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Prepublished online as a Blood First Edition Paper on June 5, 2003; DOI 10.1182/blood-2003-01-0062.
Blood, 15 September 2003, Vol. 102, No. 6, pp. 2220-2228 Localization of BCR-ABL to F-actin regulates cell adhesion but does not attenuate CML developmentFrom the Department of Pathology and Laboratory Medicine, Institute for Medicine and Engineering, Abramson Family Cancer Research Institute, Department of Bioengineering, and Department of Microbiology, University of Pennsylvania, Philadelphia; and Rosenstiel Basic Medical Sciences Research Center, Department of Biology, Brandeis University, Waltham, MA.
We have previously found that P210BCR-ABL increases the adhesion of hematopoietic cell lines to fibronectin by a mechanism that is independent of tyrosine kinase activity. To investigate the pathway(s) by which P210BCR-ABL influences cell adhesion, we used a quantitative cell adhesion device that can discern small changes in cell adhesion to assay P210BCR-ABL with mutations in several critical domains. We expressed P210BCR-ABL mutants in 32D myeloblast cells and found that binding to fibronectin is mediated primarily by the  5 1 integrin. We performed a structure/function analysis to map domains important for cell adhesion. Increased adhesion was mediated by 3 domains: (1) the N-terminal coiled-coil domain that facilitates oligomerization and F-actin localization; (2) bcr sequences between aa 163 to 210; and (3) F-actin localization through the C-terminal actin-binding domain of c-abl. We compared our adhesion results with the ability of these mutants to cause a chronic myelogenous leukemia (CML)–like disease in a murine bone marrow transplantation assay and found that adhesion to fibronectin did not correlate with the ability of these mutants to cause CML. Together, our results suggest that F-actin localization may play a pivotal role in modulating adhesion but that it is dispensable for the development of CML.
P210BCR-ABL, the oncogene formed by a balanced rearrangement between the c-abl gene on chromosome 9 and the bcr gene on chromosome 22, leads to the formation of the Philadelphia (Ph) chromosome, a diagnostic marker of chronic myelogenous leukemia (CML).1,2 Expression of P210BCR-ABL in hematopoietic progenitor cells causes deregulated cell proliferation, decreased programmed cell death, and abnormal adhesion to bone marrow (BM) stroma (for a review, see Wertheim et al3). Together, these processes are thought to lead to the defects in cell number and maturation that characterize CML. Defective cellular adhesion may enhance cell cycling and egress of leukemia cells out of the BM into peripheral blood, allowing cells to home to extramedullary sites.4-6 P210BCR-ABL is found primarily in the cytoplasm, closely associated with F-actin through a C-terminal actin-binding domain.7,8 Additionally, P210BCR-ABL phosphorylates several proteins of the focal adhesion complex.9 Modification of linkages between cancerous cells and the extracellular matrix and interactions between P210BCR-ABL and the cell cytoskeleton may be important for its oncogenic functions. P210BCR-ABL likely plays a direct role in modifying cell adhesion; however, the specific functional properties of P210BCR-ABL that cause defective adhesion have not been identified. Phosphorylation of intracellular signaling cascades by P210BCR-ABL tyrosine kinase is critical for cell transformation and development of a CML-like disease in mice.10 Clinical trials with the specific c-abl tyrosine kinase inhibitor STI-571 (imatinib mesylate [Gleevec]) show that hematologic and cytologic remission can be achieved by attenuating the P210BCR-ABL tyrosine kinase activity indicating that the tyrosine kinase domain is essential for disease pathogenesis.11 P210BCR-ABL phosphorylates several proteins involved in cell adhesion—including paxillin, FAK, and CRKL— that are associated with focal adhesion contacts.9,12 However, neither the attenuation of tyrosine kinase activity by STI-571 nor a lysine-to-arginine point mutation at amino acid (aa) 1176 rendering the tyrosine kinase domain inactive can correct for the adhesive defect caused by P210BCR-ABL expression in transformed human and murine myeloid cell lines.13 The fusion of bcr to the N-terminal portion of c-abl leads to cell transformation by deregulating c-abl functions that are normally tightly controlled.7 Recent results suggest that the N-terminal coiled-coil (C-C) domain may not be required to activate the P210BCR-ABL tyrosine kinase because mutants lacking this domain exhibit elevated tyrosine kinase activity in cell lines.14,15 Nonetheless, the C-C region contributed by bcr leads to dimerization and tetramerization16,17 of P210BCR-ABL that may enhance the c-abl tryosine kinase or that may be required for actin binding.7 Although the c-abl actin-binding function is necessary for F-actin localization and transformation of Rat-1 fibroblasts,16 the contribution of F-actin localization to either P210BCR-ABL–induced adhesion defects or CML remains unclear. Failure to oligomerize P210BCR-ABL by deletion of the bcr C-C domain severely retards the development of myeloproliferative disease (MPD) in mice, most likely because of a decrease in tyrosine kinase activity.14,15 However, loss of F-actin binding is an alternative explanation because the effect of F-actin localization on P210BCR-ABL–induced disease has not been studied in a murine BM transplantation model of CML. We used a quantitative cell adhesion device that can discriminate between changes in cell adhesion by specifically measuring the strength of binding between a population of adherent cells and fibronectin, a major component of the BM extracellular matrix.18 Using this device, we have shown that expression of P210BCR-ABL in a myeloblast cell line, 32D, leads to increased cell adhesion in accordance with several published studies using qualitative plate and wash assays.4,13,19 We also found that expression of a mutant lacking tyrosine kinase activity failed to normalize cell adhesion and that treatment of Meg-01 cells, a P210BCR-ABL–expressing cell line from a CML patient in blast crisis, with STI-571 had no effect on binding to fibronectin, suggesting that P210BCR-ABL does not enhance adhesion through inside-out signaling originating at its tyrosine kinase.13 We constructed several mutants with domain deletions or point mutations in key regions in P210BCR-ABL and expressed these mutants in 32D cells to determine the region in P210BCR-ABL that is responsible for enhancing adhesion to fibronectin. We identified 3 regions that are necessary for P210BCR-ABL to cause elevated adhesion: the N-terminal C-C domain that facilitates oligomerization and F-actin localization, the bcr sequences between aa 163 to 210, and the C-terminal actin-binding domain of c-abl. We were particularly interested in the relationship between F-actin localization, elevated cell adhesion to fibronectin, and MPD development in our mouse model. Here we report that deletion of the C-terminal actin-binding domain had no effect on development of the MPD. Together, our findings indicate that localization of P210BCR-ABL to the actin cytoskeleton is responsible for abnormal adhesion to fibronectin; however, F-actin binding, and potentially deregulated cell adhesion to fibronectin, is not a primary determinant for murine CML development.
Plasmid construction and cell preparation
To create
The (1-162) BCR-ABL mutant was cloned through the release of full-length BCR-ABL from MigP210BCR-ABL with EcoRI. The c-abl portion was isolated by digestion with HincII and was cloned into the HpaI–EcoRI site of the pK1 vector. The BCR-ABL breakpoint at aa 162 was generated through sequential PCR amplification of a fragment containing the first 486 bp bcr and 3' overlapping abl sequences (fragment 1, 5' primer 7779114 TCA CTC CTT CTC TAG GC and 3' primer GCC GCT GAA GGG CTT TGC GGA TCC GCT CGA AG, abl sequences underlined) and a second fragment (fragment 2, 5' primer CTT CGA GCG GAT CCG CAA AGC CCT TCA GCG GC and 3' primer 7779314 AGC AGA TAC TCA GCG GC) containing 5' overlapping bcr sequences. Fragments 1 and 2 were used as templates to generate a fragment that contained the BCR-ABL breakpoint at aa 162 using primers 77791 and 77793. This fragment was digested with HincII and was ligated in the correct orientation into the HpaI site of pK1 already containing the c-abl fragment. The BCR-ABL cassettes in MigP210BCR-ABL, MigRI 1-210 BCR-ABL,14 MigRI (1-63) BCR-ABL,14 and MSCV 2.2 High-titer retroviral supernatants were generated by transfection into Bosc23 cells.20 Viral titers were normalized by GFP expression of transduced NIH 3T3 cells (MigRI constructs) or colony formation of puromycin-resistant NIH 3T3 cells (pK1 constructs).14 32D cells22 were maintained in 32D culture media13 and were transduced by spinoculation.23 At 48 hours after transduction, 32D cells transduced with vectors containing the puromycin resistance gene were selected with puromycin (1.5 µg/mL) for 4 days and maintained in 32D media containing 10% WEHI supernatant. 32D cells transduced with GFP as a surrogate marker for P210BCR-ABL expression were sorted for GFP expression (MigRI) or were selected for growth in the absence of interleukin-3 (IL-3) (MigP210BCR-ABL) for 3 to 4 days, after which WEHI was reintroduced into the culture medium to maintain consistent growth rates.
Before they were spun, all cells were washed and transferred to a Tris-based buffer as described.13 Cells were treated with control or adhesion blocking reagents for 10 minutes before incubation with fibronectin. Functional blocking antibody PS/2 (anti- Adhesion assays
Circular glass coverslips (Fisher Scientific, Hampton, NH) were adsorbed with 10 µg/mL fibronectin (Becton Dickinson, Franklin Lakes, NJ) and were blocked with 1% bovine serum albumin (BSA). The cell detachment device was operated as described.27 Adhesion experiments were carried out in the absence of growth factors. Coverslips were analyzed by recording the adherent fraction of cells at various radial distances from the center. The shear stress,
The critical shear stress, Bone marrow infection/transplantation Donor BM was harvested from femurs and tibias of C57BL/6 mice that had been treated with 1 mg/kg 5-flurouracil (5-FU) 4 days previously. BM was transduced with titer-matched retroviral supernatants, recipient mice were killed when they exhibited evidence of disease, and tissues were processed for analysis as described.14,20 Western blots and flow cytometry Western blots were performed on lysates from 32D cells or from primary mouse tissue. 32D cells were incubated overnight in 32D medium with 10% WEHI, and Western blots were prepared as described using the 8E9 antibody (PharMingen, San Diego, CA) to reveal BCR-ABL.13
For flow cytometry analysis, 32D cells transduced with the control vector MigRI or MigP210BCR-ABL were handled according to the same procedure used to prepare cells for adhesion experiments except that cells were not preselected for GFP or P210BCR-ABL expression. Integrin expression was assessed in triplicate for each population. Three populations of MigRI cells and 2 populations of MigP210BCR-ABL were transduced separately. Anti- Microscopy
NIH 3T3 cells were transduced with retroviral constructs encoding P210BCR-ABL,
P210BCR-ABL leads to increased adhesion in 32D cells through fibronectin-integrin bonds
The spinning disk cell detachment device uses fluid flow to detach cells bound to a monolayer of fibronectin. Detachment force can easily be determined by the properties of the buffer and distance of the cells from the center of the coverslip (see "Materials and methods"). The spinning disk cell detachment device measures discrete changes in cell adhesion and has been used to characterize individual activation states in
To determine which integrin receptors primarily mediate adhesion in P210BCR-ABL-expressing cells, we transduced 32D cells with a retrovirus coexpressing P210BCR-ABL and GFP (Mig P210BCR-ABL) or GFP alone (MigRI). Incubation of control MigRI cells on BSA coverslips without fibronectin results in few cells remaining bound after a spin and gives a
Binding of integrins to fibronectin requires intracellular energy stores and extracellular divalent cations as cofactors. To show that cells specifically bind fibronectin, control MigRI cells were preincubated with a soluble Arg-Gly-Asp (RGD)–containing peptide that occupies the fibronectin binding site on integrins that recognize the RGD sequence. Incubation of cells with the RGD-containing peptide led to a significant decrease (P = .001) in adhesion to fibronectin. Cells were also treated with 0.02% sodium azide (NaAZ) to reduce energy stores by blocking oxidative phosphorylation or 1 mM EDTA (ethylenediaminetetraacetic acid) to chelate free cations. Both treatments decreased binding (10% of control for NaAZ and 16% of control for EDTA) to levels achieved with BSA alone (13% of control), indicating that adhesion in our system requires energy and divalent cations, properties of all integrin-fibronectin linkages.
32D cells express
The
To determine the extent to which Neither the coiled-coil domain, Y177, nor the actin-binding domain of BCR-ABL is required for factor-independent growth
We compared P210BCR-ABL mutants that lack functional C-C, Y177, or actin-binding domains to test the contribution of these regions on adhesion to fibronectin (Figure 3A). Many of these mutants were tested in our murine CML model, and we found that the deletion of the C-C domain or the mutation of Y177 was sufficient to prevent induction of the wild-type MPD.14 Expression of P210BCR-ABL in 32D cells alleviates the requirement of IL-3 for survival.34 After selection in puromycin for 4 days, each cell population was assessed for the ability to grow in IL-3–free media (Figure 3B). All mutants survived without IL-3 supplementation; however, cells transduced with the control vector pK1 rapidly died within 48 hours, as assessed by trypan blue inclusion. Growth of 32D cells transduced by all mutants except
Both the actin-binding and the coiled-coil domains are required for F-actin localization and increased adhesion to fibronectin by P210BCR-ABL
Two independently transduced populations of each mutant were repeatedly spun, and the critical shear stress of each spin is presented for 1 of the 2 populations tested (Figure 4). Of the mutations known to affect F-actin binding, both
A region in bcr between aa 163 and aa 210 is also necessary to support P210BCR-ABL cell adhesion
The (1-63) mutant, in which the C-C domain was fused to c-abl, had decreased adhesion compared with wild-type P210BCR-ABL (P < .001 and P < .001; Figure 4), but the staining pattern indicated that it retains the ability to colocalize with F-actin (Figure 5). This suggests that the mutant leads to lower adhesion through a mechanism that is distinct from One possibility is that bcr sequences downstream of the C-C domain may recruit proteins to the cell cytoskeleton that are necessary for adhesion. To investigate this possibility, we attempted to map the minimal region in bcr between aa 64 and aa 927 that facilitates P210BCR-ABL cell adhesion. We found that a (1-210) BCR-ABL mutant conferred high binding, similar to wild-type P210BCR-ABL, compared with cells expressing the (1-63) mutant (Figure 4). This suggests that aa 64-210 is a critical region in bcr that leads to increased binding to fibronectin. To narrow the critical region within aa 64-210, we constructed a (1-162) mutant that fuses the first 162 aa of bcr onto c-abl. This mutant results in transformation of 32D cells and increased whole cell phosphotyrosine levels (Figure 3B-C). When assessed for its ability to bind fibronectin in our spinning disk adhesion device, 32D cells expressing this mutant showed decreased levels of adhesion that were in the same range as the (1-63) mutant, narrowing the critical region to between aa 163 and aa 210. Tyr177 is the only tyrosine that may bind SH2-containing proteins within this region. Mutation of tyrosine to phenylalanine (Y177F) abrogates binding to GRB214,35; however, similar to P210BCR-ABL, it caused increased binding to fibronectin (Figure 4). This suggests that the P210BCR-ABL–GRB2 interaction is not responsible for increased adhesion in P210BCR-ABL–expressing 32D cells. Together, our adhesion experiments identify 3 regions in P210BCR-ABL that contribute to the ability of wild-type P210BCR-ABL to increase the magnitude of binding between 32D cells and fibronectin. The regions are the N-terminal C-C domain that facilitates oligomerization and enhances F-actin localization, the bcr sequences spanning aa 163 to 210, and the C-terminal actin-binding domain mediating F-actin localization.
Although P210BCR-ABL colocalizes with F-actin, the contribution of F-actin localization to CML development has not been characterized in a BM transplantation model of CML. Evaluation of the ability of the
All mice given
Abnormal binding between hematopoietic progenitor cells and BM stroma has been proposed, in addition to loss of programmed cell death and enhanced cell proliferation, as an important contributor to the onset of leukemia.37 Several studies using BM from CML patients5,38 or hematopoietic cell lines4,19 expressing P210BCR-ABL have documented an adhesion defect between cells and fibronectin, a major extracellular molecule of the BM microenvironment.18 Contact between P210BCR-ABL–expressing cell lines and fibronectin promotes entry into the cell cycle, suggesting that enhanced binding may promote deregulated cell proliferation and tumor formation.4 Other studies indicate that P210BCR-ABL may assist in the extravasation of leukemia cells out of the BM and into the peripheral circulation, where abnormally high levels of WBCs are typically present in untreated CML patients.5,38 Fibroblasts expressing P210BCR-ABL support this theory by exhibiting increased motility, accumulation of intracellular F-actin, and formation of filopodia compared with parental cells cultured on fibronectin.39 In vitro studies such as these provide a context for understanding the relationship between P210BCR-ABL expression and cell adhesion and motility. What remains to be determined is the contribution of defective adhesion to development of the CML disease. To investigate this relationship, we sought to understand how the P210BCR-ABL protein regulates cell adhesion and then to determine whether this process is contributory to murine CML. We used 2 experimental techniques to link cell adhesion with disease development.
We identified 3 regions in wild-type P210BCR-ABL—the N-terminal C-C domain, aa 163-210, and the C-terminal actin-binding domain—that, when deleted, lead to reduced adhesion to fibronectin compared with wild-type P210BCR-ABL. The C-C domain enhances the c-abl tyrosine kinase and the F-actin–binding domain.7 It is unlikely, however, that the depreciation in tyrosine kinase activity in the The observation that interactions between P210BCR-ABL and the cell cytoskeleton are necessary for P210BCR-ABL–mediated increased adhesion is consistent with observations in fibroblasts that indicate that P210BCR-ABL expression leads to abnormal cytoskeletal architecture, altered F-actin formation, and increased filopodia formation.39 An intact actin cytoskeleton is necessary for binding fibronectin in our system because complete depolymerization of F-actin with cytochalasin D abrogates all binding to fibronectin (Figure 2C).
Evidence from the literature shows that P210BCR-ABL expression directly alters integrin-cytoskeletal linkages by modifying F-actin. Human megakaryoblast cells, Mo7e, expressing P210BCR-ABL or CD34+ cells from CML patients are inefficient in the formation of integrin caps, or clusters, containing Our results favor a mechanism in which P210BCR-ABL binds to F-actin filaments to directly modify F-actin or, more likely, to recruit effector molecules potentially through the aa 163-210 region of P210BCR-ABL to the cell cytoskeleton. The significance of the aa 163-210 region to cell adhesion, is unclear. The Y177 binding domain is located in this region and binds GRB2, which has been suggested to activate RAS through the guanine exchange factor SOS.41 Others and we have shown that wild-type P210BCR-ABL–GRB2 binding at this region is necessary for efficient CML development.14,15,42 However, prevention of GRB2 binding through a Y177F mutation has no effect on enhanced adhesion, suggesting that GRB2 binding to P210BCR-ABL is noncontributory to cell adhesion. Recently, the P210BCR-ABL/GRB2 interaction was shown to recruit the scaffolding adapter GAB2 to the SH3 domain of GRB2. This association enhances PI3K/AKT and RAS/ERK activation and is required for efficient myeloid transformation in murine BM cells.43 Interestingly, binding of GRB2 to P210BCR-ABL is required for fibroblast transformation44 but is generally not needed to transform hematopoietic cell lines. This is likely because of alternative pathways sufficient to activate RAS in the absence of GRB2 binding at Y177 in cell lines such as 32D cells.45 One possibility not excluded by our findings is that RAS may be activated in 32D cells expressing the Y177F mutant and may enhance adhesion through pathways that do not involve GRB2 binding at this site.
Mouse models of CML using either transgenic mice or mice that underwent BM transplantation are useful tools to evaluate how P210BCR-ABL leads to leukemia. Leukemia induced by the Our findings contrast those from mice transgenic for the expression of P190BCR-ABL lacking the C-terminal F-actin–binding domain. These mice were characterized by an increase in leukemia latency and a decrease in development of acute B-cell leukemia.48 The reason for these disparate findings is likely attributed to the intrinsic differences between the 2 models that produce different diseases (MPD in our model and acute lymphoid leukemia in the model of Heisterkamp et al48). Although unlikely, differences between actin-binding mutants used for each study, such as the size of the C-terminal deletion or the additional bcr regions present in P210BCR-ABL and absent from P190BCR-ABL, may affect disease onset.
In summary, we show that the localization of P210BCR-ABL to F-actin is not required for MPD induction but is needed for the elevated adhesion to fibronectin seen in 32D cells expressing wild-type P210BCR-ABL. We used 2 independent assays to assess adhesion and oncogenicity; however, we were unable to find a consistent correlation between normalization of adhesion to fibronectin and attenuation of MPD in our murine model. This suggests that abnormal cell-fibronectin binding is unlikely to be a major independent contributor to murine CML development. Alternatively, our results implicate the dysregulated signaling caused by the P210BCR-ABL tyrosine kinase and other motifs (eg, Y177) as critical determinants of CML induction. The relative significance of the fusion of bcr sequences upstream of c-abl is best observed with the
We thank Andrea Carpenter, Bill DeMuth, Kevin Forsythe, Gladys Grays-Board, Fred Karnell, Lara Lynch, Joey Plumb, and Lanwei Xu for their technical assistance. The flow cytometry studies were performed in the University of Pennsylvania Cancer Center Flow Cytometry and Cell Sorting Shared Resource (supported in part by the Lucille B. Markey Trust and the National Institutes of Health). The Gastroenterology Cell Morphology Core at the University of Pennsylvania processed tissue sections and was supported by National Institutes of Health Center grant P30-DK50306.
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Top
Abstract
Introduction
(1-63) BCR-ABL lacking the N-terminal C-C domain, a 1.4-kb HpaI/BamHI fragment was released from MigP210BCR-ABL.
, imparted on the cells was calculated by the equation 
µ
3)1/2, where 
0.70. This is an arbitrary value to ensure that the cell distribution data conform to the numeric model; however, increasing or decreasing this value does not significantly affect the measurement of cell adhesion (data not shown). This criterion was used for all conditions with the exception of runs using cells incubated with reagents that severely block cell adhesion or when cells were incubated on matrices of BSA alone because of the few cells that remained attached after a spin. 
.050. Data are presented as mean ± SD. 
) allowed a higher fraction of cells to remain attached compared to vector control MigRI cells (
), indicating that MigP210BCR-ABL–expressing cells bound more tightly to fibronectin. Coverslips coated with BSA alone did not support significant cell binding to either MigRI (
) or MigP210BCR-ABL (data not shown) cells. Binding of MigRI or MigP210BCR-ABL cells to BSA-coated surfaces was comparable, and at times so few cells remained attached after a spin that a detachment profile could not be generated. Curves fitted to the experimental points for MigRI-expressing cells on BSA-only–coated coverslips (
), MigRI cells on fibronectin-coated coverslips (
), and MigP210BCR-ABL on fibronectin-coated coverslips (
). The dashed line represents an adherent fraction of 0.5. The critical shear stress (
) bone marrow showed evidence of disease by 14 days after transplantation. The latency between wild-type P210BCR-ABL and