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1996 87: 858-864
Suppression of apoptosis during cytokine deprivation of 32D cells is
not sufficient to induce complete granulocytic differentiation
JE Rodel and DC Link
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RAPID COMMUNICATION
Suppression of Apoptosis During Cytokine Deprivation of 32D Cells Is Not
Sufficient To Induce Complete Granulocytic Differentiation
By Jill E. Rodel and Daniel C. Link
The role of cytokines in the control of hematopoietic cell
differentiation remains controversial. Two general models
for the cytokinecontrolofhematopoieticdifferentiation
have been proposed.
In the stochastic model, cytokines
provide proliferative and survival signals to the differentiating
hematopoietic cell, but they do not provide specific lineage
commitment signals. In the instructivemodel,cytokines
transmit specific signalsto multipotent hematopoietic cells,
thereby directing lineage commitment. To distinguish between these two modelswith respectto granulocyte colonystimulating factor (G-CSFI and granulocytic differentiation,
we used the 32Dc13 cell line, which is capable
of differentiating into granulocytes in response to G-CSF.32Dcells
transfected with either bcl-2 or bcl-X, showed prolonged
survival in medium containingnocytokinesupplement.
Cells surviving in these cultures developedthe segmented
nuclei characteristicof mature neutrophils. However, no induction of myeloperoxidase
activity or increase in cathepsin
G transcripts were detected. These data support a hybrid
model for the role of G-CSF in granulocytic differentiation;
although some features
of
granulocytic
differentiation,
namely nuclear segmentation, do
not require G-CSF and appear therefore to be preprogrammed in 320 cells, the complete maturation of these cells to granulocytes appears to
be dependent on G-CSF.
0 1996 by The American Society of Hematology.
T
delayedapoptosisupon
IL-3 removal. Examination of the
cells surviving in these cultures showed multilineage hematopoietic differentiation, including granulocytic differentiation. These data suggest that FDCP-Mix cells have a preprogrammed capacity for multilineage
hematopoietic
differentiation that is independent of hematopoietic growth
factors. On the other hand, Dong et a19 recently identified
point mutations in the G-CSFR of two patients with Kostmann syndromewhodevelopedacute
myeloidleukemia.
Kostmann syndrome is a rare congenital disorder manifested
by neutropenia and an arrest of myeloid maturation at the
promyelocyte or myelocyte stage.“’.’’ The pointmutations
caused a truncation of the carboxy-terminal cytoplasmic region of the G-CSFR. Expression of the truncated receptor
in a myeloid cell line yielded cells that proliferated rather
than differentiated in response to G-CSF. These data suggest
thatthe carboxy-terminal region of theG-CSFR may be
transmittingspecificdifferentiationsignals.
In agreement
with these data, several recent reports have identified a putativedifferentiation domain in the carboxy-terminalregion
of the G-CSFR.”.”
We have chosen to examine the role
of G-CSF in granulocytic differentiation by using the 32Dc13 model of in vitro
myeloiddifferentiation.
32Dcl3(here simplydesignated
32D) cells area nontumorigenic, diploid cell line that proliferates indefinitely in the presence of IL-3.I4 In the absence
of IL-3 and in the presenceof G-CSF, this cell lineundergoes
granulocytic differentiation. We generated stably transfected
32D clones that constitutively express either bcl-2 or bclX , and examined their survival and morphology in cultures
without IL-3. We show that these cells have aprolonged
survival in such cultures, butdo not undergo complete granulocytic differentiation without G-CSF.
HE GRANULOCYTE colony-stimulating factor(GCSF) is a polypeptide growth factor that regulates the
production,differentiation,
andfunction
of neutrophilic
granulocytes.’ Its effects are mediated through its interaction
with the G-CSF receptor (G-CSFR), a member of the cytokine receptor superfamily.’Theimportance of G-CSFto
granulopoiesis wasrecently shown in mice carrying a homozygous null mutation for G-CSF; these mice had
approximately 20% of normal circulating neutrophils and a correspondingdecrease
inmyeloidprecursorsintheir
bone
marrow (BM).’ The mechanismby which G-CSF, and hematopoietic growth factors in general, regulate hematopoiesis
is controversial. Two general models for the role
of cytokines
in controlling hematopoietic differentiation have been proposed.’ In the instructive model: cytokines transmit specific
signals to multipotent hematopoietic cells directing lineage
commitment. In thestochastic model,’” cytokines support
the proliferation and survival of lineage committed cells. A
majordistinctionbetweenthese
two models is that in the
instructive model the cytokine receptors are
transmitting specific lineage commitment signals.
Two recent reports highlight the controversy with respect
to G-CSF. Fairbairn et a1’ showed that constitutive expression of the oncoprotein bcl-2 in FDCP-Mix cells (a multipotent interleukin-3 [IL-3]-dependent hematopoietic cell line)
From the Division of Hematology, the Department of Medicine,
Jewish Hospital at Washington Univer.sity Medical Center, St Louis,
MO.
Submitted September 15, 1995; accepted October 31, 1995.
Supported by the James S. McDonnell Foundation and by National Institutes of Health Grant No. K08 HL02709.
Address reprint requests to Daniel C. Link, MD, Division of Hematology, Department of Medicine, Jewish Hospital at Washington
University Medical Center, 216 S Kingshighway Blvd, St Louis, M O
63/10.
The publication costsof this article were defrayedin part by page
chargepayment. This urticle must thereforebehereby
murked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8703-0046$3.00/0
858
MATERIALS AND METHODS
Cells and cell culrure. 32Dc 13 cells were provided by Dr James
N. Ihle (St Jude Children’s Research Hospital, Memphis,
TN) and
weremaintained in RPM1 1640 medium(GIBCO,GrandIsland,
NY) supplemented with 15% WEHI conditioned medium as a source
of IL-3, 10% fetal bovine serum (Harlan, Indianapolis, IN), and Lglutamine(CM + IL-3). WEHIcells were provided by Dr Greg
Longmore (Washington University, St Louis, MO) and were mainBlood, Vol 87,No 3 (February l), 1996: pp 858-864
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ROLEOFG-CSF
IN GRANULOCYTICDIFFERENTIATION
859
rd
P4
Fig 1. bcl-2 or b d X L expression in transfected 32D clones.
bcl-2 (lanes 1 and 2) orbcl-XL
(lanes 3 and 4) immunoblots of
representative clones. The bcl-2
antiserum is specific for human
bcl-2 and therefore does not detect endogenous murine bcl-2.
Blots werestrippedandincubated with an anti-p-tubulin antibody t o control
for
protein
loading.
cp
- 55 - 31 -- 1814
BcI-Z e
2
1
Tubulin-
taincd in RPM1 1640 medium supplemented with 10% fetal bovine
serum and L-glutamine (CM).
DNA comfrucfxccnd rr-ctn.$fwiot?.y. The eukaryotic expression
vector used. pBSRaEN (a gift of Dr Andrey Shaw. Washington
University, St Louis. MO). produces a single hicistronic message
encoding the protein o f interest and the neomycin phosphotransferase
gene transcribed from the SRa promoter.I5 cDNAs encoding human
hd-2'" or murine hc/-XL(unpublished sequence) were subcloned
into pRSRaEN (both cDNAs were gifts o f Dr Stanley Korsmeyer,
Washington University, St Louis, MO).
32D cells were transfected by electroporation. Cells. S X IOfi per
sample,werewashed
in RPMI medium and resuspended i n 300
120.0%
T
-
80.0%
!
0
+
-
100.0%
0.0%
pL o f RPMI.Twenty-five micrograms o f pRSRaEN vector and
constructs containing hcl-2 or hc/-XIwere mixed with the cells and
transferred to 2-mm gap cuvettes (RTX, San Diego, CA) on ice.
Electroporation was performed using a RTX 6000 electroporation
device at 200 V, 1.200 pF capacitance. and :I resistance setting o f
R4. Samples were transferred to C M IL-3 and incubated at 37°C.
After 24 hours the cells werewashedandresuspended
in CM +
IL-3 with G418 at a concentration o f 0.8 mglmL (GIRCO). Clonal
stable transfectants were selected hy limiting dilution and culture in
96-well microtiter plates.
Antisera nnd irntnurtohlr~rfin~.Cells were washedonce with
RPM1 and lysed in Tris-buffered saline (TBS) containing 1% NP40.
Ne0
Neo + G-CSF
Bcl-2
Bcl-XL
L " 2
4
Days
"
6
8
Fig 2. Survival of 32D-bc12 or 32D-bclX cells after
cytokine deprivation. Cells were washed and cultured (8 x I O 5 cellslmL) in serum-containing medium
in the absence or presence of G-CSF at 10 nglmL.
An aliquot of cells was removed at the indicated
times and viable cells determined by trypan blue
staining. The percent cell survival was determined
by dividing the number of viable cells per milliliter
by the initial cell concentration. Data are shown as
the mean value f SD of three independent experiments.
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RODEL AND LINK
860
0 neo+G
fa bel-XL
U bel-XL+G
0 bel-2
bel-2 + G
day5
Fig 3. Survival of 32D-bc12 or 32D-bclX cells in G-CSF-containing
medium. Cells were washed and cultured (8 x 106cells/mL)in serumcontaining medium in the absence or presence of G-CSF at 10 ng/
mL for 5 days. The percent cell survival was determined by dividing
the number of viable cells per milliliter by the initial cell concentration. Data are shown as the mean value f SD of two independent
experiments.
1 mmoVL phenylmethylsulfonyl fluoride (PMSF), 0.1 UlmL aprotinin, and 10 pg/mL leupeptin and incubated at 4°C for I O minutes.
Insoluble material was removed by centrifugation at 10,OOOg for 10
minutes at 4°C. For immunoblotting, approximately 30 pg of protein
from each sample was separated on a 16.5% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred
to nitrocellulose. After blocking overnight in a solution of TBS
containing 2% nonfat dry milk and I % Tween 20, membranes were
incubated with the primary antibody in blocking buffer for 2 hours at
room temperature. The &cl-2antibody (6C8),I7a hamster monoclonal
specific for human bcl-2, and the bcl-X, rabbit antisera" were used
at dilutions of 1 :200 and 1:l ,O00 respectively (both antibodies were
a gift of Dr Stanley Korsmeyer). After washing, the bcl-2 immunoblot was sequentially incubated with a biotinylated goat-antihamster IgG antiserum (Caltag, San Francisco, CA) at a dilution of
1 :2,000 and horseradish peroxidase (HRP)-conjugated streptavidin
(Zymed, San Francisco, CA) at a 1:20,000 dilution. The bcl-XL
blot was hybridized with an HRP-conjugated polyclonal antirabbit
antibody (Amersham, Arlington Heights, IL) at a 1:5,OOO dilution.
Both blots were developed using the Enhanced Chemiluminescence
(ECL) detection system (Amersham). To control for protein loading,
the immunoblots were stripped (per protocol, ECL kit) and hybridized with an anti-p-tubulin monoclonal antibody" at a 1:500 dilution.
Cell survival assays. Cells in logarithmic growth phase were
washed three times in CM and cultured in duplicate in CM at a cell
density of 8 X 10'lmL. The number of viable cells was determined
at the indicated times by trypan blue staining. Cell counts were
performed in duplicate for each sample. Recombinant human G -
CSF was used at a concentration of I O nglmL (Amgen, Thousand
Oaks, CA).
Cell morphologyand myeloperoxidase assays. Cytospins of 1
X IO4 to 1 X 10scells were made using the Cytospin 3 cytocentrifuge
(Shandon, Pittsburgh, PA). Necrotic cells were removed by centrifugation through a cushion of Histopaque 1077 (Sigma, St Louis, MO)
at 400g for 30 minutes. Cell morphology was assessed after MayGrlinwald-Giemsa staining. Myeloperoxidase activity was determined on cytospin specimens using a leukocyte peroxidase kit, per
manufacture's recommendations (Sigma).
RNA preparation and SI nuclease protection. Total cellular
RNA was prepared from cells using a guanidinium thiocyanate miniprep, as described.2" Preparation ofthey-"P-end-labeledprobes
and S1 nuclease protection assays were performed as previously
described.2' The cathepsin G probe is a genomic fragment endlabeled at a BgnI site in exon 4, as shown (see Fig 4) (a gift from
Dr Timothy J. Ley, Washington University, St Louis,
The
murine p-actin probe is a genomic fragment end labeled at a Bgnl
site in exon 2 (a gift of Dr Timothy J. Ley, unpublished sequence).
Correctly spliced murine &actin mRNA protects a 135-nucleotide
probe fragment. Autoradiograms were exposed for 24 to 72 hours
at -70°C.
In siru detection ofapoptosis. Cells were spun onto glass slides
as described above and fixed in 1% paraformaldehyde in TBS for
30 minutes at room temperature. Endogenous peroxidase activity
was blocked by incubation of samples in 0. I % H 2 0 2 in TBS for 30
minutes at room temperature. DNA termini were labeled by incubation at 37°C for 1 hour in a solution containing S pmol/L digoxigenin-conjugated dUTP (Boehringer Mannheim, Indianapolis, IN)
and 400 UlmL terminal deoxynucleotidyl transferase in buffer supplied by the manufacturer (Promega, Madison, WI). After washing
in TBS, the slides were sequentially incubated with a sheep antidigoxigenin Fab (Boehringer Mannheim) at a 1:200 dilution and an
HRP-conjugated goat-antisheep IgG antibody (Zymed, San Francisco, CA) at a 1:120 dilution. Slides were developed with the 3amino-9-ethylcarbazole substrate per manufacturer's recommendations (Zymed, San Francisco, CA).
RESULTS
Overexpression of bcl-2 or bcl-XLin 3 2 0 cells suppresses
apoptosis induced by cyrokine withdrawal. 32D cells comprise an L-3-dependent cell line thatrapidly undergoes
apoptosis upon cytokine ~ithdrawa1.l~
Expression ofboth
hcl-2 and bcl-X, in 32D cells appears to be responsive to
L-3; mRNA levels for both genes decrease rapidlyupon
IL-3 removal.23 BC/-2 overexpression has been shown to
delay apoptosis in a number of cytokine-dependent cell lines
upon cytokine deprivati~n.*~~*~
These data suggest that bcl2, or a related family member, may mediate the survival
signal provided by cytokines. Therefore, we testedthe ability
of bcl-2 and hcl-X, to suppress apoptosis in cytokine-deprived 32D cells. cDNAs for human hcl-2 or murine hcl-X,.
were cloned into the expression vector pBSRaEN, and the
resulting constructs transfected into 32D cells. Three clones
each were isolated that contained the hcl-2, hcl-XL,or vector
alone constructs (designated 32D-bc12, 32D-bcIX, or 32Dneo, respectively). The responses of the three 32D-bc12 or
32D-bclX clones in our assays were nearly identical, therefore only representative results are shown. Immunoblots of
cell lysates demonstrated the appropriate expression of either
bcl-2 or bcl-X, (Fig l). These clones were examinedfor their
survival in serum containing medium without added [L-3.
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ROLE
DIFFERENTIATION
IN GRANULOCYTIC
861
ts
Fig 4. Morphology of 32D-bc12 cellsafter cytokine deprivation (originalmagnification x 600). May-Grlinwald-Giemsastaining was performed
on the following cells. (A) 32D-bc12 cells in IL-3-containing medium. (B and D) 32D-neo and 32D-bc12 cells, respectively,cultured for 7 days in
medium containing 10 ng/mL of G-CSF. (C) 32D-bc12 cells cultured for 7 days in medium without G-CSF. Necrotic cells were removed by
density centrifugation.
Approximately 50% of 32D-bc12 or 32D-bclX cells survived
3 days after cytokine deprivation compared with no survival
of 32D-neo cells (Fig 2). These data indicate that constitutive
expression of hcl-2 or hcl-X,. is able to prolong survival in
IL-3-deprived 32D cells; further, this survival advantage is
similar to that achieved with G-CSF stimulation of control
cells (Fig 2; neo + G-CSF). Interestingly, stimulation of
32D-bc12 or 32D-bclX cells with G-CSF had an apparent
synergistic effect on cell survival (Fig 3). A greater than
fivefold higher number of viable cells were present in GCSF-stimulated 32D-bc12 cultures when compared with either 32D-bc12 cultures without added cytokine or to 32Dneo cells cultured in G-CSF.
The phenotype of 3 2 0 cells stimulated with G-CSF is
distinct from S2D-hc12 or 332D-hclX cells cultured in the
absence of added cvtokine. The stochastic model of cytokine induced hematopoietic differentiation predicts that the
phenotype of 32D-bc12 or 32D-bclX cells surviving in culture after cytokine deprivation should be identical to the
phenotype of cells surviving in G-CSF-containing cultures.
In the presence of 1L-3, all clones had an identical myeloblastic phenotype (Fig 4A and data not shown). The morphology of cells surviving after 7 days in the indicated culture conditions is shown in Fig 4. 32D cells stimulated with
G-CSF acquire many features of more mature myeloid cells,
including nuclear segmentation and the appearance of cytoplasmic azurophilic granules (Fig 4B). 32D-bc12 cells show
a similar degree of nuclear segmentation, but the cells are
consistently smaller with no apparent granule formation (Fig
4C). 32D-bc12 cells retain the ability to differentiate in response to G-CSF (Fig 4D). Similar results wereobtained
with 32D-bcIX cells (data not shown).
G-CSF treatment of 32D cells is associated with the expression of a number of proteins foundin the primary(azurophilic)
and secondary granules of myeloid cells.” The apparent lack
of cytoplasmic granules in 32D-bc12 or 32D-bclX cells surviving cytokine deprivation suggested that expressionof granule
constituent proteins may be defective. Therefore, we examined cultured cells for their expression of myeloperoxidase,
an enzyme found in the primary granules of myelomonocytic
cells.” Over 75% of 32D-neo and 32D-bc12cells present after
7 days of G-CSF stimulation demonstrated myeloperoxidase
activity (Fig 5BandD). In contrast, lessthan 5% of 32D
cells grown in IL-3-containingmedium or 32D-bc12 cells
surviving in cultures containing no added cytokines weremyeloperoxidase positive (Fig SA and C). Similar results were
obtained with 32D-bclX cells (data not shown).
Expression of cathepsin G mRNA is induced in 3 2 0 cells
stimulated with G-CSF hut not in 32D-hcl2 or 32D-bclX
cells surviving qtokine deprivation. Cathepsin G is a he-
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862
AND LINK
RODEL
l-.
. .
..
X#
?.""".
1
.B'
Fig 5. Myeloperoxidase activity of32D-bc12cells
after cytokine deprivation (original magnification x 600). Cytochemical detection of
myeloperoxidaseactivity was performed on the following cells: (A) 32D-bc12 cellsin IL-3-containing medium. (Band D) 32D-neo and 32D-bc12
cells, respectively, cultured for 7 days in medium containing 10 nglmL of G-CSF. (C) 32D-bc12 cells cultured for 7 days in medium without GCSF. Necrotic cells were removed by density centrifugation.
matopoietic serine protease found in the primary granules
of myelomonocytic cells." Its mRNA expression is restricted
to cells at the promyelocyte stage of myelomonocytic matuTreatment of 32D-neo or 32D-bc12 cells with GCSF for 3 days resulted in the accumulation of cathepsin G
mRNA (Fig 6, lanes 2 and 5). No increase in the level of
cathepsin G transcripts is seen in 32D-bc12 cells surviving
cytokine deprivation (Fig 6, compare lanes 3 and 4). Equivalent amounts of &actin mRNA were detected in each sample, indicating thatthe differences in cathepsin G mRNA
expression observed in this assay are not caused by differences in RNA amount or integrity.
DISCUSSION
Hematopoietic cytokines are polypeptide soluble factors
that control the growth and differentiation of hematopoietic
cells. These cytokines are capable of stimulating the proliferation and enhancing the survival of the appropriate hematopoietic progenitor cell.'" However, the mechanisms by which
cytokines promote hematopoietic differentiation remain controversial. Two general models for the role of cytokines in
controlling hematopoietic differentiation have been proposed.' In the stochastic mode1,"'hematopoietic growth factors provide proliferative and survival signals to the differentiating hematopoietic cell, but they do not provide specific
lineage-commitment signals. In the instructive model:hematopoietic growth factors provide lineage commitment,
along with survival and proliferative signals. In this study,
we testedthese two models with respect to G-CSF and granulocytic differentiation. We used 32D cells, a cell line capable
of differentiating into granulocytes in response to G-CSF.I4
The stochastic model predicts that 32D cells are already
lineage committed and under permissive culture conditions
will undergo granulocytic differentiation without G-CSF.
Therefore, we generated 32D clones that constitutively expressed either bcl-2 or bcl-XLin an effort to circumvent the
cell survival signals normally provided by G-CSF. The ability of these clones to undergo granulocytic differentiation in
the absence of G-CSF was then determined.
Bcl-2 is the prototype of a family of proteins thatfunctions
to repress programmed cell death in a variety of cell lines.
Bcl-2 can suppress apoptosis in a number of IL-3-dependent
hematopoietic cell lines upon IL-3 withdrawal, including
32D ~ e l l s . * ~Our
. ~ ' data confirm that both bcl-2 and bcl-XL
are capable of suppressing apoptosis in cytokine deprived
32D cells. Prolonged survival, up to 10 days, in IL-3-deprived cultures was noted (data not shown) and was similar
to that observed in G-CSF-stimulated cultures. Interestingly, stimulation of32D-bc12 or 32D-bclX cells with GCSF had a synergistic effect on cell survival. This synergism
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863
ROLE OF G-CSF IN GRANULOCYTICDIFFERENTIATION
Bcl-2
Neo
267
-
227
-
,_
”
-
m
174
+ mCG
1
2
3
4
5
“Actin
BgI-I1
Bnl-II
mCG gene
mCG probe
212
mCGmRNA
Fig 6. Cathepsin G expression in 32D-bc12 cells after cytokine deprivation. Twenty microgramsof total cellular RNApurified from 32Dneo (neo) or 32D-bc12(bcl-2)cells stimulated with theindicatedcytokine for 3 dayswas hybridized with a murine cathepsin G probe. The
genomic organization of murine cathepsin G (exons 2 through exon
4) and the position of the probe are shown at the bottom of the
figure. Correctly spliced cathepsinG mRNA is expected to protect a
212-nucleotideprobe fragment. In thelower panel, a 5-pg aliquot of
the RNAdescribedabove
was hybridized with a murine p-actin
probe; correctly splicedmurine p-actin mRNA protects a 135-nucleotide probe fragment.
raises the possibility that G-CSF mediated repression of programmed cell death mayuse distinct molecular pathways
from bcl-2.
We have found that suppression of apoptosis in IL-3deprived 32D cells is not sufficient to induce complete granulocytic differentiation. 32D-bc12 or 32D-bclX clones were
examined for evidence of granulocytic differentiation in the
presence or absence of G-CSF. In the presence of G-CSF,
32D-bc12 and 32D-bclX cells acquired many of the features
of mature granulocytes including nuclear segmentation and
the presence of azurophilic cytoplasmic granules. The morphology of these cells in the absence of G-CSF was distinct.
The cells were consistently smaller with less abundant cytoplasm and few if any cytoplasmic granules. Interestingly,
the cells cultured in the presence or absence of G-CSF had
a similar degree of nuclear segmentation. To exclude the
possibility that the cells with segmented nuclei were undergoing apoptosis, we performed an in situ apoptosis assay.”
Apoptotic cells were detected infrequently; further, no correlation between nuclear segmentation and apoptosis was
observed (data not shown). In the presence of G-CSF, 32Dbc12 and 32D-bclX cells express myeloperoxidase and
cathepsin G,proteins found in the primary granules of myelomonocytic cells. No evidence for an increased expression
of these proteins was detected in the absence of G-CSF.
These data support a hybrid model for the role of G-CSF
in granulocytic differentiation; although some features of
granulocytic differentiation, namely nuclear segmentation,
do not require G-CSF and therefore appear to be preprogrammed in 32D cells, the complete maturation of 32D cells
to granulocytes appears to be dependent on G-CSF.
Fairbairn et al* reported thatFDCP-Mix cells constitutively expressing bcl-2 underwent multilineage hematopoietic differentiation in the absence of exogenous cytokine.
Two criteria were used to assess for granulocytic differentiation, the expression of lysozyme M and the cellular morphology after May-Griinwald-Giemsa staining. Lysozyme M is
expressed predominantly in mature myelomonoctyic cells”;
however, in this study expression was induced in FDCPMix cells undergoing predominantly erythroid differentiation, suggesting that in this system lysozyme M expression
may not be a reliable indicator of granulocytic differentiation. On the other hand, a distinguishing feature of mature
neutrophils is the segmentation of their nuclei; therefore, the
appearance of cells with segmented nuclei in their serumfree cultures is indicative of granulocytic differentiation and
consistent with the hypothesis that the process of nuclear
segmentation that occurs during granulopoiesis is independent of G-CSF.
In the present study, we show that several features associated with granulocytic differentiation in 32D cells are dependent on G-CSF, namelyinduction of myeloperoxidase activity and increased cathepsin G expression. These data suggest
that G-CSF, through its receptor, is transmitting specific maturation signals. This hypothesis is consistent withrecent
reports describing truncation mutations of the G-CSFR that
are unable to mediate differentiation responses.g”.” Work
is in progress to define the structural motifs of the G-CSFR
necessary for the differentiation response.
ACKNOWLEDGMENT
We thank Stanley J. Korsmeyer for providing the bcl-2 and bclXLcDNAs and antibodies. We alsothank Timothy J. Ley for providing the cathepsin G and &actin probes, James Ihle for providing the
32Dc13 cells, and Greg Longmore for providing theWEHl cells.
The anti-&tubulin monoclonal antibody developed by Michael
Klymkowsky“’ was obtained fromthe Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and
Molecular Sciences, John Hopkins University School of Medicine,
Baltimore, MD 21205, and the Department of Biological Sciences,
University of Iowa, Iowa City, I A 52242, under contract NOI-HD2-3 1 4 4 from the National Institute of Child Health and Human Development (NICHD).
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13. Dong F, van BC, Pouwels K, Hoefsloot LH, Lowenberg B,
Touw IP: Distinct cytoplasmic regions of thehuman granulocyte
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