells overexpressing BclXL or vector control lines. Cell lysates were
fractionated by SDS-PAGE followed by Western blotting analyses using specific
antibodies against the indicated proteins. Bak in mitochondrial extracts of
the indicated cells are shown. BD, data from the SH-SY5Y
vector line (closed diamonds) or BclXL-overexpressing SH-SY5Y line
(open diamonds) are presented. B, cells were treated with
staurosporine (solid lines) or etoposide (broken lines) for
24 h before they were harvested and stained with JC-1 for analysis of
mitochondrial potential change by flow cytometry. Numbers are percentage of
cells with green fluorescence. C, cells were treated with
staurosporine (solid lines) or etoposide (broken lines) for
48 h before they were stained with propidium iodide for DNA and analyzed by
flow cytometry. Numbers are percentage of cells with sub-G1 DNA.
D, BclXL-overexpressing SH-SY5Y cells are sensitive to
chelerythrine-induced apoptosis. Cells were treated with increasing
concentrations of chelerythrine and harvested at 24 or 48 h for mitochondrial
potential change (solid lines) or DNA content (broken lines)
assays, respectively. Error bars indicate standard deviation of data
from three independent experiments.
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Enhanced expression of antiapoptotic Bcl-2-related proteins in cancer cells
has been implicated in resistance to currently available antineoplastic agents
(2,
27). Chelerythrine has been
shown to exhibit cytotoxic activity against radioresistant and chemoresistant
squamous carcinoma cells and p53-deficient cells
(29). It delays tumor growth
in an experimental model with relatively mild toxicity to the animal
(29). Our results indicate
that chelerythrine may act as a BH3 mimetic that is able to circumvent the
upstream antiapoptotic barriers in transformed cells and thus can be explored
as a potential anticancer therapeutic.
The inhibitors of BclXL-BH3 interaction identified so far are all
proapoptotic in nature. However, the diverse structural differences among
these compounds suggest that they may act through multiple mechanisms in
affecting the Bcl-2 family proteins. Interestingly BclXL overexpression
confers slight protection against chelerythrine- and BH3I-1-induced apoptosis,
while it sensitizes the cells toward antimycin A3
(8). The identification of
chelerythrine as a novel inhibitor of BclXL-BH3 interaction adds to the
repertoire of reagents that are invaluable in defining the molecular
mechanisms by which proteins of the Bcl-2 family mediate their functions.
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FOOTNOTES
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* This work was supported by grants from the Agency for Science, Technology
and Research (A*STAR) of Singapore. 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. 
The on-line version of this article (available at
http://www.jbc.org)
contains a supplemental figure. 
¶
Adjunct staff of the Department of Pharmacology, National University of
Singapore. To whom correspondence should be addressed. Tel.: 65-68743740; Fax:
65-67791117; E-mail:
mcbyuck{at}imcb.nus.edu.sg.
1 The abbreviations used are: BH, Bcl-2 homology; CytC, cytochrome
c; H7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; GST,
glutathione S-transferase; Z, benzyloxycarbonyl; fmk, fluoromethyl
ketone; FP, fluorescence polarization; HPLC, high pressure liquid
chromatography. 
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ACKNOWLEDGMENTS
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We are grateful to Drs. Bert Vogelstein, David C. Huang, and Craig B.
Thomson for the generous supply of reagents. We thank Shugeng Cao for
technical assistance, Juan Jose Castillo (Faculty of Agronomy, University of
San Carlos, Guatemala City, Guatemala) for the collection of plants used in
this study.
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