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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3414-3422
Inhibition of BCR-ABL Expression With Antisense Oligodeoxynucleotides
Restores  1 Integrin-Mediated Adhesion and Proliferation
Inhibition in Chronic Myelogenous Leukemia Hematopoietic Progenitors
By
Ravi Bhatia and
Catherine M. Verfaillie
From the Department of Hematology and Bone Marrow Transplantation,
City of Hope National Medical Center, Duarte, CA; and the Division of
Hematology, Department of Medicine, University of Minnesota,
Minneapolis.
 |
ABSTRACT |
Chronic myelogenous leukemia (CML) is characterized by the
continuous proliferation and abnormal circulation of malignant hematopoietic progenitors. This may be related to the unresponsiveness of CML progenitors to  1 integrin adhesion receptor-mediated
inhibition of progenitor proliferation by the marrow microenvironment.
In hematopoietic cell lines, the BCR-ABL oncogene product,
p210BCR-ABL, interacts with a variety of cytoskeletal
elements important for normal integrin signaling. We studied the role
of p210BCR-ABL in abnormal integrin function in CML by
evaluating the effect of inhibition of BCR-ABL expression with
antisense oligodeoxynucleotides (AS-ODNs) on integrin-mediated adhesion
and proliferation inhibition of malignant primary progenitors from CML
marrow. Preincubation of CML CD34+HLA-DR+
(DR+) cells with breakpoint-specific AS-ODNs
significantly increased adhesion of CML progenitors to stroma and
fibronectin (FN). Pretreatment with breakpoint-specific ODNs also
resulted in significant inhibition of CML progenitor proliferation
after ligand or antibody-mediated 1 integrin engagement.
Breakpoint-specific ODNs were significantly more effective in restoring
CML progenitor adhesion and proliferation inhibition than control ODNs.
BCR-ABL mRNA and p210BCR-ABL levels in CML
CD34+ cells were significantly reduced after incubation
with breakpoint-specific AS-ODN. These studies indicate a role for
BCR-ABL in abnormal circulation and defective integrin-dependent
microenvironmental regulation of proliferation of CML hematopoietic
progenitors.
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INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML)
results from the malignant transformation of a hematopoietic stem cell
and is characterized by a massive expansion of hematopoietic
progenitors, as well as more differentiated hematopoietic cells,
originating from the malignant clone. In addition, large numbers of
circulating progenitors are seen in CML, reflecting their enhanced
release from the bone marrow cavity.1 Retention and homing
of progenitors to the marrow microenvironment is in large part
dependent on members of the 1 integrin family of adhesion receptors,
in particular the  41 receptor.2-4 Signaling through
1 integrin receptors also plays an important role in the regulation
of proliferation,5,6 differentiation,7 and
survival8 of a variety of cell types. We have shown that
signals transduced from the extracellular matrix by 1 integrins play
an important role in microenvironmental regulation of normal
hematopoietic progenitor proliferation.9,10 In contrast to
normal hematopoietic progenitors, CML progenitors show deficient adhesion to the microenvironment.11-13 Although malignant
CML CD34+ HLA-DR+ cells express normal levels
of 1 integrins on their cell surface, CML progenitors adhere
significantly less to 41 and 51 binding regions of
fibronectin (FN), indicating that 1 integrin receptor function is
abnormal in CML.13 We have shown that in addition to
defective adhesion, CML progenitors are unresponsive to 1 integrin-mediated inhibition of proliferation.14 Therefore, defects in integrin function may not only be responsible for the abnormal circulation, but also the continuous proliferation of CML
progenitors, leading to their selective expansion and growth advantage.
CML is characterized cytogenetically by a translocation between
chromosomes 9 and 22 [t(9;22)(q34;q11)], which leads to the fusion of
the c-ABL gene on chromosome 9 with the BCR gene on chromosome
22.15,16 The BCR-ABL oncogene plays a critical role in the
pathogenesis of CML. Transfection of hematopoietic cell lines with
BCR-ABL cDNA results in cellular transformation,17 and
transduction of mouse stem cells with BCR-ABL is sufficient to induce a
CML-like syndrome.18 Several studies have examined intracellular pathways leading to BCR-ABL-induced transformation. When
expressed in cell lines, p210BCR-ABL may activate the
RAS/MAPK pathway by binding to and/or phosphorylating proteins
involved in regulation of RAS function, such as RAS-GAP, SHC, and
GRB2.19,20 These observations may explain how BCR-ABL leads
to growth factor-independent proliferation of factor-dependent cell
lines. However, primary chronic phase CML hematopoietic progenitor cells do not show growth factor-independent proliferation and do not,
for the most part, differ from normal progenitors in their growth
factor responsiveness.21,22 On the other hand, recent studies suggest that p210BCR-ABL interacts with a number of
cytoskeletal proteins that are also involved in "inside-out" and
"outside-in" signaling through 1 integrins, such as F-actin,
FAK, Crkl, and paxillin.23-26 These studies led us to
hypothesize that interactions between p210BCR-ABL and
cytoskeletal elements important for 1 integrin signaling are
responsible for abnormal integrin function and disturbed
microenvironmental regulation of progenitor proliferation in CML.
Antisense oligodeoxynucleotides (AS-ODNs) complementary to the BCR-ABL
breakpoint can inhibit BCR-ABL expression, suppress CML-blast crisis
progenitor proliferation in vitro,27 and suppress CML cell
line growth in severe combined immunodeficient (SCID) mice.28 In the present study, we evaluated the effect of
inhibition of BCR-ABL expression by AS-ODNs directed against the
BCR-ABL breakpoint on 1 integrin-mediated adhesion and proliferation inhibition of CML progenitors. Our studies indicate that inhibition of
BCR-ABL expression in primary malignant CML
CD34+HLA-DR+ cells with breakpoint-specific
AS-ODNs restores integrin-mediated adhesion of CML colony-forming cells
(CFC) to stroma and FN substrates and restores inhibition of CML CFC
proliferation after contact with stroma and FN substrates or after
antibody-mediated integrin stimulation. These results indicate a role
for p210BCR-ABL in abnormal 1 integrin function in CML.
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MATERIALS AND METHODS |
Seventeen patients with CML and eight normal healthy volunteers were
evaluated after informed consent was obtained using guidelines approved
by the Committee on the Use of Human Subjects at the University of
Minnesota and the Institutional Review Board at the City of Hope
National Medical Center. Fifteen patients were in the chronic phase of
CML, whereas two were in accelerated phase. Nine patients were
receiving treatment with hydroxyurea alone, which was discontinued at
least 4 days before study. Eight patients had been previously treated
with interferon (IFN)-, which had been discontinued for at least 3 months before study. The b2a2 BCR-ABL
breakpoint was present in six patients, whereas the
b3a2 breakpoint was present in 11 patients.
Heparinized bone marrow samples were obtained by aspiration from the
posterior iliac crest. Bone marrow mononuclear cells (BMMNC) were
isolated by Ficoll-Hypaque (Sigma Diagnostics, St Louis, MO) density
gradient separation (specific gravity, 1.077) for 30 minutes at
37°C and 400g.
Selection of CD34+DR+ progenitor
populations.
CD34+ cell enriched populations were selected from CML or
normal BMMNC using sequential avidin-biotin immunoadsorbtion columns (CellPro Inc, Bothell, WA)29 or immunomagnetic column
separation (Miltenyi Biotech Inc, Auburn, CA).
CD34+HLA-DR+ (DR+) cells were then
selected by fluorescence-activated cell sorting (FACS) as previously
described.30,31
Adhesive substrates.
Bone marrow stromal layers were established from normal BMMNC in T-75
or T-150 flasks as previously described, irradiated at 1,250 cGy using
a Cesium irradiator to eliminate hematopoietic cells, and subcultured
in 24-well plates (Costar, Cambridge, MA).31 Human plasma
fibronectin was a kind gift from Dr James B. McCarthy (University of
Minnesota, Minneapolis, MN). FN was purified as a byproduct of factor
VIII production by sequential ion exchange and gelatin
chromatography32 and 75-kD tryptic fragments containing the
cell binding sequence RGDS and 33- and 66-kD COOH-terminal heparin
binding fragments were purified as previously described.32
AS-ODNs.
18 or 24 mer phosphodiester AS-ODNs to BCR-ABL junctional regions were
added to CML or normal DR+ cells as indicated in the
Results (Table 1). In addition to AS-ODNs
specific to the breakpoint present in the cells being studied, cells
were incubated with missense ODNs and ODNs to the alternate breakpoint,
eg, b2a2 if the tested cells contained the b3a2 breakpoint, as controls (control ODNs).
Incubation of DR+ cells with AS or control ODNs.
AS or control ODNs were added initially at a concentration of 40 µg/mL and a second time 18 hours later (30 µg/mL) for a total of 36 hours of incubation. To minimize AS-ODN degradation, cell incubation
was performed in a serum-free medium.33 To induce proliferation of FACS-selected normal or CML DR+ cells,
which are usually in G0,14 the serum-free
medium was supplemented with low levels of cytokines (SFM-LC), similar
to normal stroma-conditioned long-term bone marrow culture (LTBMC) medium (granulocyte colony-stimulating factor [25 pg/mL],
granulocyte-macrophage colony-stimulating factor [10 pg/mL], leukemia
inhibitory factor [50 pg/mL], stem cell factor [200 pg/mL],
interleukin-6 [1 ng/mL], and macrophage inflammatory protein-1
[200 ng/mL]),34 which we have previously used to induce
DR+ cell proliferation.14 Incubation of normal
and CML progenitors in this serum-free medium maintains CFC viability,
does not alter CFC adhesion, and induces CFC proliferation, as measured
by the thymidine suicide assay.
Coculture of DR+ cells with stromal layers.
A total of 10,000 CML and normal DR+ cells cultured in
SFM-LC were washed and resuspended in 0.5 mL LTBMC medium and either plated on irradiated normal stromal layers or kept in suspension for 4 hours, as previously described.14 After coculture, stromal layers were washed three times with warm Iscove's modified Dulbecco's medium (IMDM) and nonadherent cells
collected.30 Adherent cells were obtained using Trypsin
EDTA (Sigma). Both fractions were plated in short-term methylcellulose
assay and the percent adherent CFC was calculated as: [stroma-adherent
CFC divided by (stroma-adherent CFC + stroma-nonadherent CFC)] × 100. Thymidine suicide assays were performed to evaluate the percent of
CFC in S-phase in the different populations.
Coculture of DR+ cells with fibronectin substrates.
75-kD and 33-66-kD (60 µg/mL) proteolytic fragments from FN were
adsorbed to wells of a 48-well plate as previously
described.14 Control wells were adsorbed with 5 mg/dL
bovine serum albumin (BSA; >99% pure, fatty acid free; Sigma) alone
or with poly-L-Lysine (1:50,000; Sigma). A total of 10,000 CML or
normal DR+ cells cultured for 96 hours in SFM-LC were
washed and resuspended in 0.5 mL IMDM with 0.3% BSA and cultured
either in ligand-coated or control wells at 37°C and 5%
CO2. After 4 hours, nonadherent cells were removed by
standardized horizontal shaking of the plates followed by four to five
consecutive washings with warm IMDM. Adherent cells were removed by
trypsinization.14 Both fractions were plated in short-term
methylcellulose assay and the percent adherent CFC was calculated as:
[Adherent CFC divided by (adherent CFC + nonadherent CFC)] × 100. Thymidine suicide assays were performed on the different fractions
to determine their proliferative status.
Cross-linking of integrin receptors with monoclonal antibodies.
A total of 104 CML or normal DR+ cells were
cultured in SFM-LC for 96 hours, washed, and resuspended in 0.5 mL IMDM
with 0.3% BSA, aliquoted in 5-mL tubes (Becton Dickinson Labware,
Lincoln Park, NJ) and incubated with monoclonal antibody P4C10,
directed against the 1 integrin receptor (1:2,500; mouse ascites;
Life Technologies, GIBCO-BRL, Gaithersburg, MD), or control mouse IgG (2 mg/mL; 1:2,500; Sigma Chemical Co, St Louis, MO) for 30 minutes at
room temperature. After washing, cells were incubated with goat
antimouse antibodies (1:500; Biosource International, Camarillo, CA)
for 30 minutes at room temperature. Cells were then incubated at
37°C in a humidified atmosphere with 5% CO2 for 4 hours in the continued presence of the secondary antibodies, after
which they were washed and their proliferative status was assessed by thymidine suicide assays.14
Thymidine suicide assays.
The different cell fractions recovered after culture with stroma or
ligand-coated plates, or kept in suspension, with or without addition
of antibodies, were washed in serum-free IMDM (pH 7.3) and divided into
two equal fractions. They were then suspended in 200 µL serum-free
IMDM and incubated at 37°C with or without 5 mCi
3H-thymidine (6.7 Ci/mmol; New England Nuclear, Boston,
MA). After 20 minutes, 5 mL cold thymidine (500 mg/mL in IMDM with 20%
fetal calf serum; Sigma) was added to all samples to competitively
inhibit further 3H-thymidine uptake. Cells were washed in
IMDM and plated in methylcellulose progenitor culture for 14 to 18 days. The percentage of CFC in S-phase was calculated as the percentage
of CFC killed in the presence of 3H-thymidine {% CFC in
S-phase = [(CFC present in absence of 3H-thymidine  CFC present in presence of 3H-thymidine) × 100]
divided by CFC present in the absence of
3H-thymidine}.35 The reproducibility and
specificity of this assay have been previously
shown.9,13,35
Methylcellulose progenitor culture.
DR+ cells recovered from adhesion and thymidine suicide
assays were plated in methylcellulose culture as previously
described.36 Cultures were incubated in a humidified
atmosphere at 37°C and 5% CO2 for 14 to 18 days and
assessed for the presence of colony-forming unit-granulocyte macrophage
(CFU-GM), burst-forming unit-erythroid (BFU-E), and CFU-MIX colonies.
Polymerase chain reaction (PCR) analysis.
After incubation with AS-ODNs, CML DR+ cells were counted
and frozen at 70°C in PBS. Total cellular RNA was extracted
from thawed samples using the method of Chomczynski and
Saachi.37 After reverse transcription, amplification of
BCR-ABL mRNA was performed with the primers shown in
Table 2, using 30 to 40 cycles of
single-step PCR. Amplified samples were size separated by gel electrophoresis, ethidium bromide stained, and
photographed.38 -Actin controls were run for each
sample. The ratio of BCR-ABL to -actin was determined by
densitometry.
Western blotting.
After incubation with AS-ODNs, CML DR+ cells were counted
and protein extracts obtained by incubating with lysis buffer (1% Triton-X 100; 1 mmol/L EDTA; 150 mmol/L NaCl; 50 mmol/L Tris-HCl; 10 mmol/L sodium orthovanadate; 10 mmol/L sodium fluoride; 10 mmol/L
phenylmethyl sulfonyl fluoride [PMSF]; 0.15 U/mL
Aprotinin; 10 U/mL leupeptin, pH 7.5). Protein extracts
were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) followed by transfer to a nitrocellulose
filter. p145c-ABL and p210BCR-ABL were
visualized with an anti-c-ABL monoclonal antibody (antibody 24-11;
Santa Cruz Biotechnology Inc, Santa Cruz, CA or antibody Ab-3; Oncogene
Science Inc, Manhasset, NY), peroxidase-conjugated goat antimouse IgG
(Jackson Immunoresearch Laboratories Inc, West Grove, PA) and a Western
blot chemiluminescence reagent (Amersham Life Sciences, Arlington
Heights, IL). The ratio of BCR-ABL to c-ABL was determined by
densitometry.
Statistical analysis.
Results of experimental points obtained from multiple experiments were
reported as mean ± standard error of mean (SEM). Significance levels for differences between different samples was determined using
two-tailed Student's t-test.
| |
RESULTS |
Anti-BCR-ABL AS-ODNs restore CML CFC adhesion to stroma or FN.
In initial experiments, CML DR+ cells were incubated with
18-mer AS-ODNs specific for the BCR-ABL breakpoint
(b3a2 or b2a2) present
in the cells that were being evaluated. Negative controls included
missense AS-ODNs, eg, b2a2 missense if the
b2a2 breakpoint was present, and AS-ODNs to the
alternate breakpoint, eg, b3a2 if the tested
cells contained the b2a2 breakpoint.
Preincubation with breakpoint-specific AS-ODNs or control ODNs did not
significantly affect subsequent colony growth (data not shown).
Incubation with breakpoint-specific AS-ODNs significantly increased
adhesion of CML CFC to stromal layers (Fig
1). Control AS-ODNs had significantly less effect on CML CFC adhesion.
Breakpoint-specific AS-ODNs also enhanced CML CFC adhesion to 75-kD and
33-66-kD fragments of FN recognized by 41 and 51 integrins,
respectively,32 significantly more than control ODNs (Fig
1). These observations indicate that breakpoint-specific BCR-ABL
AS-ODNs can restore 1 integrin-mediated adhesion of CML CFC. The
effects of specific AS-ODNs on adhesion of CML cells containing either
the b2a2 or b3a2
breakpoint were equivalent.
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| Fig 1.
Anti-BCR-ABL AS-ODNs restore CML CFC adhesion to stroma
or fibronectin. A total of 10,000 DR+ cells were
incubated in serum-free medium with or without breakpoint-specific and
control ODNs after which their adhesion to BSA (A); stromal-adherent layers (B); FN 75-kD (C); and FN 33-66-kD (D) coated wells was evaluated as described in the text. Results shown represent the mean ± SEM for six (A, C, and D) or nine (B) separate experiments.
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Anti-BCR-ABL AS-ODNs restore 1 integrin-dependent inhibition of
CML CFC proliferation.
FACS selected CML DR+ cells, which are usually in
G0,13 were induced to proliferate by culture
for 4 days in serum-free medium supplemented with cytokines at
concentrations similar to those present in normal stroma-conditioned
LTBMC medium (SFM-LC).34 After 4 days, approximately 50%
of CML DR+ CFC were in S-phase as evaluated by thymidine
suicide assays. Proliferating CML DR+ cells were exposed to
AS and control ODNs during the last 36 hours of culture and then
cocultured with stroma and FN substrates for 4 hours, and the
percentage of CFC in S-phase was evaluated using thymidine suicide
assays. Consistent with previous studies, proliferation of untreated
CML CFC was similar whether cells were kept in suspension or were
cocultured with stroma or FN fragments.13 Preincubation
with either breakpoint-specific AS-ODNs or control ODNs did not alter
proliferation of CML CFC kept in suspension. However, significant
reduction in proliferation of CML CFC preincubated with
breakpoint-specific AS-ODNs was seen after coculture with stroma
(Fig 2), primarily as a result of
inhibition of proliferation of adherent CFC. CML CFC proliferation was
also significantly reduced when DR+ cells pretreated with
AS-ODNs were cocultured with FN 75-kD and 33-66-kD substrates (Fig 2).
In contrast to breakpoint-specific AS-ODNs, control ODNs did not
significantly reduce proliferation of CML CFC cocultured with either
stroma or FN (Fig 2). Inhibition of CML CFC proliferation was not a
nonspecific effect of adhesion because proliferation of CML CFC treated
with AS-ODNs was not reduced after coculture with the adhesogen
poly-L-lysine (Fig 2). In additional experiments, we examined the
effect of antibody-mediated stimulation of 1 integrins on CML CFC
proliferation. Proliferation of untreated CML CFC was not significantly
altered by cross-linking of 1 integrin receptors with blocking
anti-1 integrin mouse monoclonal antibodies and secondary antimouse
antibodies (Fig 3). However, proliferation
of CML CFC pretreated with anti-BCR-ABL AS-ODNs, but not control ODNs,
was significantly inhibited after antibody-mediated cross-linking of
1 integrin receptors (Fig 3). These observations indicate that
breakpoint-specific BCR-ABL AS-ODNs can restore 1 integrin-mediated
inhibition of CML CFC proliferation.
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| Fig 2.
Anti-BCR-ABL AS-ODNs restore inhibition of CML CFC
proliferation after coculture with stroma or FN. A total of 10,000 DR+ cells were incubated in serum-free medium for 96 hours with or without addition of breakpoint-specific and control ODNs.
After incubation, cells were either kept in suspension (A) or
cocultured with stroma (B); FN 75-kD (C); FN 33-66-kD (D); or
poly-L-lysine (E) coated wells for 4 hours, after which proliferation
was evaluated using thymidine suicide assays as described in the text.
For stroma (B), results for adherent (hatched bars), nonadherent (open
bars), and total (adherent + nonadherent) cells (solid bars) are
shown separately. The significance values in (B) represent differences in proliferation of total (adherent + nonadherent) cells. Results represent the mean ± SEM for six separate experiments.
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| Fig 3.
Anti-BCR-ABL AS-ODNs restore inhibition of CML CFC
proliferation after cross-linking with anti-1 integrin antibodies. A
total of 10,000 DR+ cells were incubated in serum-free
medium for 96 hours with or without addition of breakpoint-specific and
control ODNs. Cells were then incubated with anti-1 integrin
antibodies or control mouse antibodies for 30 minutes followed by
incubation with goat antimouse antibodies for 4 hours. Proliferation of
CFC with or without antibody treatment was assessed using thymidine
suicide assays. Results represent the mean ± SEM for four separate
experiments.
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The effect of anti-BCR-ABL AS-ODNs on CML progenitor adhesion and
proliferation is specific.
The specificity of the observed effects of breakpoint-specific AS-ODNs
on CML CFC adhesion and proliferation was further evaluated in
experiments in which CML DR+ cells were incubated with
additional breakpoint-specific and control AS-ODNs. These included
24-mer b2a2 and b3a2
breakpoint-specific AS-ODNs and a control 18-mer missense
anti-b3a2-ODN containing a TAT sequence
5 from the breakpoint, which is associated with nonspecific
effects in BCR-ABL-containing cell lines.39 Both 18- and
24-mer breakpoint-specific AS-ODNs restored CML CFC adhesion to FN and
proliferation inhibition after contact with FN, significantly more than
any of the control ODNs (Fig 4).
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| Fig 4.
Anti-BCR-ABL AS-ODNs restore CML CFC adhesion to and
proliferation inhibition by fibronectin significantly more than control ODNs. A total of 10,000 DR+ cells were incubated in
serum-free medium for 96 hours with or without addition of
breakpoint-specific and control ODNs. After incubation, cells were
either kept in suspension or were cocultured with FN substrates, and
their adhesion and proliferation were evaluated as described in the
text. Results represent the mean ± SEM for four separate
experiments.
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In another set of experiments, incubation of normal DR+
cells with anti-BCR-ABL ODNs did not alter the adhesion of normal CFC to FN (Fig 5). In addition, inhibition of
normal CFC proliferation usually seen after coculture with FN
substrates was unaltered after preincubation with BCR-ABL
AS-ODNs. These results indicate that the effects of BCR-ABL
AS-ODNs on CML CFC adhesion and proliferation are not the result of
nonspecific effects of ODNs on adhesion or
proliferation.40
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| Fig 5.
Anti-BCR-ABL AS-ODNs do not alter normal CFC adhesion or
proliferation. A total of 10,000 normal DR+ cells were
incubated in serum-free medium for 96 hours with or without addition of
b2a2 and b3a2
breakpoint-specific ODNs. After incubation, cells were either kept in
suspension or were cocultured with FN- (75 kD) coated wells for 4 hours
and their adhesion and proliferation were evaluated as described in the
text. Results represent the mean ± SEM for eight separate
experiments.
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Anti-BCR-ABL AS-ODNs inhibit BCR-ABL expression in CML
DR+ cells.
DR+ cells incubated with breakpoint-specific
AS-ODNs or control ODNs were evaluated by reverse transcriptase
(RT)-PCR for BCR-ABL mRNA levels and by Western blotting for
p210BCR-ABL levels to determine the effect of BCR-ABL
AS-ODNs on BCR-ABL expression. We show that exposure of CML
DR+ cells to breakpoint-specific AS-ODNs, but not control
ODNs, results in significant reduction of BCR-ABL mRNA levels
(Table 3 and Fig 6). Similarly, significant reduction in
p210BCR-ABL levels was observed after incubation with
breakpoint-specific AS-ODNs resulted in CML DR+ cells, but
not control AS-ODNs (Table 4 and
Fig 7). These studies indicate that, under
the conditions used, incubation with breakpoint-specific AS-ODNs can,
at least partially, inhibit BCR-ABL expression.
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| Fig 6.
Anti-BCR-ABL AS-ODNs inhibit BCR-ABL mRNA expression.
CML DR+ cells were incubated in serum-free medium with or
without addition of breakpoint-specific and control ODNs. PCR
amplification of BCR-ABL mRNA extracted from 25,000 DR+
cells was performed as described in the text, amplified samples were
size separated by gel-electrophoresis, ethidium bromide stained, and
photographed. -Actin controls were run for each sample. In the
representative experiment shown here, the b3a2
breakpoint was present in the cells studied. Cells were incubated
either without any ODNs (CONTROL), or with b3a2
antisense ODNs (B3A2AS), b3a2 missense ODNs
(B3A2MS), or b2a2 antisense ODNs (B2A2 AS).
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| Fig 7.
Anti-BCR-ABL AS-ODNs inhibit p210BCR-ABL
expression. CML CD34+ cells from a patient with a known
b3a2 BCR-ABL splice site were incubated in
serum-free medium with addition of either breakpoint-specific antisense
ODNs (AS) or with b3a2 specific missense ODNs
(MS) for 48 hours as described in the legend. mRNA obtained from 20 × 103 cells was used for RT-PCR for either the BCR-ABL mRNA
or -actin (top panel). Protein extracts obtained from 1 × 106 cells were subjected to SDS-PAGE
and Western blotting with an anti-c-ABL antibody (Ab3) as described in
Materials and Methods (bottom panel).
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| |
DISCUSSION |
Adhesion receptor-mediated interactions with the extracellular matrix
play an important role in the regulation of cellular proliferation,
differentiation, and survival.5-8 Neoplastic transformation is often associated with altered cell-matrix interactions and perturbation of normal microenvironmental regulation of cell growth and
function.41-43 The abnormal circulation and unregulated
expansion of malignant hematopoietic cells in CML may be related to
defective function of 1 integrin adhesion receptors. We hypothesized
that the BCR-ABL oncogene may induce abnormal 1 integrin function in
CML progenitors through its interactions with cytoskeletal proteins
critical for normal integrin function. We show here that downregulation
of BCR-ABL expression with breakpoint-specific AS-ODNs results in
restoration of 1 integrin function in CML progenitors. These studies
suggest a possible mechanism by which the BCR-ABL oncogene may
contribute to abnormal unregulated proliferation and abnormal
circulation of malignant hematopoietic progenitors in CML.
In previous studies, BCR-ABL AS-ODNs have inhibited clonogenic growth
of CML-blast crisis progenitors but have failed to consistently inhibit
growth of CML-chronic phase progenitors.27,44 In the present studies, the growth of chronic phase CML CFC was not altered after 48 hours of exposure to BCR-ABL AS-ODNs. In addition, BCR-ABL AS-ODNs did not by themselves alter the percentage of CML progenitors in S-phase. However, the present studies indicate that downregulation of BCR-ABL expression with BCR-ABL breakpoint-specific AS-ODNs is
associated with restoration of progenitor-microenvironmental interactions in CML. Malignant CML progenitors show deficient adhesion
to stroma and FN through 41 and 51 integrins. Treatment with breakpoint-specific AS-ODNs resulted in enhanced CML progenitor adhesion to stroma and 41 and 51 integrin binding fragments of FN. In normal progenitors, 1 integrin-mediated adhesion to stroma
and FN, or engagement of the integrin receptors with anti-1 antibodies, leads to inhibition of progenitor proliferation. CML hematopoietic progenitors, in contrast, are unresponsive to 1 integrin-mediated inhibition of proliferation.13
Breakpoint-specific AS-ODNs, besides restoring CML CFC adhesion, also
restore inhibition of CML progenitor proliferation after coculture with
stroma, FN, or anti-1 integrin antibodies. Reduced BCR-ABL
expression does not seem to represent increased numbers of normal cells
given the lack of reduction in CFC numbers after culture, the tiny
fraction of BCR-ABL-negative cells in the starting CD34+
population, and the lack of significant expansion in normal CFC number
after 36 hours of culture in medium with low cytokine concentrations. These observations support the hypothesis that p210BCR-ABL
is responsible for abnormal 1 integrin-mediated adhesion and inhibition of proliferation in CML progenitors.
When interpreting the results of experiments using AS-ODNs, it is
important to recognize that cellular responses observed after exposure
to AS-ODNs may not necessarily be the result of specific inhibition of
the mRNA targeted by that ODN.45 In previous studies,
missense ODNs, and AS-ODNs to the breakpoint not present in the cells
being evaluated, have had nonspecific effects on BCR-ABL-transduced
cell lines.46 In the present studies, however, significant
inhibition of BCR-ABL expression at the protein and mRNA level in CML
CD34+ cells was observed with breakpoint-specific BCR-ABL
AS-ODNs, but not control ODNs. In addition, 18- and 24-mer
breakpoint-specific AS-ODNs restored 1 integrin-mediated adhesion
and proliferation inhibition of CML CFC significantly more than control
missense ODNs, control AS-ODNs to the breakpoint not present in the
cells being studied, and a control b3a2
missense ODNs containing the TAT sequence present in
anti-b2a2 AS-ODNs,39 which is
particularly associated with nonspecific effects. Our studies indicate
that even partial inhibition of BCR-ABL expression in CML primary
progenitors is sufficient to allow restoration of integrin-dependent
microenvironmental regulation. Although AS-ODNs can nonspecifically
alter cell adhesion,40 this does not appear to be the case
here because normal CFC adhesion or adhesion-mediated proliferation
inhibition was not affected by BCR-ABL AS-ODNs.
BCR-ABL-expressing cell lines express several-fold more
p210BCR-ABL than primary CML hematopoietic
cells.47 As a result, in previous studies using CML cell
lines, high concentrations of phosphorothioate AS-ODNs, which are more
likely to have nonspecific effects, have been required to achieve
inhibition of BCR-ABL expression. The selective effects of
breakpoint-specific BCR-ABL AS-ODNs on BCR-ABL expression and CML CFC
adhesion and proliferation in our studies may thus be related to the
use of phosphodiester instead of phosphorothioate AS-ODNs, the use of
serum-free medium to prevent phosphodiester AS-ODN degradation, and the
use of relatively low concentrations of AS-ODNs, all of which reduce
the likelihood of nonspecific effects.45 Higher
concentrations or prolonged exposure to AS-ODNs, or the use of
phosphorothioate AS-ODNs, may possibly have resulted in more complete
inhibition of BCR-ABL expression in our studies, but would probably
have caused increased nonspecific effects.
p210BCR-ABL may induce abnormal integrin function in CML by
associating with and/or abnormally phosphorylating cytoskeletal
proteins or cytoskeleton-associated signaling proteins important for
normal integrin function. We have recently shown that 1 integrin
receptor capping after antibody-mediated receptor engagement and
cross-linking, which is an indicator of the ability of the integrin
receptor to interact with the cytoskeleton,10 is
significantly impaired in BCR-ABL-transformed cells, indicating
abnormal receptor cytoskeletal interactions.48 Inhibition
of BCR-ABL mRNA and protein expression by insertion of antisense gene
constructs restores 1 integrin capping in these cells, supporting a
role for BCR-ABL in defective capping.49 Integrin
cytoplasmic domains do not have intrinsic enzymatic activity, but
mediate "inside-out" and "outside-in" signaling through
interactions with cytoskeletal elements such as -actinin, talin,
vinculin, paxillin, and tensin in focal adhesion complexes.50-52 These proteins, besides connecting integrin
receptors to actin microfilaments, also serve as a framework for the
association of other signaling proteins, including protein tyrosine
kinases such as FAK and SRC and adapter proteins such as GRB-2 and
CRK,52,53 thereby linking integrin receptor stimulation to
various intracellular signaling pathways. Integrin stimulation is also
associated with cytoskeletal translocation and activation of the
phosphoinositide 3-kinase (PI-3 kinase),54 which may also
play an important role in modulation of integrin function. The focal
adhesion-associated proteins paxillin, FAK, vinculin, talin, and tensin
are constitutively phosphorylated in BCR-ABL-transfected cell
lines.24,26,55 Recent studies suggest that the adapter
proteins CRKL and CBL are prominent p210BCR-ABL substrates
in primary CML cells25 and may form a complex with p210BCR-ABL and link it with paxillin and the p85
regulatory subunit of PI-3 kinase.56-58 Finally, other
studies have shown that the COOH terminus of c-ABL has F- and G-actin
binding domains with actin bundling activity, that
p210BCR-ABL shows enhanced actin binding through this
COOH-terminal domain, and that this interaction may be important for
the transforming ability of BCR-ABL.23,59 Some or all of
these interactions of p210BCR-ABL with integrin-related
proteins may contribute to abnormal integrin function in CML.
Some other recent observations also support the concept that BCR-ABL
may alter integrin-mediated adhesion, as well as signaling from the
extracellular matrix (ECM). Proliferation of normal fibroblasts requires signals generated both by cytokine stimulation, as well as
cell attachment via integrins. BCR-ABL transfection has been reported
to abrogate the anchorage requirement, but not the growth factor
requirement, for fibroblast proliferation.60 Cytokine and
serum starvation of hematopoietic cell lines or primary hematopoietic progenitors is associated with failure of integrin-mediated adhesion to
FN, but is reversed after readdition of cytokines.61,62 In
contrast, the same cell lines transfected with BCR-ABL and serum and
cytokine starved continued to adhere to FN, but adhesion could not be
upregulated by readdition of cytokines.62 The proadhesive effect of BCR-ABL therefore mimics the effect of cytokines in BCR-ABL-negative cell lines. These studies suggest that BCR-ABL can,
in different cellular contexts, alter inside-out signaling pathways
that regulate integrin-mediated adhesion and growth signaling. The
differences between the effects of BCR-ABL in these studies and our own
may be related to the differences in the cell types and the assay
conditions used. In the former studies, fibroblasts and cell lines
rather than primary hematopoietic progenitors were used. Further, the
cell lines were serum and cytokine starved before adhesion studies,
which may explain their relatively deficient adhesion. It is possible
that BCR-ABL may prevent decrease in cell adhesion after cytokine
withdrawal by preventing downregulation of Rho and Rac,63
required for actin stress fiber formation, through constitutive
activation of intracellular signal molecules such as p62DOK
and pl20GAP.64,65 In addition, BCR-ABL protects cells that are cytokine starved from apoptotic cell death.21 The
decrease in adhesion of BCR-ABL-negative cell lines, but not
BCR-ABL-containing cell lines, after cytokine starvation may therefore
be related to increased stress on these "normal" cells. Reduced
adhesion of CML progenitors in our studies is not the result of reduced growth of adherent progenitors, as the total number of colonies, adherent + nonadherent, generated from CML DR+ cells after
coculture with control BSA and FN fragments was similar, and
proliferation of the CML progenitors that adhered to FN was not reduced
when measured in thymidine suicide assays.
In conclusion, we show that inhibition of BCR-ABL expression with
breakpoint-specific AS-ODNs restores 1 integrin-mediated adhesion
and proliferation inhibition in CML CFC. Our studies link the BCR-ABL
oncogene with altered integrin-dependent microenvironmental regulation
and abnormal circulation of hematopoietic progenitor proliferation in
CML. Future studies will be directed towards understanding the specific
molecular mechanisms underlying p210BCR-ABL-induced
disruption of integrin function in CML progenitors.
| |
FOOTNOTES |
Submitted September 16, 1997;
accepted December 30, 1997.
Supported in part by National Institutes of Health (Bethesda, MD)
Grants No. R29 CA74455, PO1 CA 45814, PO1 CA65493, and R01 HL 49930;
and the University of Minnesota Hospitals and Clinics (Minneapolis).
R.B. is a recipient of an American Society of Clinical Oncology Young
Investigators Award (Alexandria, VA). C.M.V. is a Scholar of the
Leukemia Society of America (New York, NY).
Address reprint requests to Ravi Bhatia, MD, Department of Hematology
and Bone Marrow Transplantation, City of Hope National Medical Center,
1500 E Duarte Rd, Duarte, CA 91006.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors would like to acknowledge the excellent technical
assistance of Heidi Munthe, Denise Malone, and Brad Anderson.
| |
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R. C. Zhao, Y. Jiang, and C. M. Verfaillie
A model of human p210bcr/ABL-mediated chronic myelogenous leukemia by transduction of primary normal human CD34+ cells with a BCR/ABL-containing retroviral vector
Blood,
April 15, 2001;
97(8):
2406 - 2412.
[Abstract]
[Full Text]
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E. Laurent, M. Talpaz, H. Kantarjian, and R. Kurzrock
The BCR Gene and Philadelphia Chromosome-positive Leukemogenesis
Cancer Res.,
March 1, 2001;
61(6):
2343 - 2355.
[Full Text]
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N. Heisterkamp, J. W. Voncken, D. Senadheera, I. Gonzalez-Gomez, A. Reichert, L. Haataja, A. Reinikainen, P. K. Pattengale, and J. Groffen
Reduced oncogenicity of p190 Bcr/Abl F-actin-binding domain mutants
Blood,
September 15, 2000;
96(6):
2226 - 2232.
[Abstract]
[Full Text]
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Y. Jiang, R. C. H. Zhao, and C. M. Verfaillie
Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34+ cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27Kip, which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity
PNAS,
September 5, 2000;
(2000)
190104497.
[Abstract]
[Full Text]
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K. H. Kain and R. L. Klemke
Inhibition of Cell Migration by Abl Family Tyrosine Kinases through Uncoupling of Crk-CAS Complexes
J. Biol. Chem.,
May 4, 2001;
276(19):
16185 - 16192.
[Abstract]
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Y. Jiang, R. C. H. Zhao, and C. M. Verfaillie
Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34+ cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27Kip, which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity
PNAS,
September 12, 2000;
97(19):
10538 - 10543.
[Abstract]
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Abstract
Introduction
Abstract