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NEOPLASIA
From the Department of Bioengineering, Institute for
Medicine and Engineering, Department of Pathology and Laboratory
Medicine, and Department of Microbiology, University of Pennsylvania,
Philadelphia; and Division of Hematology and Medical Oncology,
Department of Cell and Developmental Biology and Biochemistry and
Molecular Biology, Oregon Health Sciences University, Portland.
The t(9;22) chromosomal translocation results in expression of
P210BCR-ABL, a fusion protein necessary for the development
of chronic myelogenous leukemia (CML). The constitutive activation of
the P210BCR-ABL tyrosine kinase results in phosphorylation
of multiple signaling pathways leading to the transformed phenotype.
Additionally, extracellular interactions between
P210BCR-ABL-expressing progenitor cells and bone marrow
stroma may provide external signals that facilitate CML
development. In contrast to the intracellular signaling pathways
involved in CML, little is known about how P210BCR-ABL
expression modifies cell-cell and cell-substratum interactions. To investigate the role of P210BCR-ABL in
modulating cellular adhesion, we used a highly sensitive and quantitative cell detachment apparatus that measures the strength of
association between a population of cells and an adhesive matrix. Our
findings show that P210BCR-ABL expression increased
adhesion nearly 2-fold between the myeloblastic cell line, 32D, and
fibronectin compared to a control vector. We then investigated whether
abnormal adhesion due to P210BCR-ABL expression was caused
by its tyrosine kinase activity. A quantitative analysis of
cell-fibronectin adhesion found that neither expression of a
kinase-inactive P210BCR-ABL mutant in 32D cells or
attenuation of kinase activity by STI571 (imatinib mesylate)
in 32D cells transduced with wild-type P210BCR-ABL could
correct the nearly 2-fold increase in cell-fibronectin adhesion.
Similarly, STI571 treatment of Meg-01 cells, a
P210BCR-ABL-expressing cell line derived from a patient in
blast crisis, failed to inhibit adhesion to fibronectin.
Together, our results indicate that changes in adhesion
induced by P210BCR-ABL are independent of its tyrosine
kinase activity.
(Blood. 2002;99:4122-4130) P210BCR-ABL is a 210-kd nonreceptor
tyrosine kinase that is the product of the t(9;22) balanced
translocation between the bcr gene on chromosome 22 and the
abl gene on chromosome 9.1 The translocation results in the formation of the Philadelphia chromosome (Ph), which is
the diagnostic marker for chronic myelogenous leukemia (CML), a disease
characterized by abnormal accumulation of hematopoietic cells in the
bone marrow, elevated peripheral white blood cell counts, and defective
progenitor-stromal adhesion in vitro.2,3 There is
intense interest in the observation that P210BCR-ABL
expression in cell lines and bone marrow progenitor cells leads to defective cell binding to fibronectin and stromal cells because abnormal adhesion in transformed cells may contribute to loss of
contact inhibition and proliferation in the bone marrow of patients
with CML.3-5 However, the mechanism by which
P210BCR-ABL alters cellular adhesion is poorly understood.
P210BCR-ABL is primarily found in the cytoplasm and
colocalizes with actin through a C-terminal actin-binding
domain.6,7 It is unknown if P210BCR-ABL
directly modifies actin or recruits signaling molecules to the cytoskeleton. However, transformed cells exhibit increased motility and
enhanced membrane ruffling and proteins of the focal adhesion complex,
such as focal adhesion kinase and paxillin, are phosphorylated in
P210BCR-ABL cells.8,9 Together, these
observations suggest that P210BCR-ABL induces abnormal
cell-substratum binding that is likely involved in the pathogenesis of
CML. One possible mechanism is that P210BCR-ABL regulates
an inside-out signaling pathway that stimulates integrin-mediated adhesion in a manner similar to growth factors, such as interleukin 3 (IL-3).10 The tyrosine kinase activity is critical for
the oncogenic effects of P210BCR-ABL because
expression of P210BCR-ABL mutants with either
temperature-sensitive tyrosine kinase activity or an inactive tyrosine
kinase domain are not oncogenic in vitro or in vivo.11,12
Accordingly, the P210BCR-ABL constitutively activated
tyrosine kinase is a plausible focal point for inside-out signaling.
Differentiation of the hematopoietic compartment from stem cells
through progenitors is a tightly regulated process that relies on
specific interactions between stromal and hematopoietic cells for
proper cues governing proliferation and
differentiation.13,14 In the case of CML, defective
progenitor-stromal interactions have been documented using both bone
marrow from CML patient donors3,4 and transformed cell
lines5,15; yet these studies have yielded equivocal
results. It is unclear if these disparate findings are due to
methodologic differences or the nature of the cells assayed. Most
adhesion studies are limited to panning-style assays that use an
uncharacterized and inconsistent washing technique to detach cells. To
overcome the inherent variability of panning-style assays to measure
adhesion, we adapted a quantitative adhesion assay that relies on fluid
flow to remove bound cells using a measurable detachment
force.16 This assay provides a quantitative analysis of
cell-ligand interactions, and thus provides the capability to measure
small changes in adhesion.17 The cell detachment device
has previously been used to examine the role of
To investigate the role of P210BCR-ABL in cellular
adhesion, we used the cell detachment device to measure adhesive
changes in 32D cells following transduction by
P210BCR-ABL-expressing retroviruses. The 32D cells
expressing P210BCR-ABL were 1.7 times more adhesive to
fibronectin than cells transduced with a control vector. We then used
either a tyrosine kinase dead P210BCR-ABL mutant with a
Lys1176Arg (K1176R) amino acid substitution12 or
wild-type P210BCR-ABL cells treated with the specific
P210BCR-ABL tyrosine kinase inhibitor, STI571 (imatinib
mesylate, Gleevec),21 to evaluate the role of tyrosine
kinase activity in regulating cellular adhesion. The importance of the
P210BCR-ABL catalytic activity in contributing to CML is
underscored by recent studies indicating that the
P210BCR-ABL tyrosine kinase inhibitor STI571 normalizes
white blood cell counts in patients with CML.22 We found
that neither the tyrosine kinase inactive mutant nor treatment of
wild-type P210BCR-ABL with STI571 could normalize the
increased adhesion observed on expression of
P210BCR-ABL. We also found that localization of
P210BCR-ABL to F-actin filaments is independent of tyrosine
kinase activity. These findings suggest that both altered adhesion to
fibronectin and actin localization are tyrosine kinase-independent
activities of P210BCR-ABL. These tyrosine
kinase-independent properties of P210BCR-ABL may synergize
with its tyrosine kinase-dependent activity to induce the transformed phenotype.
Plasmid construction and cell culture
Fifteen to 17 hours prior to assessing adhesion, pK1 and/or
BCR-ABL-expressing 32D cells were diluted into 32D medium containing 1% WEHI with or without 10 µM STI571, as indicated. Meg-01 cells were treated with 10 µM STI571 for 3.5 hours or an equal volume of
the drug carrier (phosphate-buffered saline [PBS]) for 3.5 hours or
1.5 hours prior to assaying binding. Cells were washed 2 times in PBS
and resuspended in adhesion buffer (24 mM Tris, 137 mM NaCl, 1 mM
MgCl2, 3 mM KCl, 2 mM glucose) at 1 × 106
cells/mL. In some control experiments, Meg-01 cells were treated with
2.125 mM RGD peptide (catalogue no. 03-34-0035, Calbiochem, San Diego,
CA) for 10 minutes prior to seeding on fibronectin substrates.
Adhesion assays were carried out in the absence of growth factors and
were completed within 2 hours of washing. Controls showed that adhesion
levels did not change during the period in which multiple samples were
assayed (data not shown).
Adhesion assays
Western blots and flow cytometry Western blots were performed on lysates from 32D cells expressing BCR-ABL or pK1 that were incubated overnight in 32D media with 10% WEHI with or without decreasing concentrations of STI571. Meg-01 cells were treated with 10 µM STI571 or an equal volume of PBS alone for 3 hours. Equal volumes of lysates were assayed on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels to detect P210BCR-ABL, 12.5% SDS-PAGE gels to detect grb2, or 10% SDS-PAGE gels to detect tyrosine phosphorylated proteins. Proteins were transferred from gels to nitrocellulose membranes and were blotted with an anti-abl antibody (24-11, Santa Cruz Biotechnology, Santa Cruz, CA), an antiphosphotyrosine antibody (PY20, Pharmingen, San Diego, CA), or an anti-grb2 antibody (81, Transduction Laboratories, Lexington, KY). Anti- 4 (R-2, Pharmingen) and
anti-5 (5H10-27, Pharmingen) antibodies were used to
assess integrin expression by flow cytometry. All samples and controls
were run in triplicate. The mean fluorescence index (MFI) was
calculated as the difference in geometric mean fluorescence between
experimental samples stained with primary and secondary antibodies and
negative control samples stained with the secondary antibody alone;
this value was normalized to the geometric mean fluorescence of the
negative control to give the MFI.
Microscopy NIH3T3 or 32D cells were transduced with retroviral constructs encoding P210BCR-ABL, K1176R, or the pK1 control plasmid and selected for 4 days with puromycin. NIH3T3 cells were cultured overnight on fibronectin coverslips with or without 10 µM STI571 as indicated. 32D cells were plated onto fibronectin coverslips at a concentration of 1 × 105 cells/mL by following the same procedure outlined above for adhesion assays. Cells were fixed with 2% paraformaldehyde, permeablized with 0.1% Triton-X, and blocked with 1% denatured BSA (NIH3T3 cells) or 5% normal goat serum diluted in 1% denatured BSA (32D cells). Coverslips were stained with 5 µg/mL anti-abl antibody (8E9, Pharmingen). Bound anti-abl antibody was revealed with goat antimouse Texas red antibody (NIH3T3 cells) or goat antimouse Cy3 antibody (32D cells, Jackson ImmunoResearch, West Grove, PA). Actin was stained with 0.1 µg/mL fluorescein isothiocyanate-phalloidin (Sigma, St Louis, MO) and the nucleus was revealed with 4',6-diamidino-2-phenylindole (DAPI) staining (1:10 000 dilution). Cells were imaged using confocal microscopy at the Bioengineering Confocal and Multiphoton Imaging Facility at the University of Pennsylvania.
The cell detachment device quantitatively measures the strength of binding between cells and fibronectin The cell detachment device presents a 2-dimensional matrix of fibronectin and BSA to a monolayer of cells seeded at a constant surface density throughout the coverslip (data not shown). This well-characterized surface is mounted onto a rotating circular stage that creates convective fluid flow within the chamber that dislodges bound cells (Figure 1A). The detachment force is calculated (equation in "Materials and methods") from the properties of the buffer, the dimensions of the coverslip, and the rotational speed that is measured by a digital tachometer. The detachment force is zero at the center of the matrix and increases linearly to reach a maximum near the circumference, allowing for a range of detachment forces to be tested during a single experiment. Accordingly, few cells remained near the edge due to the higher force at this location than closer to the center (Figure 1B).
The fraction of cells that remain on the surface after a spin
(normalized to the count at the center where r = P210BCR-ABL expression leads to increased adhesion The advantage of the spinning disk adhesion assay is its ability to quantitatively measure the strength by which cells bind fibronectin over a range of defined forces. Accordingly, we asked whether expression of P210BCR-ABL influenced cell adhesion to fibronectin compared to pK1 control cells expressing an empty vector. In the presence of fibronectin, expression of P210BCR-ABL in 32D cells increased the fraction of bound cells at nearly all shear stresses tested compared to pK1 control cells, indicating that expression of P210BCR-ABL led to increased cell adhesion (Figure 2A). The critical shear stress, 50, (ie, adhesion strength) is the force required to dislodge half of the cells and is a measurement of the strength of
association between cells and fibronectin.29,30 The
50 is averaged for a set of identical experiments and
can be compared over different conditions, such as with and without
P210BCR-ABL expression (Figure 2B). Expression of
P210BCR-ABL led to an increase in the average critical
shear stress, 50, from 35.1 dyne/cm2 to 59.6 dyne/cm2, indicating that adhesion increased approximately
1.7-fold. We also observed nearly a 2-fold increase in adhesion when
P210BCR-ABL is expressed from a retroviral construct
coexpressing green fluorescent protein (data not shown).
The constitutive tyrosine kinase activity is not required for P210BCR-ABL-mediated adhesion The finding that P210BCR-ABL expression leads to increased cell adhesion in our system may potentially be explained by inside-out signaling initiated by the P210BCR-ABL tyrosine kinase. To assess the role of tyrosine kinase activity on cell adhesion to fibronectin, a P210BCR-ABL mutant lacking tyrosine kinase activity, K1176R, was transduced into 32D cells. Similar to wild-type P210BCR-ABL 32D cells, K1176R 32D cells bound more tightly to fibronectin than control pK1 cells (Figure 3A). Moreover, we found that the critical shear stress of a series of spins using 2 separate populations of K1176R-expressing cells were not statistically different from P210BCR-ABL (P = .14, P = .89, not shown), but were significantly elevated compared to the control pK1 cells (P < .001, P = .003, Figure 3B).
One possible mechanism by which P210BCR-ABL increases
adhesion is by influencing integrin expression. The
Treatment of P210BCR-ABL cells with STI571 fails to normalize P210BCR-ABL-mediated adhesion To confirm our previous finding using the tyrosine kinase-inactive K1176R mutant, we treated P210BCR-ABL-expressing cells with STI571 as an additional method to evaluate the contribution of kinase activity on adhesion. STI571 is a specific inhibitor of the P210BCR-ABL tyrosine kinase by nature of the unique adenosine triphosphate-binding cleft in the catalytic domain.21,33 Although STI571 is effective at inhibiting the kinase of the platelet-derived growth factor receptor and the c-kit receptor,34 we do not expect these receptors to play a role in our assay because their ligands are absent from the adhesion buffer.P210BCR-ABL expression dramatically increases
phosphotyrosine levels in hematopoietic cells lines leading to cell
transformation and IL-3 independence.35 We confirmed that
K1176R expression or treatment of P210BCR-ABL cells with
STI571 abrogated both the kinase activity and transforming ability of
P210BCR-ABL. Specifically, the K1176R mutant or overnight
treatment of P210BCR-ABL-expressing cells with 10 µM
STI571 or less reduced the total cellular phosphotyrosine level in a
dose-dependent manner, even though P210BCR-ABL expression
was unchanged (Figure 4A). Furthermore,
expression of K1176R was also comparable to untreated or STI571-treated
wild-type P210BCR-ABL in our system (Figure 4A). K1176R
failed to rescue 32D cells from IL-3 withdrawal (data not shown) and
treatment with STI571 reversed the growth factor independence of
wild-type P210BCR-ABL-expressing 32D cells leading to
apoptosis in the absence of IL-3 (Figure 4B). However, STI571-treated
P210BCR-ABL cells survived in 10% WEHI medium containing
IL-3, showing that STI571 was not cytotoxic at the concentrations used
in this study.
As an independent method to confirm our finding that K1176R exhibits
increased adhesion in the absence of tyrosine kinase activity, we
treated P210BCR-ABL-expressing 32D cells with STI571. We
compared the adhesion of P210BCR-ABL cells that were
untreated or treated with STI571 overnight at the same conditions used
to evaluate proliferation and tyrosine kinase activity. We found that
attenuation of catalytic activity not only failed to decrease cell
adhesion compared to untreated cells, but may have slightly increased
the ability of these cells to bind fibronectin (Figure
5). Taken together, these results suggest
that the kinase activity is not a major contributor to the increased
cell adhesion to fibronectin brought about by P210BCR-ABL
expression.
STI571 treatment of a Ph+ human megakaryoblastic cell line, Meg-01, abrogates kinase activity but fails to alter cell adhesion To determine whether human Ph+ cells also exhibit increased adhesion to fibronectin, we investigated the role of P210BCR-ABL kinase activity in modulating adhesion in Meg-01 cells. The Meg-01 cell line was derived from the bone marrow of a 55-year-old man in the blast crisis phase of CML.26 Treatment of these cells with 10 µM STI571 led to a marked reduction in cell viability by 48 hours following treatment initiation (Figure 6A). Similar to expression of wild-type P210BCR-ABL in 32D cells, STI571 treatment did not effect expression of P210BCR-ABL but led to a reduction in the whole cell phosphotyrosine levels compared to Meg-01 cells treated with PBS (Figure 6B). Attenuation of tyrosine kinase activity was observed as early as 3 hours after treatment of Meg-01 cells. At this time, binding of kinase attenuated Meg-01 cells to fibronectin was assessed and was found to be unchanged (P = .81) when compared to cells treated with an equal volume of PBS.
Localization of BCR-ABL in the cytoplasm to F-actin is unchanged by K1176R expression or in response to STI571 We sought to determine other characteristics of P210BCR-ABL that may be kinase-independent. Because adhesion to fibronectin did not correlate with kinase activity, we asked if the kinase activity of P210BCR-ABL affects its subcellular localization in NIH3T3 fibroblasts or 32D cells when detected by confocal microscopy. Like wild-type P210BCR-ABL, abrogation of the kinase activity by either the K1176R point mutation or STI571 treatment did not alter the cytoplasmic localization of P210BCR-ABL in either cell line (Figure 7A,B). In NIH3T3 cells, P210BCR-ABL was found along membrane ruffles, actin filaments, and strongly stained close to the cellular membrane in the presence or absence of kinase activity regardless of the method by which it was attenuated (Figure 7A). The 32D cells remained rounded and did not spread on fibronectin during the 15-minute incubation period used in both the immunohistochemistry analysis and the adhesion assays. P210BCR-ABL and actin both displayed a cortical staining pattern along the membrane of 32D cells, and this pattern did not vary in the presence or absence of tyrosine kinase activity (Figure 7B).
To investigate the role of P210BCR-ABL in modulating cell adhesion to fibronectin, a major component of the bone marrow extracellular matrix,36 we used a highly sensitive and quantitative adhesion assay to measure the hydrodynamic forces used to detach bound cells. This device has advantages over conventional plate and wash assays by directly measuring the strength by which a population of cells is anchored to the extracellular matrix. The standardized detachment forces used in this system eliminate operator bias and variability in washing techniques that may occur in studies using panning-style adhesion assays. In contrast to long-term culture-initiating cell assays,37 or similar adhesion assays that rely on secondary colony formation to enumerate adherent and nonadherent progenitor cells in a heterogeneous population of primary bone marrow,3 our adhesion apparatus directly measures the magnitude of binding between a homogeneous population of cells and fibronectin. Due to the unique geometry of the detachment device, we assay a range of shear stresses during each spin and observe changes in adhesion throughout the entire range of forces that are tested. The cell detachment device generates a range of detachment forces (0-100 dyne/cm2) on a single fibronectin-coated coverslip, allowing our data to be fitted to known models of cell adhesion,16,17,29,30,38 and recapitulates shear stresses commonly found throughout the human body. It should be noted that the precise hydrodynamic environment of the bone marrow is unknown; however, it is likely that some of the forces generated by the detachment device exceed forces found in the bone marrow. Nevertheless, we observe differences in adhesion between P210BCR-ABL-expressing 32D cells and control cells at all shear stresses assayed, suggesting that increased adhesion induced by P210BCR-ABL occurs at physiologic forces. We expressed P210BCR-ABL in the myeloblastic cell line, 32D, which affords a homogenous population of cells to study changes in adhesion in the predominant cell type affected by CML. Using this model system, we found that P210BCR-ABL expression led to a 1.7-fold increase in adhesion. Consistent with our findings, there have been 2 other reports demonstrating that P210BCR-ABL promotes increased adhesion to fibronectin.5,15 Our results are also consistent with studies showing that adhesion between P210BCR-ABL-expressing cells and fibronectin promotes cell proliferation, cell cycle progression, and protects cells from DNA damage-induced apoptosis.15,39 In this scenario, increased adhesion may both promote the accumulation of progenitor cells and enhance their survival, 2 features associated with the pathogenesis of CML. P210BCR-ABL phosphorylates paxillin, focal adhesion
kinase, and other members of the focal adhesion complex, suggesting a
link between the catalytic activity of P210BCR-ABL and
regulation of integrin-mediated adhesion.9 To assess
whether the tyrosine kinase activity of P210BCR-ABL
initiates inside-out signaling leading to increased adhesion, we tested
the binding of 32D cells transduced with both the K1176R mutant or
wild-type P210BCR-ABL cells treated with STI571 at
concentrations shown to attenuate tyrosine kinase activity, reverse 32D
cell transformation, and kill human leukemia cells over a period of
days. Using these 2 independent methods to attenuate kinase activity in
32D cells transduced with P210BCR-ABL, we found that
P210BCR-ABL-mediated increased adhesion remains intact in
the absence of tyrosine kinase activity. Adhesion to fibronectin was
also shown to be independent of tyrosine kinase activity in a second
cell line, Meg-01, a Ph+ cell line derived from a patient
in the blast crisis phase of CML. Together, these results support our
hypothesis that altered adhesion due to P210BCR-ABL
expression is kinase-independent. Consistent with our results describing tyrosine kinase-independent functions of
P210BCR-ABL, a kinase inactive mutant of Drosophila abl
rescued pupal lethality and defects in eye development of flies lacking
abl function.40 Also in support of our findings,
P210BCR-ABL has been reported to localize to F-actin in a
noncatalytic manner.7,41 Furthermore, expression of a
kinase dead c-src mutant restored cell spreading of
src Two previous studies found an inverse relationship between P210BCR-ABL-induced transformation and adhesion. p3T3 fibroblasts, which become anchorage independent on transformation by P210BCR-ABL, return to the adherent layer of cells after treatment with STI571.43 Bhatia and colleagues found that treatment of CML bone marrow with tryphostin AG957, a tyrosine kinase inhibitor, enriched adherent cells with progenitors that populated colony-forming cell assays, suggesting that P210BCR-ABL decreased adhesion in a kinase-dependent manner.44 In contrast to these assays, our rotating disk system directly measures the magnitude of cell-fibronectin binding, which is independent of the transformation status of the cells and does not require a read-out step that may be sensitive to processes that are known to require tyrosine kinase activity such as proliferation or differentiation. One significant implication of our results is that altered binding to fibronectin is likely to be a direct consequence of P210BCR-ABL expression and not a side effect of cellular transformation because increased adhesion does not correlate with cell transformation in our system. Two functions of P210BCR-ABL, actin binding and activation of Ras signaling, are potential mechanisms by which P210BCR-ABL may modulate adhesion. P210BCR-ABL is found in the cytoplasm, colocalized with F-actin filaments,6 and cannot be removed from F-actin filaments by treatment with either 10 µM STI571 alone or by complete abrogation of kinase activity by the K1176R mutant. Using confocal microscopy we show that P210BCR-ABL is found along membrane ruffles, actin filaments, and close to the cellular membrane irrespective of its kinase activity or the method by which the tyrosine kinase activity was attenuated. This suggests that F-actin localization is kinase independent, and a recent report has shown that the Abl-related gene-1 (Arg-1) also binds F-actin even after deletion of its kinase domain.45 An intact actin cytoskeleton is required for cell adhesion in our system because treatment with cytochalasin D severely attenuates binding of 32D cells and other cells to fibronectin (data not shown).46 One possibility is that P210BCR-ABL may modify the actin cytoskeleton and influence binding. Alternatively, the role of P210BCR-ABL as a docking protein to recruit downstream molecules to actin, rather than its tyrosine kinase activity, may contribute to defective adhesion. Transgenic mice expressing P190BCR-ABL with a deletion of the C-terminal actin-binding domain survive up to 3 times longer and develop hematologic disease with lower frequency compared to wild-type P190BCR-ABL transgenic mice.47 As shown in our immunohistochemistry studies, the association of P210BCR-ABL with actin is independent of its kinase activity. Thus, cell adhesion and proper subcellular localization of P210BCR-ABL may be prerequisites for P210BCR-ABL tyrosine kinase to initiate CML. Our results are surprising in light of the wealth of evidence showing that the kinase activity of P210BCR-ABL is critical to disease initiation and in vitro cell transformation. Thus, it is unlikely that elevated adhesion due to P210BCR-ABL expression alone can initiate the cellular changes that lead to CML. The K1176R mutant retains the ability to modulate adhesion to fibronectin exhibited with wt P210BCR-ABL. However, mice receiving bone marrow transduced with the K1176R mutant fail to develop a CML-like disease under conditions that would normally support disease development with wild-type P210BCR-ABL.12 This raises the question of which functions of P210BCR-ABL are required for CML induction. To this end, murine retroviral transduction models of CML have been particularly insightful. Although tyrosine kinase activity is required, mutants that retain tyrosine kinase activity but lack either the oligomerization domain or grb2-binding site are markedly inhibited in their ability to induce CML in murine models.48-50 This suggests that the activated tyrosine kinase activity of P210BCR-ABL is insufficient for CML induction. Furthermore, we have found that even though the oligomerization domain of P210BCR-ABL is unnecessary for tyrosine kinase activity, it is required to maintain actin colocalization and to obtain wild-type levels of binding to fibronectin (J.A.W., manuscript in preparation). One possibility is that tyrosine kinase activity, signaling through grb2 binding, and tyrosine kinase-independent regulation of oligomerization effecting actin localization and cell adhesion, are all required for efficient CML induction in murine models. Utilization of the cell detachment device on additional P210BCR-ABL mutants and primary CML bone marrow should allow us to investigate this and other possibilities.
We thank Martin Carroll, Alan Gewirtz, Ruibao Ren, and members of the Pear Lab for critical reading of the manuscript and helpful suggestions. We are particularly grateful to Andrés García and Laura Lynch for their assistance with the spinning disk system and Ruibao Ren and Karen Ehrmann for providing useful reagents.
Submitted September 20, 2001; accepted January 30, 2002.
Supported by National Institutes of Health grants HL18208 (D.A.H.) and CA77570 and a Scholar Award from the Leukemia and Lymphoma Society (W.S.P.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Warren S. Pear, University of Pennsylvania, 611 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104-6160; e-mail: wpear{at}mail.med.upenn.edu.
1. Nowell PC, Hungerford DA. A minute chromosome in human chronic granulocytic leukemia. Science. 1960;132:1497-1499.
2.
Sawyers CL.
Chronic myeloid leukemia.
N Engl J Med.
1999;340:1330-1340 3. Verfaillie CM, McCarthy JB, McGlave PB. Mechanisms underlying abnormal trafficking of malignant progenitors in chronic myelogenous leukemia. Decreased adhesion to stroma and fibronectin but increased adhesion to the basement membrane components laminin and collagen type IV. J Clin Invest. 1992;90:1232-1241[Medline] [Order article via Infotrieve]. 4. Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF. Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia. Nature. 1987;328:342-344[CrossRef][Medline] [Order article via Infotrieve]. 5. Bazzoni G, Carlesso N, Griffin JD, Hemler ME. Bcr/Abl expression stimulates integrin function in hematopoietic cell lines. J Clin Invest. 1996;98:521-528[Medline] [Order article via Infotrieve].
6.
McWhirter JR, Wang JY.
Activation of tyrosinase kinase and microfilament-binding functions of c-abl by bcr sequences in bcr/abl fusion proteins.
Mol Cell Biol.
1991;11:1553-1565 7. McWhirter JR, Wang JY. An actin-binding function contributes to transformation by the Bcr-Abl oncoprotein of Philadelphia chromosome-positive human leukemias. EMBO J. 1993;12:1533-1546[Medline] [Order article via Infotrieve]. 8. Salgia R, Li JL, Ewaniuk DS, et al. BCR/ABL induces multiple abnormalities of cytoskeletal function. J Clin Invest. 1997;100:46-57[Medline] [Order article via Infotrieve]. 9. Salgia R, Brunkhorst B, Pisick E, et al. Increased tyrosine phosphorylation of focal adhesion proteins in myeloid cell lines expressing p210BCR/ABL. Oncogene. 1995;11:1149-1155[Medline] [Order article via Infotrieve].
10.
Schofield KP, Rushton G, Humphries MJ, Dexter TM, Gallagher JT.
Influence of interleukin-3 and other growth factors on alpha4beta1 integrin-mediated adhesion and migration of human hematopoietic progenitor cells.
Blood.
1997;90:1858-1866 11. Carlesso N, Griffin JD, Druker BJ. Use of a temperature-sensitive mutant to define the biological effects of the p210BCR-ABL tyrosine kinase on proliferation of a factor-dependent murine myeloid cell line. Oncogene. 1994;9:149-156[Medline] [Order articl |
Top
Abstract
Introduction
1 integrins in facilitating adhesion
between the K562 erythroleukemia cell line and fibronectin to show that
antibody-mediated integrin activation stimulates adhesion and increases
the force needed to detach adherent cells.
µ
0.80, with the exception of runs using
matrices consisting of BSA alone or when Meg-01 cells were treated with
the RGD peptide due to the few cells that remain attached in the
absence of fibronectin. The critical shear stress, 
.05. Data are presented as mean ± SD.
) or in the presence of
fibronectin
(
).
(-
-) represents a curve fitted to the experimental points. The
dashed line represents an adherent fraction of 0.5. The critical shear
stress (
). A higher proportion of P210BCR-ABL
cells remain attached at nearly every given shear stress compared to
pK1 cells suggesting that P210BCR-ABL leads to increased
binding to fibronectin. (-
)
expressing 32D cells is depicted along with pK1 control cells for
comparison (
,
) or free (
/