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2. disease
3. cancer

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Effect of Interleukin-lp Converting Enzyme Inhibitor on Acute
Myelogenous Leukemia Progenitor Proliferation
By Zeev Estrov, Roy A. Black, Paul R. Sleath, David Harris, Quin Van, Ruth LaPushin,
Elihu H. Estey, and Moshe Talpaz
Interleukin-1P (IL-IP) converting enzyme (ICE) is a cysteine
protease that specifically cleaves precursor IL-1p to itsbiologically active form. Recent studies have also implicated
apoptosis in vertebrate cells. Because
ICE in the induction of
!L-l plays a major role in acute myelogenous leukemia (AML)
blast proliferation, we sought t o investigate the effect of ICE
inhibition on AML progenitors. To do this, we used bocaspartyl (benzyl) chloromethylketone (BACMKI an inhibitor
designed t o penetrate cells and bind covalently to theactive
site ofICE. Our preliminary experiments showed thatincubation of activated peripheral blood cells with 2.5 pmol/L of
BAMCK downregulated production of matureIL-lp but had
no effect on tumor necrosis factor-&. To test the effects of
the inhibitor onAML cells, we firstused the OCI/AML3 cell
line. We found that these cells produce IL-1p and bind the
biotinylated cytokine and that IL-l inhibitors, such as IL-l
neutralizing antibodies, IL-l receptor antagonist, and soluble
IL-l receptors, specifically inhibit OCI/AML3 proliferation,
indicating that IL-Ip isan autocrine growth factor for OCI/
AMW cells. The ICE inhibitor suppressed OCI/AMW growth
in a dose-dependent manner (at 0.4 t o 4 pmol/L) and downregulated mature IL-lp production, as assessedby Western
immunoblotting. Similar results wereobtained with marrow
aspirates from 16 AMLpatients. The ICE inhibitor suppressed proliferation ofAML precursors (by up t o 78%;
mean, 44%) in a dose-dependent fashion at concentrations
ranging from0.4 t o 5 pmol/L but not proliferationof normal
marrow progenitors; the suppressive effect was reversed by
IL-Ip. Furthermore, incubation of AML cells with 4 pmol/
L BAMCK downregulated the production of mature IL-Ip,
suggesting that the growth-inhibitory effect is mediated
through suppression of thebiologically active cytokine. Our
data indicate that inhibition of ICE suppresses AML blast
proliferation andsuggest that ICE inhibitors mayhave a role
in futuretherapies for AML.
0 1995 by The American Society of Hematology.
A
tivities, IL- lp is the predominant secreted form of IL- 1, and
the amount of IL-10 mRNA found in activated cells is usually 10- to 50-fold greater than that of IL-la." The protein
products of both the IL-la and IL-Ip genes are 3 I - k D precursor (pro) forms. Pro-IL-la is biologically active and
binds to IL- 1R without further processing, whereas IL-10
must first be processed from its inactive cytoplasmic precursor to an active 17.5-kD form.Ix Pro-IL-Ip is cleaved to its
mature form by a unique cell-associated cysteine protease
termed IL-10 converting enzyme (ICE)."-" Selective inhibition of this enzyme blocks production of mature IL-lp,22
offering a potentmeans for inhibition of IL-1 and hence
suppression of AML Proliferation. However, surprisingly,
ICE was recently reported to induce programmed cell death
in various cellular
suggesting that ICE also
serves as a human cell-suicide gene.
These observations prompted us to investigate the effect
of ICE inhibition on AML progenitor growth. We used an
ICE inhibitor, boc-aspartyl (benzyl) chloromethylketone
(BACMK), which is designed to penetrate cells, covalently
bind to the active site of ICE, and form an irreversible complex that inactivates its enzymatic activity.26We found that
the ICE inhibitor downregulated production of mature ILIO and suppressed the proliferation of both the OCUAML3
myeloid leukemia cell line and freshleukemic BM cells from
AML patients.
CUTE MYELOGENOUS leukemia (AML) is a clonal
hematologic malignancy characterized by abnormal
proliferation of myeloid leukemia cells.' In recent years,
extensive research has shown that, similar to normalhematopoietic precursors, AML progenitors respond to hematopoietic growth factors, produced by thebonemarrow
(BM)
stroma, accessory cells, or the leukemic cells themselves.'"'
One of these factors, interleukin-l (IL-l), is a polypeptide
hormone that possesses a wide spectrum of biologic activities. We and othersI4"' have found that IL-1 plays a major
role in stimulating the proliferation of AML progenitor cells
by modulating autocrine and paracrine growth-stimulating
pathway^.'".'^.'^ Therefore, the inhibition of IL-l activity either by blocking the interaction of this cytokine with its
cellular receptor or by neutralizing IL-1 has been hypothesized to suppress the proliferation of leukemic cells. Indeed,
specific IL-l inhibitors such as IL-1 receptor antagonist (IL1RA) and soluble IL-l receptors (sIL-1R) potently inhibit
the growth of AML progenitors in vitro.14"'
Recently, a novel strategy for inhibiting IL-I by suppressing its synthesis became available. Although the two
distinct forms of IL- I , IL-la and IL-l/?, share identical ac-
From the Departments of Bioimmunotherapy and Hematology,
The University of Texas M. D. Anderson Cancer Center, Houston,
m;and Immunex Corp, Seattle, WA.
Submitted April 27, 1995; accepted August 8, 1995.
Supported in part by National Cancer Institute Grant No. POI
CA 55164.
Address reprint requests to Zeev Estrov, MD, Departmentof Bioimmunotherapy, Box 302, U.T. M. D. Anderson Cancer Center, 1515
Holcombe Blvd, Houston, TX 77030.
The publication costsof this article were defrayed in part by page
chargepayment. This article must therefore be hereby marked
"advertisement" in accordance with I8 U.S.C. section 1734 solely to
indicate this fact.
0 I995 by The American Society of Hematology.
0006-4971/95/8612-0034$3.00/0
4594
MATERIALSANDMETHODS
ICE inhibitor. BACMK
was
synthesized as previously described," starting with boc(henzy1)-asparticacid. BACMK was dissolved in dimethyl sulfoxide (DMSO; Sigma Chemical co, St Louis,
MO) and further diluted in culture medium so that the final concentration of DMSO would not exceed 0.1%.
Cell lines. The OCI/AML3 cell line, originally established from
an AML patient's sample? was kindly provided by M.D. Minden
(Ontario Cancer Institute, Toronto, Ontario, Canada). The cells were
maintained in RPMI-1640 (GIBCO, Grand Island, NY) culture medium supplemented with 10% fetal calf serum (FCS; Flow LaboraBlood, Vol 86, No 12 (December 15). 1995: pp 4594-4602
M
M
M
M
M
M
M
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4595
EFFECT OF ICE INHIBITOR ON AML CELLS
Table 1. Clinical Data on AML Patients
Patient
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Age
(yr)/Sex
Cytogenetic
Abnormality
Misc
65F
-5,-7
Misc
Dip,-Y
t(15;17)
Misc
Misc
Dip,-Y
-5,-7
5lM
39lF
67lM
75lF
7
54lM
39lF
57lM
Dip,-Y
-5,-7
Misc
Dip,-Y
-5,-7
i 8
Dip,-Y
FAB
Category
(g/dL)
W BC
(XlOB/L)
M2
MO
M2
MI
M3
M2
10.0
9.7
8.5
6.8
11.1
9.5
2.8
1.3
29.6
99.4
17.2
89.7
58
49
18
19
25
808
MI
M4
MO
M5
MO
M2
MI
MI
M 5 81
MI
9.5
10.1
7.9
8.9
4.7
8.5
9.4
8.0
10.6
8.5
62.4
28.6
42.0
67.1
32.0
29.6
2.7
11.0
77 61.9
123.4
64
60
91
Hb
71
Platelets
(~1091~)
76
81
61
18
72
46
41
65
%
Blasts
12
6
94
84
89
50
91
75
35
85
94
44
54
93
% Blasts
in B M
86
84
73
73
69
57
86
74
67
40
73
82
73
95
Abbreviations: FAB, French-American-British;Hb, hemoglobin; WBC, white blood cells; BM, bone marrow; Misc, miscellaneous; Dip, Diploid.
tones, McClear, VA) or RPMI-1640 supplemented with 50 nglmL
insulin-like growth factor-l (IGF-1; Boehringer Mannheim Biochemicals, Indianapolis, IN) and 1% (wt/vol) human albumin (Sigma
Chemical CO).The HL60 (promyelocytic leukemia) and K562
(chronic myelogenous leukemia blast crisis) cell lines were obtained
from the American Type Culture Collection (ATCC; Rockville, MD)
and maintained in RPMI-1640 medium containing 10% FCS. The
growth factor-dependent AML cell line M07eZ8was kindly provided
by A. Ciarletta (Genetics Institute, Cambridge, MA) and the erythroleukemia line *lz9 was obtained from ATCC. Both lines were
maintained in 20% FCS, 2 mmoVL glutamine, and 10 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor
(rhGM-CSF Immunex Corp, Seattle, WA).
Subjects. BM aspirates were obtained from 16 AML patients
with high marrow blast counts (14 patients with >66% and 2 patients
with 57% and 40% BM blasts, respectively). Clinical data on the
patients are shown in Table 1. For control studies, we used peripheral
blood cells from healthy donors and BM samples from seven hematologically normal volunteers. These studies were performed with
the patients' informed consent and were approved by the Human
Experimentation Committee of our institution.
Analysis of mature IL-lp production by peripheral blood cells.
Human peripheral blood mononuclear cells were isolated and stimulated with lipopolysaccharide (LPS) as previously de~cribed.~'
The
cells were then resuspended in medium containing [3sS]-labeledmethionine and cysteine with either 1% DMSO or DMSO plus 2.5
pmoVL BACMK. After a further 1 hour of stimulation, l-mL aliquots of the medium were taken, and IL-lp or tumor necrosis factora (TNFa) was precipitated with rabbit antisera (Immunex Corp) and
protein A-Sepharose. The precipitated material was then analyzed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) followed by fluorography."
IL-Ip enzyme-linked imrnunosorbent assay (ELISA). The ELISA
was performed using an IL-lp ELISA kit (Cistron Biotechnology,
Pine Brook, NJ), as previously described." Cell lysates and standard
dilutions of IL-lp (20, 50, 100, 200, 500, and 5000 pg1mL) were
added to test wells in duplicate and incubated for 2 hours at 37°C.
The test wells were then washed three times with phosphate-buffered
saline (PBS), incubated with rabbit IL-1p antiserum for 2 hours,
washed as previously described, and incubated for 30 minutes with
goat antirabbit IgG conjugated to horseradish peroxidase. The test
wells were vigorously washed, and a substrate (0-phenylenediamine
dissolved in 3% hydrogen peroxide solution) and 4 N sulfuric acid
were added. The color intensity wasread within 15 minutes at a
wavelength of 490 nm using a microplate autoreader (model EL309; Biotek, Vinooski, VT). The average net optical density (OD)
of the standard IL-10 concentration was then plotted, and the amount
of IL-1p in each sample was determined by interpolation from the
standard curve.
Receptor binding assay. To detect IL-1R on OCVAML3 cells,
we used a flow cytometric assay.32We first biotinylated IL-lp using
a modification of a previously described method.33Briefly, 10 pg
IL-1p (Boehinger Mannheim Biochemicals) was dissolved in 0.1
mL of 50 mmom sodium bicarbonate buffer, pH 8.5, and added to
a test tube containing 12.5 pL of NHS-LC-Biotin (Pierce Chemical
CO, Rockford, L)dissolved in water. The test tube was incubated
at room temperature for 30 minutes. The unreacted biotin was then
removed by dialysis using Slide-A-Lyzer Cassette (Pierce Chemical
CO) and the biotinylated IL-1p was stored at 4°C. OCVAML3 cells
were washed twice in PBS and lo5 cells were incubated with biotinylated IL-lp for 60 minutes. After 30 minutes of incubation with
streptavidin-phycoerythrin (PE; Caltag Laboratories, South San
Francisco, CA), the cells were washed again in 2% bovine serum
albumin in PBS. In blocking experiments with unlabeled IL-l@,cells
were incubated with the biotinylated IL-lp and a 100-fold molar
excess of the unlabeled cytokine. To assess background fluorescence,
an additional sample was simultaneously processed without the addition of the biotinylated cytokine. All incubations were performed on
ice to prevent internalization of the receptors. After staining, the
cells were evaluated on a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Cell line clonogenic assay. The clonogenic assay was performed
as previously de~cribed.'~
Briefly,OCVAML3 cells were cultured
in 0.8% methylcellulose (Fluka Chemical Corp, Ronkonkoma, NY),
10% FCS, and RPMI or under serum-free conditions in1% (voll
vol) methylcellulose, 50 ng/mL of IGF-l (Boehringer Manneheim
Biochemicals), and1%human
albumin (Sigma) at 2 to 4 X IO4
cellslmL. HL60 and K562 cells were cultured in a similar fashion
in methylcellulose supplemented with 10% FCS inRPMI, and M07e
and TFl cells were cultured with 20% FCS in the presence of 10
nglmL of rhGM-CSF. The following reagents were added at the
initiation of culture: IL-1p neutralizing antibodies (100 nglmL; Genzyme, Boston, MA), sIL-1R (500 nglmL; Immunex Corp), IL-IRA
( 1 0 0 ng/mL; Synergen Inc. Boulder, CO), increasing concentrations
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
ESTROV ET AL
4596
Precursor 11-1
Mature 11-1p
-
-
1 2 3 4
5 6 7 8
*TNF
Fig 1. Effect of BACMK on the release of mature IL-1p and TNFby LPS-stimulated peripheral blood leukocytes. Cells were cultured
and cytokine production was analyzed as described in the Materials
and Methods. The fluorogram shows the analysis for IL-lp fromcells
exposed to DMSO (lanes 1 and 2) and 2.5 pmol/L BACMK (lanes 3
and 41 and the analysis for TNF-a fromcells exposed to DMSO (lanes
5 and 6 ) and 2.5 WmollL BACMK (lanes 7 and 8).
(I
of BACMK (described above), Z-phe-chloromethyl-ketone(ZPCK;
Bachem Bio Science Inc. King of Prussia, PA) dissolved in DMSO
(Sigma) and further diluted in RPM1 so thatthe concentration of
DMSO would not exceed 0.1%. and/or 10 U/mL rhlL-lb (molecular
weight 17.500: Boehringer Mannheim Biochemicals). The culture
mixture wasplaced in 35-mmPetri dishes (Nunc Inc. Naperville,
IL) in triplicate and maintained at 37°Cwith 5% COz in air in a
humidified atmosphere. Colonies were counted after 7 days using
an inverted microscope. A colony was defined as a cluster of more
than 40 cells.
Western immunohlotting. Cell lysates were assayed for protein
Concentration using the BCA Protein Assay Reagent (Pierce Chemical CO). Each set of paired samples was then adjusted to have the
same protein concentration. The SDS-PAGE analysis was performed
by using a modification of the method of Laemmli.'s Antigens were
dissolved in Laemmli sample buffer at room temperature. Electrophoresis was conducted at constant wattage (10 W) in running buffer
cooled to 4°C. Stacking gels contained 4 9 (wt/vol) acrylamide, and
separating gels contained I 2 8 (wt/vol) acrylamide. Approximately
200 pL of sample protein was loaded into each of the appropriate
lanes. Proteins separated by using the SDS-PAGE technique were
transferred to nitrocellulose membranes. Transfers were performed
overnight at 30 V in a cooled (4°C) reservoir containing 25 mmol/
L Tris, 192 mmollL glycine, and 20% methanol (pH 8.3) transfer
buffer.'" Nitrocellulose membranes were removed from the blot apparatus and placed in a solution of Ponceau S stain (0.5% Ponceau
S and 1 % glacial acetic acid in H,O) to verify equal loading of
protein in control and treated samples." After staining for 5 minutes,
membranes were rinsed for 2 minutes and examined. Equal loading
of protein was verified and the membranes were then rinsed for an
additional IO minutes and immunoscreened. The membranes were
blocked in BLOTTO (5% dried milk dissolved in S0 mmol/L PBS)
for at least I hour at room temperature. The membranes were then
washed three times in PBS plus 0.5% Tween 20. Next, the membranes were incubated for I hour with polyclonal rabbit anti-IL-IP
antibodies (Endogen Inc.Boston. MA) or withnormal rabbit IgG
(used as a control) diluted 1:200 in PBS containing 0.5% Tween 20.
After incubation, the membranes were subjected to three IS-minute
rinses in PBS containing 0.5% Tween 20. Detection of bound antibody was accomplished using the ECL Western Blotting Detection
System (Amersham Corp. Arlington Heights, IL). The membranes
were incubated with antirabbit horseradish peroxidase-labeled antibody at a concentration of 1:2,000 in PBS plus 0.5% Tween 20 at
room temperature for I hour. After this incubation, the membranes
were washed in PBS containing 0.5% Tween 20 and bound antibody
was detected according to the ECL protocol. Chemiluminescence of
the membranes was detected byuseof X-OMAT ARS x-rayfilm
(Kodak, Rochester. NY) in stainless steel exposure cassettes (Sigma
Chemical CO).
Ar//~erent-ce//froctio~~uti~~tt.
Low-density BM mononuclear cells
obtained by Ficoll-Hypaque (Pharmacia. Piscataway, NJ) fractionation were incubated in plastic tissue culture dishes or flasks (Falcon
Plastics, Becton Dickinson, Oxnard. CA) with 10% FCSin a-medium (GIBCO). The fractionation procedure was repeated until no
cells adhered to the tissue culture dishes. Nonadherent cells harvested
in this way contained less than 3% monocytes as confirmed by the
following techniques: ( I ) microscopic differential count of at least
1 0 0 cells prepared with Wright's stain and (2) nonspecific (a-naphthy1 butyrate) esterase staining and immunocytochemical analysis
using CD14 (My-4) monoclonal antibodies (Coulter Immunology.
Hialeah, FL) to identify monocyte-promonocyte cells. as previously
described.'X.'"
T-cell depletion. T cells were depleted fromthe nonadherent
fraction by negative immunomagnetic selection."' In this modification of the negative immunomagnetic selection technique, nonadherent BM cells were incubated with CD3 monoclonal antibodies (Becton Dickinson) at a concentration of I pgll0" cells in PBSwith
0.25% FCS for 30 minutes at 4°C. The labeled cells were washed
three times and then incubated with goat antimouse IgG-conjugated
immunomagnetic beads (Advanced Magnetics, Cambridge, MA) at
4°C for 60 minutes in an end-over-end rotation at 20:l bead:cell
ratio. lmmunomagnetic bead-rosetted cells were removedusing a
magnetic particle concentrator (Advanced Magnetics). andunrosetted cells remaining in suspension wereharvested by a Pasteur
pipette. In some experiments, this procedure wasrepeatedtwice.
The T-lymphocyte-depleted population contained less than 3%CD3
cells as assessed byan immunocytochemical technique performed
on cytospun cells.'x.20
I
i
a
: c
Fluorescence intensity
Fig 2. Binding of biotinylated IL-lp to OCllAMUcells and competition with unlabeled cytokine. Background fluorescence was determined by incubation with streptavidin-PE alone. Blocking experiments were performed with a 100-fold molar excessof unlabeled
cytokine. la1 Background; (b) IL-lp; (c) blocking.
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EFFECT OF ICE INHIBITOR ON AML CELLS
4597
500
T
400
U)
P)
"
C
0
6c 300
0
2
4
Fig 3. Effect of IL-l inhibitors
on OCllAMU proliferation. The
E
means 2 SD of colony numbers
C
m
from triplicate cultures are depicted. As described in the Materials andMethods, 100 ng/mL of
IL-1p neutralizing antibodies(ILlp Ab.), 500 nglmL of slL-1R IdL1R).100
nglmLof IL-1RA (IL1RA). and 10 UlmL of IL-lp were
added at the initiation culture.
of
Results from one of three identical experimentsthatshowed
identical resultsare presented.
200
S
100
0
IlI
AML blast colony assay. A previouslydescribedmethodwas
used to assayAMLblastcolony
f~rmation?'.~'Briefly, 1 X IOs
nonadherent T-cell-depleted BM cells were plated in 0.8% methylcellulose in a-medium supplemented with 10% FCS either alone or
with the following growth factors: 15 ng/mL of rhGM-CSF. 15 ng/
mL of rhlL-3 (Immunex Corp), IO ng/mL of rhG-CSF (Amgen Inc.
Thousand Oaks, CA),or 50 nglmL of recombinant human stem cell
factor (rhSCF; Amgen Corp). BACMKwas dissolved in DMSO
as describedaboveandaddedattheinitiation
of the cultures at
concentrations ranging from 0.1 to 5 pmol/L in the absence or presence of I O UlmL of IL-IP. The cultures were incubated in 35-mm
Petri dishes in duplicate or triplicate for 7 days at 37°C in a humidified atmosphere of 5% CO? in air. AML blast colonies were microscopically evaluated on day7 of culture. A blast colony was defined
as a cluster of 20 or more cells. Individual colonies were plucked,
-
IL-1RA
SlL-l
R
IL-10
IL-1Ab.
IL-1RA
SlL-lR
+IL-lO
+11-10
+IL-10
smeared on glass slides, and stained to confirm leukemic cellular
composition (that the AML blast colony assay identifies blasts rather
than normal progenitors has been previously shown by cytogenetic
analysis of colonies obtained using this assay4').
Normal BM progenitor colonv culture assay. A modification of
a previously described assay was used.@
In brief, 2 X 10' nonadherentlow-density BM cells werecultured in 0.8% methylcellulose
with Iscove's modified Dulbecco's medium (IMDM; GIBCO), 30%
FCS, I O ng/mL of rhGM-CSF, and I .O UlmL of human erythropoie-
"""
c
0
h
I
I
\
1
2 300 -
Q
E
-- -
"
17.5 kDa +
Fig 4.
Effect of the ICE inhibitor on the production of matureIL-
10 by OCI/AMU cells. Cells were incubated in the presence or absence of 4 pmol/L BACMK for 24 hours, and the amount of mature
IL-1p produced by these
cells was assessed by Western immunoblotting as described in the Materials and Methods.
P2 200 -
m,
l
3
0
,
0
1.5
01
ICE Inhib.
* Inhib. +11-18
100 -
0.5
2
2.5
0 ZPCK
3
3.5
4
ICE Inhibitor @M)
Fig 5. Effect of ICE inhibitionon OCI/AMW proliferation. The
means of colony numbersfrom triplicate culturesare depicted. Cultures containing 10% DMSO (DMSO control) grew colonies at numbers that were not different from control
cultures. ICE inhibitor 2 ILl p and ZPCK was added at the initiation of culture, as described in
the Materials and Methods. One of four experiments with identical
results ispresented.
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4598
ESTROV ET AL
HL60
I
I
1
120
l
I
l
M07e
Y
400
200
100
0
300
Mean Mumber of Colonies
500
Control
DMSO
0.4
1 0
2.0
4.0
ICE Inhibitor (JIM)
5.0
11-10 11-10
+4 #M
ICE Inh.
Fig 6 . Effect of ICE inhibitor on the proliferation of
HL60,K562,
Fig 8 . Effect of ICE inhibition on the proliferation of AML marrow
TFI, and M07e cells. The means of colony numbers from triplicate
progenitors. The means? SD of AML colony numbers from triplicate
cultures are depicted. Colonies were
grown in methylcellulose as
cultures of patients 10 through 14 are presented as a percentage of
described in the Materials and Methods (Control) and in the presencecontrol. The means of colony numbers for cultures grown
in the
of either 5p m o l / L ICE inhibitor (BACMK) or DMSO at a concentration presence of GM-CSF (control) were428, 1,014,210, and 141, respecidentical to that used to dissolve
BACMK.
tively.
of 40 or more granulocytc or tnonocyIc-macrclphage cclls o r both).
I n d i v i d u a l colonies were plucked from the cullurcs wilh a micropipclte and analy/.cd f o r cellular composition.
RESULTS
RACMK ,supprrsscs tlzc prorluc.liorz of I ~ N ~ L II L~- PI P by
peripherd Idood cel1.s. To test the efficacy of the ICE inhibitor, we used LPS-stimulated peripheral blood lcukocytcs
and measured the production o f the mature form of L I P
and TNF-LYby these cells, a s described abovc. As shown i n
Fig 1, exposurc o f thc cells to 2.5 pInol/L BACMK suppressed the production of rnaturc IL- 10 by 85%. However,
incubation of these cells with BACMK did not result in a
decrease i n either the level of p~-o-Il,-Ipor thc production
of TNF-n, indicatingthat, at this concentration,BACMK
docs not exert a nonspccific cffect o n protcin secretion.
IL- l o is an riutocrirw grnwthjucfr~rfi,~OCIIAML-.I c r l l s .
To investigatetheeffect of ICE inhibition on AML cells,
we first used the OCVAML3 cell line. Using ELISA, we
found that the lysates of OCI/AML3 cells andtheir supernatant, harvested at a
log phase of growth, contain ILat
489 pg/2 X I O 7 cells and 49 pg/mL, respcctively. We then
used hiotinylated ILIP to evaluate whether OCUAML3
cells express 11,- 1R. Our Iluorocytomctric studiesshowed
that OCVAML3 cells bind biotinylated L I P and that the
binding is reversed by thcunlabeled cytokinc, indicating
its specificity (Fig 2). I n ;I clonogcnicassay, OCI/AML3
proliferation was suppressed by IL- 1 P-neutraliLing antihodics, lI,-IRA, 0 1 slI,-ll<, and the growthinhibition was reversed by L I P (Fig 3). Taken together, these data suggest
that 11,- lo acts as a n autocrinc growth factor lor OCVAML3
cells.
HACMK s u p p t - c s s c s p r o o t ~ t c ~ i oofn mcrturr' IL-ID by O C I /
AMLS c~clls. To evaluate whetherthe ICE inhibitor can
penetrate AMI, cclls and inhibit the production of hiologically active IL- ID, we inculxltcd OCliAML3 cells with
BACMK for 24 hoursand then measured theamount of
Io
-
~~
GM-CSF
G-CSF
FCS
~~~
~
r
P
,
"
IL-3
1-
SCF
Fig 7. Effect of ICE inhibition on AML progenitor proliferation. The
means of colony numbers from duplicate or triplicate cultures are
presented as a percentage of control (the mean number of colonies
grown in the presence of 10% DMSO butin the absence of BACMK).
B M cells of patients 1 through 5 were grown in the presence of FCS
alone (the means of colony numbers in control cultures were 340,
600, 408,602, and 564, respectively), G-CSF (the means of colony
numbers in control cultures were 392,1,422,
938,796, and 1,501,
respectively), and rhSCF (the means of colony numbers
in control
cultures were414,831, 480,904, and 1,703, respectively); of patients
3 through 8 in the presence of IL-3 (means of colony numbers
in
control cultures were 487,1,341,986,801,15,090,
and 321, respectively); and of patients 5 through
9,15, and 16 in the presence of
GM-CSF (the means of colony numbersin control cultures were 781,
1,909, 928, 1,019, 1,887, 183,
and 146, respectively).
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4599
EFFECT OF ICE INHIBITOR ON AML CELLS
... ... ... ... ... ... ... ... ... ... .......................... ... ... ... ... ...
. . . . . . . . . . . . . . . . . . .
.....
...
.:::::::::::-.:::.::
+Control
OWMK
200-
I
'?
1,000,000
z
'LlsO0
L
100,000
n
10,000
I
E
2100-
3
50-
1,000
0-
..
100
10
1
....................................
0.1
I
l
l
l
l
l
l
l
l
l
l
t
1 2 3 4 5 6 7 0 9 101112131415
Days
Patiom 2
m o m3
Fig 9. Effect of ICE inhibitor on normal BM cell viability (AI and progenitor growth (B).(A) Normal BM low-density cells from two different
individuals (samples1 and 2) were maintained in duplicate in RPMI-1640 supplemented with 10% FCS in the presence and absence of either
BAMCK or DMSO. Viable cells were enumerated using
the trypan blue exclusion test. The means of viable cell numbers from duplicate cultures
are depicted. (B1 The means f SD of CFU-GM (top) and BFU-E (bottom) colony numbers of triplicate cuttures obtained from
3 different
hematologically normal individuals (designatedas patients 1, 2, and 3) are presented.
mature IL- 10 produced by these cells. We found that 4 ymoV
L of the inhibitor (the highest concentration we used in most
experiments) significantly reduced the amount of mature IL1 0 produced by OCUAML3 cells (Fig 4).
BACMK suppresses OCI/AML3 colony
proliferation.
We then tested the effect of the ICE inhibitor on the proliferation of OCUAML3 cells. As shown in Fig 5, BACMK suppressed OCUAML3 colony growth in a dose-dependent fashion at concentrations ranging from 0.4 to 4 ymoVL (by up
to 60%); ZPCK, a similar compound designed to inhibit
chymotrypsin-like proteases, did not affect OCUAML3 proliferation. The addition of 10 U/mL of IL-10 at the initiation
of the culture substantially reversed this inhibitory effect,
indicating that the inhibition was due to a reduction in the
level of mature IL-10. Identical results were obtained when
the same experiments were performed in the presence of
FCS (data not shown). In contrast, BACMK did not affect the
growth of either GM-CSFOL-3-dependent (TF1 and M07e)
or-independent (HL60 and K562) AML cell lines (Fig 6).
Interestingly, although IL-1 is a strong inducer of GM-CSF,"
neither IL-10 (with or without GM-CSF) nor its inhibitors
significantly affected the proliferation or viability of the two
GM-CSFflL-3-dependent cell lines (data not shown).
BACMK suppresses AML progenitor proliferation. The
next experiments were performed to examine the effect of
ICE inhibition on AML progenitor proliferation. We used
BM cells from 16 newly diagnosed AML patients (Table 1).
We found that 4 ymoVL BACMK suppressed AML colony
proliferation when AML blast progenitors were grown either
in the presence of FCS alone (mean inhibition, 36%; range,
12% to 65%) or with the addition of G-CSF (mean inhibition,
43%; range, 25% to 60%), GM-CSF (mean inhibition, 62%;
range, 48% to 78%), IL-3 (mean inhibition, 37%; range,
20% to 55%), or rhSCF (mean, 44%;range, 48% to 78%)
(Fig 7). When GM-CSF wasused as a growth factor,
BACMK suppressed AML colony proliferation in a dosedependent manner at concentrations ranging from 0.4 to 5
ymoVL (at a higher concentration, BACMK's solvent,
DMSO, was toxic), and BACMKs suppressive effect was
reversed by IL-lP (Fig 8). In sharp contrast, 5 ymoVLof
BACMK affected neither the viability of low-density normal
BM cells maintained in RPMI-1640 supplemented with 10%
FCS (Fig 9A) nor the number of normal marrow CFU-GM
and BFU-E colony-forming cells (Fig 9B).
BACMK suppressed production of mature IL-10 by AML
cells. To delineate the mechanism by which the ICE inhibi-
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
ESTAOV ET AL
4600
A
17.5 kDa -+
B
4
17.5 kDa -b
Fig 10. Effect of ICE inhibitor on the production of mature lL-l/3
by patients' AML marrow cells. B M cells from patients 15 (A) and 16
(B1were incubated for 24 hours in thepresence or absence of4 prnoll
L BACMK, and the amount of mature IL-lp produced by these cells
was measured as described in the Materials and Methods.
tor suppresses AML progenitor proliferation, AML cells obtained from patients 15 and 16 were incubated for 24 hours
in the presence or absence of 4 pmol/L of BACMK, and the
production of mature IL- I D was then measured using Westem immunoblotting, as described above. As shown in Fig
IO, we found that BACMK significantly suppressed the production of mature IL-Io in cells obtained from patient 15
(Fig IOA) and from patient 16 (Fig IOB).
DISCUSSION
Human ICE is a substrate-specific cysteine protease that
cleaves the 3 I-kD pro-IL-Ip at the A~p'"-Ala"~bond. ICE
has no homology to other known Cys or Ser proteases."
Active ICE consists of a 20-kD subunit, containing the active
site, and a IO-kD subunit, bothofwhich derive from an
inactive 45-kD p r e ~ u r s o r . ~ ~Recently,
~ ' ~ ~ ~ "ICE
~ ~ was found
to share 28% sequence homology with one of the "death
genes" (ced-3) in the nematode Caenorhabditis elegans.
Furthermore, overexpression of ICE in rat cells causes cell
death; this effect depends on its protease activity.'"''l In addition, specific inhibition of ICE prevents cell death in neurons
deprived of growth factors," suggesting that ICE may also
be a human cell-suicide gene. In a recent study, Wanget
a147isolated and characterized a ced-3-related gene termed
Ich-I. Ich-l mRNA was found to splice into two different
forms. One encodes the protein ICH-IL, which contains
amino acid sequences homologous to both the p20 and p10
subunits of ICE, and the second form encodes ICH-ls, a
3 12 amino acid truncated version of the ICH-IL protein.
Whereas overexpression of Ich-I,. induces programmed cell
death, the overexpression of Ich-ls suppresses Rat-l cell
death induced by serum depri~ation.4~
In contrast to these
results, thymocytes and macrophages from ICE-deficient
mice were found to undergo apoptosis normally, but the
mice had a major defect in the production of mature IL-lp
and were resistant to endotoxic
Because IL-10 is produced by AML BM cells and provides AML progenitors with a growth advantage,'"'" we
hypothesized that inhibition of ICE, resulting in suppression
of mature IL-10 production, would inhibit AML progenitor
proliferation. However, the recent data showing that ICE can
induce apoptosis in human cells2"2ssuggested that inhibition
of ICE would stimulate AML cell proliferation.
To investigate the effect of ICE inhibition on AML cell
proliferation, we used a small synthetic molecule, which may
have clinical utility, instead of the cowpox virus crmA gene,
which encodes a specific inhibitor of ICE:'
BACMK was
designed to penetrate cells, form an irreversible complex
with active ICE, and block its activity.'" These properties of
BACMK have been shown in a recent study in which inhibitionof ICE prevented apoptosis ofmammary epithelial
cells?" In our preliminary experiments, we found that the
inhibitor indeed suppressed the production of mature IL-I@
but not of TNF-a by LPS-stimulated peripheral blood cells.
Similar results were recently reported by Miller et al." who
examined the effect of the ICE inhibitor WIN 67694 in
murine macrophages and a murine model of inflammation.
We therefore used BACMK to examine the effect of ICE
inhibition on AML cell proliferation.
Our initial studies were performed with the OCI/AML3
cell line. These cells produce IL-lp and express IL-IR, and
the cells' growth is inhibited by IL-l -inhibitory molecules.
These findings suggest that IL-Ip is an autocrine growth
factor for the OCI/AML3 cell line. We therefore tested the
effect of the ICE inhibitor on OCUAML3 proliferation and
on the production of mature IL-ID. We found that BACMK
significantly suppressed OCI/AML3 colony formation in a
dose-dependent manner and inhibited the cleavage of proIL-ID to its biologically active form. The colony-inhibitory
effect was reversed by IL-10 and was not obtained with
ZPCK, a compound with an enzymatic activity similar to
that of BACMK. Furthermore, BACMK did not suppress
the proliferation of two GM-CSFAL-3-dependent cell lines
whose growth was not affected by either IL-Io or its inhibitors and by two growth factor-independent cell lines (HL60
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
EFFECT OF ICE INHIBITOR ON AML CELLS
and K562). Taken together, these results suggest that the
effects induced by the ICE inhibitor are specific and mediated through the inhibition of biologically active IL-10.
We then studied the effect of the ICE inhibitor on AML
progenitors. Similar to other IL-1 inhibitors such as IL-1RA
and s I L - ~ R , ' ~BACMK
"~
suppressed AML progenitor proliferation either in the presence of FCS alone or with the addition of G-CSF, GM-CSF, IL-3, or rhSCF. Interestingly, the
antiproliferative effect exceeded that of sIL-1R and IL-1RAL4
and, although IL-3, GM-CSF, and SCF were shown to induce
a similar effect on the production of IL-10 by AML blasts,52
BACMK was more effective in the presence of GM-CSF
than in the presence of other growth factors. Similar to its
effect on OCVAML3 cells, BACMK suppressed the production of mature IL-10 by patients' AML cells, and its antiproliferative effect was reversed by IL-1, indicating that the
inhibitory effect is mediated through blocking the production
of biologically active IL-10. Similar to other IL-1 -inhibitory
molecule^,^^"^ BACMK did not affect either the in vitro
viability of normal BM cells or the proliferation of normal
marrow CFU-GM and BFU-E colony-forming cells.
In conclusion, our data indicate that BACMK is capable
of penetrating hematopoietic cells and effectively suppressing the biologic activity of ICE. In addition, these data
support previous studies showing that inhibition of IL-1 suppresses AML cell proliferation. Finally, our study suggests
that small synthetic ICE inhibitors may prove to be effective
agents for the treatment of AML, particularly in the setting
of minimal residual disease in which conventional chemotherapy is i n e f f e ~ t i v e . ~ ~
ACKNOWLEDGMENT
We thank Melissa Burkett for editing the manuscript.
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1995 86: 4594-4602
Effect of interleukin-1 beta converting enzyme inhibitor on acute
myelogenous leukemia progenitor proliferation
Z Estrov, RA Black, PR Sleath, D Harris, Q Van, R LaPushin, EH Estey and M Talpaz
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