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Oligodeoxynucleotides Antisense to c-ab1 Specifically Inhibit Entry Into
S-Phase of CD34+ Hematopoietic Cells and Their Differentiation
to Granulocyte-Macrophage Progenitors
By Vittorio Rosti, Gaetano Bergamaschi, Claudia Lucotti, Marco Danova, Carmelo Carlo-Stella, Franco Locatelli,
Laura Tonon, Giuliano Mazzini, and Mario Cazzola
A number of experimental observations suggest that the
hemaproto-oncogene c-ab1participates in the regulation of
topoietic cell growth. We used an antisense strategy t o
study therelationship between c-ab1 expression and hematopoietic cell proliferation and differentiation. Purified normalhuman bone marrow-derived CD34+ cells were obtained by immunomagnetic selection and incubated with
18-base-unmodified antisense oligodeoxynucleotides comalternative first
plementary t o t hfirst
e six codons of the two
of incubation, an aliquot
exons of c-&l, la andlb. At the end
of cells was assayed for clonogenic growth and the
remainder was used for flow cytometric
analyses. Cell kinetics were
evaluated by means of bothsingle parameter DNA and bivariate DNA/bromodeoxyuridine (BrdU) flow cytometry.
Apoptosis was routinely studied by DNA flow cytometric
analysis and, in some cases, also through DNA agarose gel
electrophoresis for detectionof oligonucleosomalDNA fragments. Expression of differentiation markers was studied
by flow cytometry.Exposure t o antisense oligonucleotides
specifically inhibited the accumulationof c-ab1 mRNA in
CD34' cells. Preincubation with the c-ab1 antisense oligomers reduced the proportion of cells in S-phase from 19%
f 5% (mean f SD) t o 7% f 4% ( P < .05), and BrdU labeling
from 13% f 6% t o 6% f 3% ( P < .05). Flow cytometry and
DNA agarose gel electrophoresis showed that treated
CD34'
cells accumulated in the Go/G1region of theDNA histogram
with no evidence of either differentiation or apoptosis. By
contrast, both growth factor deprivation and exposure of
CD34+ cells t o t h etyrosine kinase inhibitor tyrphostinAG82
clearly induced apoptosis. When cells were preincubated
with antisense oligonucleotides and then plated for
evaluation of colony formation,this resulted in a significant inhibition of colonyforming unit granulocyte-macrophage growth
(from 44 f 15 t o 22 k 9; P < .01) but had noeffect on burstforming unit erythroid growth (24 f 11 v21 k 11; P < .05).
These results suggest that c-ab1 expression is critical for
entry of human CD34+ hematopoietic cells into S-phase and
for their differentiation
t o granulocyte-macrophage progenitors. They also indicate that other tyrosine kinases besides
p145c-"b'are active in the prevention of apoptosis, so that
inhibition of c-ab1 RNA accumulation arrests CD34' cells in
Go/G, without activating programmeddeath.
0 1995 by The American Societyof Hematology.
T
30 minutes at 400g; interface.cells were washed three times and
resuspended at 2 X 106/mLin Iscove's modified Dulbecco's medium
(IMDM; Irvine Scientific, Santa Ana, CA) with 20% fetal bovine
serum (FBS;HyClone, Logan, UT). Adherent cell depletion was
obtained by incubation in 25-cm2 tissue culture flasks for two sequential l-hour periods at 37"C, 5% COz. LDBMCs depleted of
adherent cells (LDBMCs AC-) were further depleted of T lymphocytes (LDBMCs AC- T-) by rosetting with sheep red blood cells
pretreated with 2-aminoethylisothiouronium bromide. LDBMCs
AC- T- were then depleted of CD19+ cells (LDBMCs AC- TCD19-) by beads coated with a monoclonal antibody (MoAb) directed against the CD19 antigen (Immunotech SA, Marseille,
France). CD34' cells were obtained from the LDBMC AC- TCD19- fraction by positive selection using beads coated withthe
HE C-ABL PROTO-ONCOGENE encodes for a nonreceptor tyrosine kinase, ~ 1 4 5 " " ~whose
',
biologic function is still unknown.' The protein is ubiquitously expressed
and is mainly localized in the nucleus, although it may also
be found in the cytoplasm.*Recent results from biochemical
and molecular studies have indicated that c-ab1 may be involved in cell cycle regulation.' The carboxy-terminal portion of c-ab1contains two distinct domains that can bind to Factin' and to DNA,4 respectively. The c-ab1 protein becomes
hyperphosphorylated during metaphase, resulting in an inhibition of its DNA-binding activity! More recently, it has
been found that c-ab1 and the retinoblastoma gene protein
form a complex in vitro, and that this complex regulates the
cell cycle-related activity of ~ - a b l Taken
.~
together, these
data suggest a role for c-ab1 in signal transduction as well
as in cell-cycle regulation.'
The biologic role of c-ab1 in normal hematopoiesis is unclear. Studies based on the use of antisense oligomers have
shown that c-ab1 function is important for the in vitro proliferation and differentiation of granulocyte-macrophage colony-forming units (CFU-GM).6,7Animal models have provided little information so far regarding the role of c-ab1 in
hematopoietic cell proliferation and differentiation.','
In this work we used an antisense strategy to study the
relationship betweenc-ab1 expression and proliferation of
normal human bone marrow (BM)-derived CD34+ cells.
MATERIALS AND METHODS
Normal subjects. After informed consent, normalBMwas obtained from eight BM donors.
Cell separation procedures. Mononuclear light-density BM
cells (LDBMCs) were separated by centrifugation on a Ficoll-Paque
(Pharmacia Biotech, Uppsala, Sweden) gradient (1,077 g/mL) for
Blood, Vol 86,No 9 (November l), 1995:pp 3387-3393
From the Department of Internal Medicine and Medical Therapy,
Section of Internal Medicine and Medical Oncology, the Department
of Pediatrics, University of Pavia, and Istituto di Ricovero e Cura
a Carattere Scientific0 (IRCCS) Policlinico S. Matteo, Pavia; the
Department of Hematology, University of Parma, Parma; and Consiglio Nazionale delle Ricerche (CNR) Study Centerfor Histochemistry, Pavia, Italy.
Submitted December 30, 1994; accepted June 29, 1995.
Supported by grants to M.C. from Associazione Italiana per la
Ricerca sui Cancro, CNR (Progetto Applicazioni Cliniche della Ricerca Oncologica [ACRO]),IRCCS Policlinico S. Matteo, and Fondazione Ferrata Storti.
Address reprint requests to Mario Cazzola. MD, Clinica Medica
2, Policlinico S. Matteo, 27100 Pavia, Italy.
The publication costs of this article were defrayed in parr by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section I734 solely to
indicate this fact.
0 I995 by The American Society of Hematology.
0006-4971/95/8609-0008$3.00/0
3387
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3388
QBEND 10 MoAb directed against the CD34 antigen (Immunotech).
LDBMCs AC- T- CD1 9- wereincubated with theanti-CD34 immunobeads at a ratio of I mgof beads per IO' LDBMCs AC- TCD19- for 15 minutes at 4°C. Cells were then exposed to a cobaltsamarium magnet for 10 minutes at room temperature and thenonadherent fraction was discarded. Bead-conjugated cells were recovered,
washed, and evaluated for their purity byflow cytometry using a
fluorescein isothiocyanate (F1TC)-conjugated anti-HPCA-2 CD34
MoAb (clone 8G12; Becton Dickinson, San Jose, CA). The percentage of CD34' cells was greater than 90% in all experiments.
Oligodeoxynucleotides. Synthetic nonmodified oligodeoxynucleotides were purchased from Med Probe (Med Probe, Oslo, Norway).Four types of oligomers were used: ( l ) 18-base antisense
oligodeoxynucleotides complementary to either the 6.0 kb (exon Ia)
or 7.0 kb (exon Ib) c-ab1 mRNA, beginning from the second codon
of each mRNA (the sequences are 5'-CTTCAGGCAGATCTCCAArespectively) and (2) 183' and 5'-TTJTCCAGGCTGCTGCCC-3',
base sense oligodeoxynucleotides, corresponding to the IaandIb
antisense sequences andusedas
control (the sequences are 5'7TGGAGATCTGCCTGAAG-3' and 5'GGGCAGCAGCCTGGAAAA-3', respectively).
Pre-incubafionwitholigomers.
CD34' cells (lo5) were incubated with both anti Ia and anti Ib oligomers for 17 to 20 hours at
37°C in liquid culture (IMDM with 10% FBS). The final concentration for each oligomer was 14 pmol/L, whichhad proven to be
optimal in previous ~tudies.~
Control cultures were performed both
with sense oligomers and with culture medium only. At the end of
the incubation, an aliquot of the CD34' cells was used for the
clonogenic assay and the remainder was used for the flow cytometric
analysis (see below). In the experiments in which bromodeoxyuridine (BrdU) incorporation was evaluated, 30 minutes before the end
of preincubation CD34' cells were pulse-labeled with 30 pmol/L
BrdU (Sigma Chemical CO, St Louis, MO); the medium was then
washed out and thecells were rinsed twice with fresh medium before
fixing with cold 70% ethanol. When preincubation with oligomers
was protracted for 48 to 72 hours (according to cell availability),
the appropriate oligomers were added to the culture medium at a
concentration of 7 pmol/L every 24 hours.
In some experiments, CD34+ cells were preincubated withthe
tyrosine kinase inhibitor tyrphostyn AG82 at a concentration of 200
pmol/L for 24 hours, washed three times, and evaluated by DNA
electrophoresis and flow cytometric DNA analysis to detect
apoptosis as previously described in detail."
Reverse transcription polymerasechain reaction (RT-PCR) analysis of c-ab1 mRNA. To evaluate the effects of oligonucleotides on cab1 mRNA expression, total RNA was isolated according to standard
procedures" from 2 to 4 X lo4 CD34' cells after a 20-hour treatment
with the oligonucleotides and from control CD34' cells and analyzed
by a competitive RT-PCR in which c-ah1 and p-actin cDNAs were
simultaneously amplified. Briefly, total RNA wasreverse transcribed
in a final volume of 20 pL using 250 ng each of c-ab1 and p-actin
3' antisense primers complementary to the third exon of c-abl', and
to nucleotides 1,032 to 1,055 of the p-actin c-DNA." The reaction
mixture contained: 100 U of Moloney-murine leukemia virus reverse
transcriptase (GIBCO-BRL, Gaithersburg, MD), 20 U RNasin, 0.2
mg/rnL bovine serum albumin (Promega Biotec, Madison, Wl), 0.01
m o w dithiothreitol (D'IT), 50 mmol/L TRIS-HCI, pH 8.3.75 mmol/
L KCI, 3.0 mmoVL MgCI, and 2.5 mmol/L deoxynucleotide triphosphates (dNTPs). RNA and primerswere initially heated for 3 minutes
at 80°C followed by 30 minutes at 69°C. After addition of the enzyme, the reaction mixture was incubated for 60 minutes at 42°C
and 10 minutes at 95°C. Ten microliters of cDNAs as subsequently
amplified in a final volume of 50 pL containing 125 ng of the two
3' primers used in the reverse transcription reaction and 250 ng each
of the 5' sense primers corresponding to the second exon of c-abl"
ROSTI ET AL
and to nucleotides 819 to 842of the p-actin cDNA." Amplification
was performed in 50 mmol/L KC], 10 mmol/L TRIS-HC1, pH 8.3,
I .5 mmol/L MgCI,, 0.01% gelatin, 0.4 mmol/L dNTPs and 1 U of
Taq polymerase (Perkin-Elmer, Branchburg, NJ). After denaturation
for 2 minutes at 94"C, samples underwent 40 cycles of amplification
involving 1 minute at 94°C 25 seconds at61°Cand 2 minutes at
72°C. In the final cycle, temperature was held at 72°C for 7 minutes.
Amplification products were run through a 2% agarose gel in I X
TBE buffer. The 0-actin PCR product was directly visualized on
the ethidium bromide-stained gel. Because the c-ab1 gene is much
less expressed than the p-actin gene, its evaluation after simultaneous c-ab1 and@-actinRT-PCR required the gel to beSouthern blotted
onto a nylon filter thatwas hybridized with a "P-labeledprobe
spanning the second and the third exons of c-abl.
Colony assays. Clonogenic assays were performed as previously
described.' Briefly, 5 X IO3 CD34' cells were plated in 35-mm Petri
dishes in 1 mL aliquots of IMDM containing 30% FBS, 5 X
M 2-mercaptoethanol, 2 IU erythropoietin, 10 ng interleukin-3, 10
ng granulocyte-macrophage colony-stimulating factor and 0.9% (wt/
vol) methylcellulose. Assays were performed in duplicate. After
incubation for 14 days at 37°Cin a fully humidified atmosphere
supplemented with 5% COz. the number of colonies was scored
using an inverted microscope.
Flow cytometricanalysis of cell-cyclephasedistribution
and
BrdU uptake. Monoparametric conventional cell-cycle analysis
was performed on control and treated nuclei using a Partec PAS I1
flow cytometer (Partec SPA, Basel, Switzerland). After preincubation with oligomers, ethanol-fixed cells were centrifuged, washed,
and stained (30 minutes at room temperature) with 50 pg/mL propidium iodide (PI) (Calbiochem, San Diego, CA) in phosphate-buffered
saline (PBS). The staining solution also contained 0.l % Triton X 1 0 0
(Calbiochem) and 1% wt/vol RNAse A (Sigma).'4 The percentage
frequencies of cells in the different cell-cycle phases were calculated
using a mathematical model adapted for a dedicated Partec computer."
The presence of apoptotic cells was also investigated withthe
same analysis, according to theprinciple that thereduced stainability
of apoptotic cells withDNA fluorochromes is a consequence of
partial loss of low-molecular-weight DNA.'"I7
For biparametric BrdUlDNA analysis, after a pulse-label with 30
ymol/L BrdU, CD34' cells were washed andfixedwithice-cold
70% ethanol. Then cells were washed with PBSandDNA
was
denaturated with 2 N HCI (20 minutes at room temperature). BrdU
uptake was detected after 30-minute incubation with 100 pL of a
1 :10 dilution in PBS of anti-BrdU mouse MoAb (Becton Dickinson,
San Jose, CA), followed by incubation with 100 pL of a 1 5 0 dilution
in PBS of a FITC-labeled goat antimouse MoAb (Sigma). Finally,
cells were washed twice with PBS at room temperature and counterstained for 3 hours at room temperature with 5 pg/mL PI."
Bivariate measurement of green fluorescence (BrdU-labeled cells)
versus red fluorescence (DNA content) was performed with a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA) as previously described."
Gel electrophoresisfor detection of oligonucleosornal DNA fragments. DNA fragmentation was evaluated by DNA electrophoresis
on 2% agarose gel as previously described.'"
Immunophenotyping. Fluorescence-activated cell sorting (FACS)
analysis of differentiation markers was performed on purified CD34'
cells after incubation for 20 hours in liquid culture (IMDMwith
10% FBS) without (control) and with anti-ab1 oligonucleotides.
The expression of lineage-associated antigens on CD34' cells was
determined by incubation with antibody against CD34 together with
antibody to the specific antigen. Briefly, cells were incubated for 30
minutes at 4°C with a phycoerythrin-conjugated CD34 MoAb (antiHPCA-2, clone 8G12) alone or with one of the following F I X -
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3389
c-ab1FUNCTION IN CD34'CELLS
bp
C S AS
Fig 1. Effect of anti-ab1 oligonucleotides on RT-PCR amplification
of c-ab1 mRNA in CD34' cells. The ethidium bromide-stained gel
shows the237-bp bands corresponding t o p-actin c-DNA amplification, whereas the autoradiographicbands correspond to the218-bp
products of c-ab1 mRNA RT-PCR. C, control; S, sense oligonucleotide;
AS, antisense oligonucleotide. Distance between c-ab1 and p-actin
bands does not reflect the realbase pair difference.
conjugated MoAbs: LeuM9 (CD33). Leu18 (CD45RA), Leu12
(CD19), and Leu9 (CD7). all supplied by Becton Dickinson.
Statistical ana/ysis. Data were analyzed with the statistical package Extatix (Select Micro System Inc. Yorktown Heights, N Y ) run
on a Macintosh Quadra 650 (Apple Computer Inc, Cupertino, CA)
personal computer. The results were expressed as means ? 1 SD,
and the Student's t-test for paired data was used to verify the probability of significant differences between means.
RESULTS
We first evaluated the effect of anti-ab1 oligodeoxynucleotides on c-ab1RNA accumulation in CD34' cells using a
RT-PCR approach. The effect on CD34' cell proliferation
and differentiation was then studied using four different approaches that all give different information: (1) in vitro committed progenitor cell growth was studied using classical
14-day semisolid methylcellulose cultures; (2) direct cell
kinetics were evaluated by means of single parameter DNA
and bivariate DNA/BrdU flow cytometric analyses; (3)
apoptosis was investigated by bothDNAflow cytometric
analysis and gelelectrophoresis for detection of oligonucleosoma1 DNA fragments; (4) differentiation was assessed by
FACS analysis of lineage-associated antigens.
Inhibition of c-ab1 mRNA accumulation by antisense oligodeoxynucleotides. The aim of antisense strategy is to
inhibit gene expression. The binding of an antisense oligonucleotide to its complementary portion of the mRNA molecule
may result in either degradation of mRNA through RNAse
H or inhibition of translation without altering RNA stability.2" To evaluate whether the antisense oligomers used provided specific gene inhibition, total cellular RNA from dif-
ferently treated samples was reverse transcribed
and
amplified using primers specific for sequences derived from
the c-ab1 and P-actin genes. The two sequences were simultaneously amplified in the same reaction tube. A representative
experiment involving control cells and cells exposed to antisense and sense oligomers, respectively, is illustrated in Fig
I . The similar level of the P-actin amplification product in
different samples indicates that equal amounts of total RNA
were used for amplification: in turn, this implies that the
reduction of c-ab1 levels in the antisense-treated samples was
caused by specific inhibition ofc-ab1mRNA
expression.
This is in agreement with previous observations that oligomers targeted against the two c-ab1 mRNAs induce RNA
degradation and are effective in inhibiting the expression of
p I 45c.ah'?
Effect of oliRodeoxynucleotides onin vitro CD34' cell
proliferation and differentiation in 14-day semisolid methylcellulose cultures. Results of studies on the clonogenic
growth of CD34' cells are summarized in Table I. After 14
days, approximately 50 to 100 colonies were obtained from
5 X IO' control CD34' cells plated under the experimental
conditions previously described. Abouttwo thirds were
CFU-GM and one third erythroid burst forming units (BFUE) (Table l).
When CD34' cells were exposed to sense oligomers, the
number of CFU-GM- and BFU-E-derived colonies did not
differ from that of control cultures. Preincubation with the
antisense oligomers significantly inhibited CFU-GM (P <
. O l ) buthadno effect on BFU-E growth. In most of the
samples exposed to antisense oligomers, not only the number
but also the size of CFU-GM was reduced.
Cell-cycle studies. Results of both single parameter
DNA and bivariate DNA/BrdU flow cytometric analyses are
shown in Table 2 and in Fig 2.
The majority of fresh CD34' cells isolated from normal
human BM was in the quiescent state. In fact, evaluation of
nuclear DNA content by staining with PI showed that 12%
? 3% of cells were in S-phase. Incubation for 17 to 20 hours
in liquid culture (IMDM with 10%FBS) induced recruitment
of cells into the cell cycle; as shown in Table 2, the percentage of cells in S-phase increased to 19% ? 5%, whereas
BrdU uptake (BrdU labeling index) was 12% ? 6%.
Treatment with antisense oligomers resulted in a signifi-
Table 1. Effect of Sense and Antisense Oligomers Targeted t o c-ab1
la and Ib mRNA on theIn Vitro Growth of BFU-E and CFU-GM
From NormalHuman BM-derived CD34' Cells
CRU-GM (no./dishl
BFU-E (noJdish1
Control
44 z 15
24 -C 11
Preincubation with sense
42 t 14' (5 2 4)24
t 13' (0 2 5)
oligomer (inhibition %)
Preincubation with antisense
22 -c 9t (51 t 11) 21 ? 11' (12 t 5)
oligomer (inhibition%)
After preincubation with theoligomers, cells were plated in methylcellulose for the evaluation of colony formation. Values represent
mean 2 1 SD I n = 7).
P > .05, Student's t-test (comparison with control).
t P < .01, Student's t-test (comparison with control).
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ROSTI ET AL
3390
to antisense oligomers for up to 72 hours, no evidence of
oligonucleosomal-size fragments was found on DNA electrophoresis, nor wasthere any sign of anapparently hypodiploid cell population on DNA flow cytometry.
To learn whether this simply reflected methodologic problems. weused established procedures for apoptosis induction."' First, growth factor deprivation using serum-free cultures induced apoptosis in CD34' cells (data not shown).
Second, weusedthe
tyrosine kinase inhibitor tyrphostin
AG82, which induces apoptosis in hematopoietic cells and
thereby reduces the fraction of cells in the S-phase of the
cell cycle."' When CD34' cells were incubated for 24 hours
in the presence of tyrphostin AG82 at a final concentration
of 200 pmol/L, clear evidence of apoptosis was found (Fig
3). This was indicated by the appearance of both DNA degradation on agarose gel electrophoresis and an apparently hypodiploid region, the A" peak, below the Go/GI region on
DNA flow cytometry.
Effect qf anti-ah1 oligonucleotides on the immunophenov p e qf CD34' hematopoietic cells. The immunophenotype
ofBM CD34' cells is shown in Table 3. There was no
substantial difference between control cells and cells exposed to antisense oligomers with respect to the expression
of lineage-associated antigens. In particular, therewas no
significant change in the proportion of CD34' cells expressing CD33.
Table 2. Effect of Sense and Antisense Oligomers Targeted to c-ab1
la and Ib mRNA on the Cell-Cvcle Kinetics of CD34' Cells
FCM DNA S-phase
BrdU Uptake.
(% of total cells)
(% of total cells)
Control
Preincubation with sense
19 f 5
12
oligomer
Preincubation with antisense
18 2 6 t
11 lr 5 t
7 -c 4*
oligomer
6
?
z
6
3*
Values represent means t 1 SD (n = 8).
Abbreviation: FCM, flow cytometry.
* Values are expressed as percentage of BrdU' cells, ie, as BrdUlabeling index.
t P > .05, Student's t-test (comparison with control).
P c ,005, Student's t-test (comparison with control).
*
cant decrease in both DNA S-phase and BrdU uptake with
respect to the control, whereas no effect was observed when
sense oligomers were used (Table 2). After exposure to antisense oligomers, there was an accumulation of cells in the
Go-GIregion of the DNA histogram (Fig 2) with no evidence
of arrest during S-phase: in fact, the bivariate cytogram
DNNBrdU failed to show significant aliquots of cells with
a DNA content typical of the S-phase (Fig 2, window a),
but not incorporating BrdU (Fig 2, window b). This indicates
that entry into S-phase was inhibited.
When antisense treatment was prolonged for 48 or 72
hours, no additional significant changes in the S-phase of
CD34' cells were observed (data not shown).
Evaluation of apoptosis in CD34' cells following exposure to anti-ah1 oligonucleotides andthe tyrosine kinase
inhibitor tyrphostyn AG82. We then explored the possibility that inhibition of S-phase entry resulted in the apoptosis
of CD34' cells. The presence of apoptosis was established
according to DNA electrophoresis and flow cytometry."' In
several experiments in which CD34' cells were exposed
-
-800
DISCUSSION
Our present knowledge of c-ab1 function derives directly
from studies of the normal gene and indirectly from investigations on constitutively activated genes, eg, bcr-ab1 fusion
genes and v-abl.
Overexpression of c-ab1 has been found to inhibit in vitro
growth of fibroblasts by causing cell-cycle arrest," suggesting that c-ab1 acts as a negative regulator of cell growth.
By contrast, oligodeoxynucleotides antisense to c-ab1have
-m
800
*
Mx)
i
. ...
3
. .
Z -
- 200
c -
h
i
0
3
200
200
s I
I
400
200
0
D N A
A00
A
I
I
0
200
AS
Y
I
I
A00
C O N T E N T
Fig 2. Flow cytometric analysis of CD34' cellsbefore (C) and after treatment with sense (S) and antisense (AS) oligomers. DNA distribution
histogramsclearly show that treatment with antisense oligomers results in reduced percentage of cellsbelonging to the S-phase region with
an accumulation in the GJG, region. No effect was observed when sense oligomers were used. Bivariate BrdU/DNA cell-cycleanalysis
confirmed this pattern as shown in the upper-right boxes. Windows show how gates were set for determination of actively proliferatingcells
(a) and S-phase cells not incorporating BrdU Ib).
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3391
c-ab1 FUNCTION IN CD34- CELLS
A
~
C
T
DNA CONTENT
B
C T
Y
Fig 3. Detection of apoptosis in control CD34' cells (C) and CD34' cells after 24-hour incubation with 200 pnol/L tyrphostin AG82 (TI. (A)
Flow cytometric DNA histograms of CD34+ cells.The arrow on the left histogram indicates the population of apoptotic cells, ie, the & peak.
(B)Corresponding lanes of DNA agarose gel electrophoresis.
been shown to inhibit myeloid colony formation in vitro.".'
Tybulewicz et alx and Schwartzberg et al" investigated c-ab1
function by examining the phenotypes of mice with homozygous disruption of the gene. These animals had no detectable
c-ab1 proteinand were severely affected, displaying increased perinatal mortality and multiple and variable phenotypes. Most animals had thymus and spleen atrophy, lymphopenia, foreshortened heads and eye defects. These findings
are not consistent with the negative role of c-ab1 suggested
by Sawyers et al"; this discrepancy, however, may reflect a
cell-type dependent modulation ofc-ab1 function, with the
ab1 tyrosine kinase stimulating or inhibiting growth depending on the cell context.
Studies on bcr/abl transgenic mice or mice transplanted
with hematopoietic cells infected withbcr/abl retroviruses
have shown that these animals develop several hematologic
malignancies, ranging from a myeloproliferative syndrome
to acute myeloid or lymphoid leukemia.""" Abrogation of
growth factor-dependence has been observed in murine and
Table 3. Effect of Anti-ab1 Oligonucleotideson the
lmmunophenotype of CD34' Hematopoietic Cells
Control CD34' Cells
CD33 (%)
CD45RA (%)
CD19 (%l
CD7 (%l
37.6
36.8
3.1
6.1
2 6.8
-c 3.6
? 0.7
2
2.0
CD34' Cells After Exposure to
Anti-ab1 Oligonucleotides
39.1 2 5.5
36.4 2 3.2
0.9
3.9
5.4 2 3.3
Valuesrepresentpercentages of cellscoexpressingtherelevant
antigen (means c 1 SD, n = 3). Student's t-test showed no significant
difference between control cells and cells exposed to antisense oligomers.
human myeloid cell lines after introduction and expression
of the p2 IOkr'"h'
However, studies on
fresh
murine
multipotent cells'" have provided more subtle results. When
multipotent progenitor cells were infected with a retrovirus
that carried bcr-abl. the mixed colonies that resulted were
responsive to growth factors and could differentiate; these
cells later became growth-factor independent but were not
leukemogenic.'" A decreased rate of programmed cell death
has been observed in Philadelphia-positive cells expressing
the bcr-ab1 fusion protein.""."'Taken together, these observations suggest thatthe presence ofbcr-ab1 alone does not
cause growth factor independence. although it initiates a
stepwise process."'
In the present work, inhibition of c-ab1 mRNA expression
resulted in growth arrest of CD34' cells, as indicated by
the reduced fraction of cells actively synthesizing DNA ( S phase) and the higher proportion in G&,. Growth arrest
may have different outcomes, including entry into Goreversibility or quiescence, programmed cell death anddifferentiation. CD34' cells treated with c-ab1 antisense oligonucleotides for 24 to 72 hours apparently did not undergo apoptosis.
After inhibition of all tyrosine kinases with tyrphostin AG82,
the appearance of both the typicalDNA degradation on agarose gel electrophoresis and the A,, peak on DNA flow cytometry (Fig 3) clearly indicated the presence of apoptotic cells.
Therefore, hematopoietic progenitor cells contain tyrosine
kinases other than p14S""h' that are active in preventing
apoptosis, and inhibition of p14S''"h' production alone does
not allow cells to proceed to programmed cell death.
Induction of differentiation was also a very unlikely outcome of growth arrest. In fact, the expression of lineageassociated markers such as CD33 was notmodified after
exposure to anti-ab1 oligonucleotides (Table 3). CD34' cells
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3392
ROSTl ET AL
that lack CD33 are considered to be more primitive hematopoietic cells that are precursors to clonogenic cells, whereas
CD34+ cells expressing CD33 have been shown to be committed
If growth arrest induced differentiation in CD34+ hematopoietic cells, the number of CD33'
committed progenitors wouldbe expected to increase; the
lack of any change in theproportion of CD33' cells indicates
that this was not the case. This was indirectly confirmed also
by the observation that the number of CFU-GM decreased
after exposure to antisense oligomers.
Our results suggest that c-ab1 function is critical for entry
of CD34+ hematopoietic cells into the S-phase of the cell
cycle. The kinetic data reported in Table 2 and Fig 2 are
consistent with the hypothesis that ~ 1 4 5 " " plays
~'
a specific
role in the transition of cells from G o G I to S phase. They
are in agreement with the observation thatc-ab1 activates
c-myc tran~cription'~
and that this effect might be cell lineage- or tissue-specific.'s The different effect of anti-ab1 oligonucleotides on CFU-GM and BFU-E growth might be
explained by a different induction of c-myc mRNAby
p 14Yab'in granulocyte-macrophage versus erythroid progenitors.
These findings may be helpful in understanding the pathogenesis of chronic myelogenous leukemia (CML) and may
provide a potentially useful tool for ex vivo cell manipulation. CML is a condition with an activated c-ab1 proto-oncogene whose product, p210b"""h',has markedly increased tyrosine kinase activity, and a residual normalc-ab1gene."
Because tyrosine kinase activity is essential for the stimulation of cell growth by oncogenic ab1 proteins,' it can be
speculated that p210kr-""very actively induces entry into S
phase of CML immature hematopoietic progenitors, a higher
proportion of which, therefore, are cycling." This involves
a greater likelihood of genetic errors whose frequency is
directly related to the number of cell divisions.38Therefore,
CML stem cells are more likely to develop those genetic
lesions that afford them growth factor independence and an
aberrant differentiation profile; this would lead to acceleration and blast crisis.
REFERENCES
1. Wang JYJ: Ab1 tyrosine kinase in signal transduction and cell
cycle regulation. Curr Opin Genet Dev 3:35, 1993
2. van Etten RA, Jackson P, Baltimore D: The mouse type IV cab1 gene product is a nuclear protein, and activation of transforming
ability is associated with cytoplasmic localization. Cell 58:669, 1989
3. McWhirter JR, Wang JYJ: Activation of tyrosine kinase and
microfilament binding functions ofc-ab1by bcr sequences in bcr/
ab1 fusion proteins. Mol Cell Biol 11:1553, 1991
4. Kipreos ET, Wang JYJ: Cell cycle-regulated binding of c-ab1
tyrosine kinase to DNA. Science 256:382, 1992
5. Welch PJ, Wang JYJ: A C-terminal protein binding domain in
the retinoblastoma protein regulates nuclear c-ab1 tyrosine kinase in
the cell cycle. Cell 75:779, 1993
6 . Caracciolo D, Valtieri M, Venturelli D, Peschle C, Gewirtz
AM, Calabretta B: Lineage specific requirement of c-ab1 function
in normal hematopoiesis. Science 245:1107, 1989
7. Rosti V, Bergamaschi G , Ponchio L, Cazzola M: c-ab1 function
in normal and chronic myelogenous leukemia hematopoiesis: In vitro
studies with antisense oligomers. Leukemia 6:1, 1992
8. Tybulewicz LJ, Crawford CE, Jackson PK, Bronson RT, Mulli-
gan RC: Neonatal lethality and lymphopenia in mice with homozygous disruption of the c-ab1 proto-oncogene. Cell 65:1153, 1991
9. Schwartzberg PL, Stall AM, Hardin JD, Bowdish KS, Humaran
T, Boast S, Harbison ML, Robertson EJ, GoffSP: Mice homozygous
for the abl"' mutation show poor viability and depletion of selected
B and T cell population. Cell 65: I 165. 1991
IO. Bergamaschi G, Danova M, Ponchio L,Rosti V, Lucotti C,
Cazzola M: Inhibitors of tyrosine phosphorylation induce apoptosis
in human leukemic cell lines. Leukemia 7:2012, 1993
1 1. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162: 156, 1987
12. Hermans A, Cow J, Selleri L, von Lindem M, Hagemeijer
A, Wiedemann LM, Grosveld G: bcr-ab1 oncogene activation in
Philadelphia chromosome-positive acute lymphoblastic leukemia.
Leukemia 2:628, 1988
13. Ponte P, Ng S-Y, Engel J, Gunning P, Kedes L: Evolutionary
conservation inthe untranslated regions of actin mRNAs: DNA
sequence of a human beta-actin cDNA. Nucleic Acids Res 12: 1687,
1984
14. Danova M, Riccardi A, Ucci G, Luoni R, Giordano M, Mazzini G: Ras oncogene expression and DNA content in plasma cell
dyscrasias: A flow cytofluorimetric study. Br J Cancer 62:781, 1990
15. Fried J: Method for quantitative evaluation of data from flow
microfluorimetry. Comput Biomed Res 9:263, 1976
16. Telford WC, King LE, Fraker PJ: Comparative evaluation of
several DNA binding dyes in the detection of apoptosis-associated
cromatin degradation byflow cytometry. Cytometry 13: 137. 1992
17. Bergamaschi G, Rosti V, Danova M, Lucotti C, Cazzola M:
Apoptosis: Biological and clinical aspects. Haematologica 79:86,
1994
18. Wilson GD, McNally NJ, Karcher H, Pfragner R:Thelabelling index of human and mouse tumors assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry. Cytometry
6:6427, 1988
19. Giordano M, Danova M, Mazzini G, Gobbi P, Riccardi A:
Cell kinetics within vivo brornodeoxyuridine assay, proliferating
cell nuclear antigen expression and flow cytometry: Prognostic significancein acute non-lymphoblastic leukemia. Cancer 71 :2739,
1993
20.WagnerRW:
Gene inhibition using antisense oligodeoxynucleotides. Nature 372:333, 1994
21. Sawyers CL, McLaughlin J, Goga A, Havlik M, Witte 0:
The nuclear tyrosine kinase c-ab1 negatively regulates cell growth.
Cell 77:121, 1994
22. Daley GQ, van Etten R, Baltimore D: Induction of chronic
myelogenous leukemia in miceby the P210kr'"'' gene of the Philadelphia chromosome. Science 247:824, 1990
23. Kelliher MA, McLaughlin J, Witte ON, Rosenberg N: Induction of a chronic myelogenous leukemia-like syndrome in mice with
v-ab1 and bcr-abl. Proc Natl Acad Sci USA 87:6649, 1990
24. Heisterkamp N, Jenster G, ten Hoeve J, Zovich D, Pattengale
PK, Groffen J: Acute leukemia in bcr/abl transgenic mice. Nature
344:251, 1990
25. Daley GQ, Baltimore D: Transformation of an interleukin
3-dependent hematopoietic cell line by the chronic myelogenous
leukemia-specific p210kr'""' protein. Proc Natl
Acad
Sci
USA
85:9312, 1988
26. Laneauville P, Sun G, Timm M, Vekemans M: Clonal evolution in a myeloid cell line transformed to interleukin-3 independent
growth by retroviral transduction and expression of p210k""b' Blood
80:1788, 1992
27. Carlesso N,Griffin JD, Druker BJ:Use of a temperaturesensitive mutanttodefinethe
biological effects ofthep210k"""
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
c-ab1 FUNCTION
IN CD34' CELLS
tyrosine kinase on proliferation of a factor-dependent murine myeloid cell line. Oncogene 9:149, 1994
28. Sirard C, Laneuville P,Dick JE Expression of bcr-ab1 abrogates factor-dependent growth of human hematopoietic M07E cells
by an autocrine mechanism. Blood 83:1575, 1994
29. Gishizky ML, Wit& ON: Initiation of deregulated growth by
multipotent progenitor cell by bcr-ab1 in vitro. Science 256:836, 1992
30. McGahon A, Bissonetted R, Schmitt M, Cotter KM, Green
DR, Cotter TG: BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death. Blood 83:1179, 1994
31. Bedi A, Zehnbauer BA, Barber JP, Sharkis SJ, Jones RI:
Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia.
Blood 83:2038, 1994
32. Andrews RG, Singer JW, Bemstein ID: Precursors of colonyforming cells in humans can be distinguished from colony-forming
cells by expression of the CD33 and CD34 antigens and light scattering properties. J Exp Med 169:1721, 1989
33. To LB. Haylock DN, Dowse T,Simmons PJ, Trimboli S ,
33s3
Ashman LK, Juttner CA: A comparative study of the phenotype
and proliferative capacity of peripheral blood (PB) CD34+ cells
mobilized by four different protocols and those of steady-state PB
and bone marrow CD34+ cells. Blood 84:2930, 1994
34. Arcinas M, Sizer KC, Boxer LM: Activation of c-myc expression by c-ab1 is independent of both the DNA binding function of
c-ab1 and the c-myc EP site. J Biol Chem 269:34, 1994
35. WongKK, Hardin JD, Boast S, Cooper CL, Merrell KT,
Doyle TG, Goff SP, Calame KL: A role for c-ab1 in c-myc regulation.
Oncogene 10:705, 1995
36. Bergamaschi G , Rosti V: Pathogenesis of chronic myelogenous leukemia. Haematologica 79:1, 1994
37. Eaves AC, Cashman JD, Gaboury LA, Kalousek DK, Eaves
C: Unregulated proliferation of primitive chronic myeloid leukemia
progenitors in the presence of normal marrow adherent cells. Proc
Natl Acad Sci USA 83:5306, 1986
38. Luzzatto L: Leukemia is a genetic disorder of somatic cells.
Haematologica 75: 105, 1990
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1995 86: 3387-3393
Oligodeoxynucleotides antisense to c-abl specifically inhibit entry
into S-phase of CD34+ hematopoietic cells and their differentiation to
granulocyte-macrophage progenitors
V Rosti, G Bergamaschi, C Lucotti, M Danova, C Carlo-Stella, F Locatelli, L Tonon, G Mazzini and
M Cazzola
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