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[Cancer Research 64, 5322-5331, August 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Effect of Mutational Inactivation of Tyrosine Kinase Activity on BCR/ABL-Induced Abnormalities in Cell Growth and Adhesion in Human Hematopoietic Progenitors

Pandurangan Ramaraj1, Harjeet Singh1, Ning Niu1, Su Chu1, Melissa Holtz1, Jiing Kuan Yee2 and Ravi Bhatia1

Divisions of 1 Hematology and Bone Marrow Transplantation and 2 Virology Research, City of Hope National Medical Center, Duarte, California


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic myelogenous leukemia (CML) results from transformation of a primitive hematopoietic cell by the BCR/ABL gene. The specific BCR/ABL signaling mechanisms responsible for transformation of primitive human hematopoietic cells are not well defined. Previous studies have suggested that constitutively activated tyrosine kinase activity plays an important role for in abnormal proliferation of CML progenitors but has not clearly defined its role in abnormal adhesion and migration. We established a human progenitor model of CML by ectopic expression of BCR/ABL in normal CD34+ cells using retrovirus-mediated gene transfer. CD34+ cells expressing BCR/ABL demonstrated several features characteristic of primary CML progenitors including increased proliferation in committed and primitive progenitor culture, reduced adhesion to fibronectin, and reduced chemotaxis to stroma-derived factor-1{alpha}. We expressed a kinase-inactive BCR/ABL gene to directly investigate the role of kinase activity in abnormal progenitor function. Abnormalities in proliferation were completely reversed, whereas defects in adhesion and migration were significantly improved but not completely reversed in cells expressing a kinase-inactive BCR/ABL. Furthermore, the BCR/ABL kinase inhibitor imatinib mesylate markedly inhibited proliferation of BCR/ABL-expressing progenitors but did not fully correct the adhesion and migration defects. Expression of BCR/ABL genes with deletions of either the COOH-terminal actin binding or proline-rich domains resulted in enhanced adhesion and chemotaxis compared with wild-type BCR/ABL but did not affect progenitor proliferation. We conclude that abnormal kinase activity is essential for abnormal proliferation and survival of CML progenitors but that abnormal adhesion and migration result from both kinase-dependent and -independent mechanisms.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic myelogenous leukemia (CML) is a lethal hematological disorder resulting from transformation of a primitive hematopoietic cell. Malignant cells in CML are characterized by a balanced translocation between chromosomes 9 and 22, fusing a truncated BCR gene to sequences upstream of the second exon of ABL(1 , 2) . The resulting BCR/ABL fusion oncogene encodes a protein tyrosine kinase with elevated and dysregulated enzymatic activity (3) . The BCR/ABL gene has been shown to play a critical role in the pathogenesis of CML (4, 5, 6) . Clinically, CML presents with a myeloproliferative disorder characterized by a vast expansion of hematopoietic progenitor, precursor, and mature cells (7) . Other important features of CML are the presence of increased numbers of circulating progenitors and extramedullary hematopoiesis.

CML committed and primitive progenitors demonstrate increased sensitivity to growth factor (GF)-induced proliferation and maturation (8, 9, 10, 11) . Primary CML hematopoietic progenitors have also been reported to be GF independent for proliferation and survival, although this has not been observed consistently (12, 13, 14, 15) . In addition, CML progenitors are insensitive to inhibition by chemokines that inhibit normal progenitor proliferation (16 , 17) . CML progenitors demonstrate deficient integrin-mediated adhesion and impaired integrin-mediated inhibition of proliferation, although normal levels of {alpha}4, {alpha}5, and ß1-integrin receptors are expressed (18, 19, 20, 21, 22, 23) . CML progenitors also demonstrate increased spontaneous motility on fibronectin (23 , 24) . However, directed migration to a gradient of the chemokine stromal cell-derived factor 1 (SDF-1{alpha}) is reduced (23 , 25 , 26) . These abnormalities may explain in part some of the clinical features of CML, including abnormal myeloproliferation, peripheral circulation of hematopoietic progenitors, and extramedullary hematopoiesis.

Improved understanding of the molecular mechanisms that lead to human progenitor transformation is required for a better understanding of CML pathogenesis and to assist development of additional mechanism-based therapies. Studies performed in immortalized cell lines have identified several potentially important BCR/ABL signaling domains and downstream signaling pathways (27) . However, the contribution of different mechanisms to transformation can vary considerably from one cell type to the other, and the role of these mechanisms in transformation of primitive human hematopoietic cells in which CML arises is unclear. One approach has been to express the BCR/ABL gene in murine stem cells using retroviral transduction followed by transplantation to irradiated hosts, resulting in induction of a myeloproliferative disorder (5 , 6 , 28) . This murine CML model has been used to study of the role of different BCR/ABL signaling domains in hematopoietic cell transformation (29, 30, 31, 32) . However, murine disease differs from clinical CML in being highly aggressive and fulminant and in often progressing to acute T-cell leukemia. These differences may be related to species-specific differences between human and murine stem and progenitor cells. An approach that is highly relevant to clinical disease is to study primary progenitor cells from CML patients. However, studies of molecular mechanisms are limited by the low numbers of such cells that can be obtained, considerable intersample variability related to the acquisition of additional genetic abnormalities and prior therapeutic exposures, the presence of both Ph– and Ph+ cell populations, and difficulty in performing structure-function correlations. A human model of CML based on expression of the p210BCR/ABL transgene in primary human CD34+ cells could be potentially very useful to investigate specific mechanisms resulting in progenitor transformation relevant to clinical disease and for preclinical evaluation of new therapeutic interventions for CML.

Several laboratory and clinical observations suggest that abnormal kinase activity plays a critical role in BCR/ABL-induced transformation (3 , 33 , 34) . However, recent reports indicate that inactivation of kinase activity does not reverse abnormal adhesion of BCR/ABL-expressing cell lines and that this abnormality results from kinase-independent mechanisms. In contrast, in previous studies we found that treatment of primary CML progenitors with a pharmacological BCR/ABL kinase inhibitor led to significant reversal of the adhesion defect. However pharmacological inhibitors could potentially affect cellular adhesion through effects on targets other than the BCR/ABL kinase (33 , 35) . In the present study we established a human progenitor model of CML based on retroviral expression of the p210BCR/ABL transgene in primary human CD34+ cells and used this model to directly investigate the role of kinase activity as well as kinase-independent mechanisms in BCR/ABL-induced abnormalities in proliferation and adhesion of human hematopoietic progenitors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Human umbilical cord blood samples were obtained using protocols approved by the Institutional Review Board of the City of Hope National Medical Center. Mononuclear cells were isolated using Ficoll-Hypaque density gradient separation and CD34+ cell enriched populations selected using immunomagnetic column separation as described previously (11) .

Vectors
The MIGR1 and MIG-210 vectors were gifts from Dr. Warren Pear (University of Pennsylvania, Philadelphia, PA; Ref. 5 ). The control vector, MIGR1, has a 5' murine stem cell virus-based long terminal repeat, an internal ribosome entry site sequence downstream of a multiple cloning site, and the green fluorescent protein (GFP) gene downstream of the internal ribosome entry site. The MIG-210 vector has the p210 BCR-ABL oncogene (7.1 Kb) inserted into the EcoRI site in the multiple cloning site, upstream of the internal ribosome entry site. A vector containing a kinase inactive p210BCR-ABL gene (MIG-210KI) was generated by excision of the K1176R p210BCR-ABL mutant from a pGDP210KI vector (a kind gift from Dr. Brian Druker, Oregon Health Sciences University, Portland, OR; Ref. 36 ) and cloning into the MIG R1 vector. Vectors containing p210BCR/ABL genes with deletions of the COOH-terminal actin-binding domain (MIG-210dA) and proline-rich region were generated by excising the respective mutant genes from pBabe-BCR-ABL-AD (a kind gift from Dr. Ruibao Ren, Brandeis University, Waltham, MA; Ref. 37 ) and a pGDP210{Delta}P1P2 (a kind gift from Dr. Brian Druker; Ref. 38 ) vectors and ligation into the EcoRI site of the MIG R1 vector.

Infectious virus particles were produced by transient transfection of 293 cells with retroviral vector plasmid and pCL-ampho plasmid (a kind gift from Dr. Martin Haas, University of California San Diego, San Diego, CA; Refs. 39 , 40 ). Supernatants were collected 24–48 h after transfection and the number of infectious particles titrated by measuring efficiency of transduction of HT1080 cell lines.

Retroviral Transduction
CD34+ cells were cultured in serum-free medium (Stem Cell Technologies, Vancouver, British Columbia, Canada) supplemented with the following GFs: Flt-3 ligand (100 ng/ml), stem cell factor (50 ng/ml), thrombopoietin (100 ng/ml), interleukin-6 (10 ng/ml), and interleukin-3 (25 ng/ml) at 37°C with 5% CO2 for 48 h in plates that had been precoated with Fibronectin CH-296 (Retronectin; Pan Vera, Madison, WI). Subsequently, cells were resuspended in virus containing supernatants (multiplicity of infection = 10), in the presence of the same GFs, and plated on fibronectin-coated plates pre-exposed to viral supernatants for 30 min to allow binding of virus particles and thereby increase the cell exposure to virus. This was repeated after 24 h. Cells were harvested 48 h after the second virus exposure, washed, and labeled with anti-CD34-APC antibodies or isotype controls (Becton Dickinson, San Jose, CA), and CD34+GFP+ cells were collected using a MoFlo flow cytometer (Cytomation Inc., Fort Collins, CO).

Western Blotting
Western blotting was performed to detect BCR/ABL expression and protein tyrosine phosphorylation. CD34+GFP+ cells were cultured in medium supplemented with low concentrations of GF similar to those present in stroma-conditioned medium [granulocyte-macrophage colony stimulating factor (200 pg/ml), granulocyte colony stimulating factor (1 ng/ml), stem cell factor (200 pg/ml), leukemia inhibitory factor (50 pg/ml), macrophage inhibitory protein 1{alpha} (200 pg/ml), and interleukin-6 (1 ng/ml; Ref. 41 )] for 16 h. For some studies cell numbers were expanded by culture in GF containing medium. Cells were lysed in buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% NP40, and 0.5% sodium deoxycholate, supplemented with protease and phosphatase inhibitors. Primary CML CD34+ cells were also analyzed for comparison. Protein extracts were resolved on 5% SDS-PAGE gels, transferred to nitrocellulose membranes, blocked in 10% nonfat milk in PBS with 0.1% Tween, and labeled with an appropriate dilution of primary antibody [anti-Abl (Ab-3), anti-BCR (Ab-2; both from Oncogene Science, Cambridge, MA), antiactin (AC-15; Sigma), and antiphosphotyrosine (4G10; a kind gift from Dr. Brian Druker)], followed by horseradish peroxidase-conjugated secondary antibody (1:8000; Jackson). Antibody detection was performed using the Superfemto kit (Pierce Biotechnology, Rockford, IL).

Evaluation of Progenitor Growth
Colony Forming Cell (CFC) Assays.
CD34+GFP+ cells were plated in methylcellulose progenitor culture and assessed for the presence of CFU-Mix, CFU-GM, and BFU-E colonies as described previously (11) . Where indicated, increasing concentrations of imatinib mesylate were added to the culture medium.

Liquid Culture with GFs.
CD34+GFP+ cells were cultured in IMDM with 30% FCS, and identical GF conditions to those used in CFC assays and the number of cells generated were counted after 14 days. Where indicated, increasing concentrations of imatinib mesylate were added to the culture medium.

Single Cell Analysis.
Single CD34+GFP+ cells were sorted into individual wells of a 96-well plate and cultured as described above. After 14 days, plates were scanned to identify wells containing cells, and the number of cells was counted in individual wells.

Long-Term Bone Marrow Culture.
CD34+GFP+ cells were plated in triplicate in long-term bone marrow culture medium on M2-10B4 murine fibroblast feeders subcultured in 24-well plates as described previously (11) . After culture for 2, 4, and 6 weeks, cells were harvested, pooled, and plated in CFC culture and the number of colonies evaluated.

Adhesion Assays
Plates (96-well) were adsorbed with Fibronectin CH-296 at a concentration of 8 µg/cm2. Control wells were adsorbed with BSA (Sigma). CD34+GFP+ cells were incubated in serum-free medium with low concentrations of GF as described above for 24 h. Where indicated, increasing concentrations of imatinib mesylate were added to the culture medium. Cells were then washed and resuspended in IMDM+BSA and plated in fibronectin-coated wells for 2 h. Subsequently, nonadherent and adherent fractions were separated as described previously (42) . Both fractions were plated in methylcellulose progenitor culture, and the percentage of adherent CFC was calculated.

Migration Assays
Progenitor migration was assayed by evaluating the movement of progenitors from the upper to the lower chamber of 6.5-mm Transwells with filters of 5-µm diameter pore size, as described previously (43) . The membranes were coated on both sides using a Fibronectin CH-296 solution (8 µg/cm2). CD34+GFP+ cells were incubated in serum-free medium with low concentrations of GF as described above for 24 h. Where indicated, increasing concentrations of imatinib mesylate were added to the culture medium. Cells were then washed and placed in the upper chamber of the transwell. Assays were performed with or without the addition of SDF-1{alpha} (100 ng/ml) to the lower chamber. Cells that migrated to the lower chamber of the transwell after incubation at 37°C for 6 h were assayed for CFC in methylcellulose progenitor culture. The percentage of migrating CFC was calculated as: (migrating CFC/total CFC) x100%.

Adhesion-Mediated Signaling
CFC proliferation was evaluated using thymidine suicide assays. CD34+GFP+ cells were cultured in serum-free medium with low concentrations of GF for 48 h. Subsequently, cells were washed and resuspended in IMDM with 0.3% BSA and incubated for 4 h in wells coated with fibronectin CH-296 or control wells coated with BSA. The proliferation of CFC after culture with or without fibronectin was evaluated in thymidine suicide assays as described previously (20) .

Statistics
Results of data obtained from multiple experiments were reported as the mean ± 1 SE. Significance levels were determined by Student’s t test analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CD34+ Cell Transduction with BCR/ABL and Kinase-Inactive BCR/ABL Expressing Murine Stem Cell Virus Vectors.
Supernatants containing infectious murine stem cell virus retrovirus particles carrying the p210BCR/ABL gene (MIG210), a kinase-inactive p210BCR/ABL (MIG210KI), and controls containing GFP alone (MIG) were generated by transient transfection of 293 cells. The kinase-inactive BCR/ABL mutant has a point mutation changing the lysine residue at position 1176 to arginine, referred to as K1176R, that inactivates the tyrosine kinase activity of BCR/ABL This mutant has been well characterized in previous in vitro studies and in the murine CML model (28 , 36 , 44) . Human CD34+ cells were transduced by culture with virus-containing supernatants using an optimized protocol (Fig. 1A)Citation . The transduction results are summarized in Table 1Citation and a representative experiment shown in Fig. 1BCitation . Transduction efficiency was lower for MIG210 and MIG210KI vectors, possibly related to the presence of the large BCR/ABL insert upstream of the internal ribosome entry site and GFP. The efficiency of CFC transduction was also significantly lower for MIG210- and MIG210KI-exposed cells compared with MIG (P < 0.05; n = 5). Significantly greater cell expansion was seen during transduction culture with MIG210 compared with MIG vectors (P < 0.02; n = 10). Expansion of MIG210KI vector-exposed cells was less than that for MIG210-exposed cells (P = 0.06) and did not differ significantly from cells exposed to MIG vectors. The yield of CD34+GFP+ cells collected after flow cytometric sorting was lower for MIG210- and MIG210KI-exposed cells compared with MIG-exposed cells (P < 0.05; n = 10). This notwithstanding, this procedure resulted in consistent and reliable collection of sufficient numbers of transduced cells for subsequent analyses.



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  		Fig. 1. Retroviral transduction of CD34+ cells with BCR/ABL genes. A, human CD34+ cells were transduced by culture in the presence of virus-containing supernatants as shown and described in "Materials and Methods." B, the results of a representative experiment are shown. C, cell lysates were made from transduced CD34+green fluorescent protein (GFP)+ cells and primary chronic myelogenous leukemia (CML) CD34+ cells, and BCR/ABL expression was assessed by Western blotting (WB). Blots were reprobed with an antiactin antibody to confirm equal sample loading. D, assessment of protein tyrosine phosphorylation was performed by probing blots with antiphosphotyrosine (anti-PY) antibodies. FACS, fluorescence-activated cell sorter; IL, interleukin; SCF, stem cell factor; TPO, thrombopoietin; FL, Flt-3 ligand.

 

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  		Table 1 Results of transduction of human CD34+ cells with MIG, MIG210, and MIG210KI vectors

The percentage of transduction of total cells and CD34+ cells was determined by flow cytometry analysis. The percentage of transduction of CFCa was determined by comparing the number of GFP+ colonies detected by immunofluorescence microscopy with total number of colonies detected by light microscopy. The fold cell expansion was determined by comparing the total number of cells present after transduction culture with the number of cells initially placed in culture. The total number of CD34+GFP+ cells obtained after flow cytometry sorting is also shown. Results represent mean ± SE of multiple experiments.

 
BCR/ABL expression in transduced CD34+GFP+ cells was assessed by Western blotting. The p210BCR/ABL protein was detected in MIG210- and MIG210KI-transduced CD34+ cells at similar levels of expression and, as expected, was not seen in control MIG-transduced cells (Fig. 1C)Citation . BCR/ABL expression levels in transduced CD34+ cells appeared to be increased compared with CD34+ cells obtained from CML patients. Phosphotyrosine blotting confirmed increased levels of protein tyrosine phosphorylation in MIG210-transduced CD34+ cells but not in MIG210KI-transduced cells, confirming kinase inactivation in the mutant (Fig. 1D)Citation .

Proliferation and Survival of BCR/ABL and Kinase-Inactive BCR/ABL-Expressing CD34+ Cells.
Selected CD34+GFP+ cells were plated in methylcellulose progenitor assays to evaluate committed progenitor growth. Cultures initiated with MIG210-transduced cells were striking for the presence of very large colonies, which included CFU-GM, BFU-E, and CFU-Mix colonies (Fig. 2, A and B)Citation . These large colonies were not observed in cultures initiated with MIG210KI-transduced CD34+ cells. There was no significant difference in total CFC cloning efficiency of CD34+ cells transduced with the different vectors (Fig. 2C)Citation . However, the numbers of CFU-Mix were significantly increased in cells transduced with MIG210 vectors [CFU-Mix per 1000 cells (mean ± SE) of 2.8 ± 1.1 for MIG, 14.5 ± 5.1% for MIG210, and 2.2 ± 1.1 for MIG210KI-transduced CD34+ cells, respectively; P < 0.05 comparing MIG210 with MIG or MIG210KI; n = 8]. The total number of cells generated during committed progenitor culture was assessed by growing CD34+ cells in similar serum and GF culture conditions as for methylcellulose progenitor culture, but substituting IMDM for methylcellulose. CD34+ cells transduced with MIG210 vectors generated significantly greater numbers of cells than MIG-transduced cells, and this abnormality was reversed in cells expressing kinase-inactive BCR/ABL (Fig. 2D)Citation . These results confirm the observation of increased colony size (Fig. 2E)Citation and indicate that BCR/ABL-induced increased clonal proliferation of individual CD34+ cells was reversed with kinase inactivation. This was confirmed by analysis of single CD34+GFP+ cells sorted into individual wells of a 96-well plate and cultured as described for bulk cultures. The number of cells/clone (mean ± SE) after 14 days of culture was 925 ± 381 for controls, 18,300 ± 5,134 for wild-type BCR/ABL-expressing cells, and 1,757 ± 505 for kinase-inactive BCR/ABL-expressing cells. The percentage of clones that generated >10,000 cells was 2%, 16%, and 3.5% for control, BCR/ABL-expressing, and kinase-inactive BCR/ABL-expressing cells, respectively.



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  		Fig. 2. Growth of BCR/ABL-expressing CD34+ cells in progenitor culture. CD34+green fluorescent protein (GFP)+ cells selected by flow cytometry were plated in methylcellulose progenitor culture for 14 days at 37°C and 5% CO2 and assessed for the presence of colony forming cell (CFC) as described in "Materials and Methods." A, cultures initiated with MIG210-transduced cells were striking for the presence of very large colonies. B, fluorescent microscopy confirmed GFP expression in cells composed of the colony. C, the total CFC frequency per 1000 CD34+ cells is shown (n = 8). D, the total number of cells generated during committed progenitor culture was assessed by plating CD34+GFP+ cells in liquid culture using identical serum and growth factor conditions as used in CFC assays. The number of cells generated per 1000 CD34+ cells is shown (n = 5). E, the ratio of cells generated in liquid culture to colonies generated in semisolid methylcellulose culture from 1000 CD34+ cells is shown (n = 5). Results of data obtained from multiple experiments are reported as the mean; bars, ±SE. Significance levels determined by Student’s t test analysis are shown: *, P < 0.05; **, P < 0.01, MIG versus MIG210 vector-transduced cells; {dagger}, P < 0.05; {dagger}{dagger}, P < 0.01, MIG210 versus MIG210KI vector-transduced cells.

 
The ability of committed progenitors cells to withstand GF deprivation was assessed by culture of CD34+GFP+ cells in serum-free medium in the absence of GF for 96 h followed by assessment of CFC capacity. Increased survival of CFC was seen with p210BCR/ABL-transduced cells, compared with controls (55.3 ± 27.0% of input number of CFC for BCR/ABL-expressing cells, compared with 13.6 ± 7.4% for controls; P = 0.05; n = 8). However, survival of CFC-expressing kinase-inactive p210BCR/ABL was significantly lower than BCR/ABL-expressing CFC and similar to those of control CFC (15.1 ± 4.0% of input number of CFC).

The primitive progenitor potential of transduced CD34+ cells was assessed by evaluating their ability to generate CFC after culture on stromal adherent layers for 2–6 weeks. p210BCR/ABL-transduced cells generated significantly increased numbers of CFC after 2, 4, and 6 weeks of long-term bone marrow culture compared with controls (Fig. 3)Citation . Enhanced growth in long-term bone marrow culture was reversed in cells transduced with kinase-inactive p210BCR/ABL.



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  		Fig. 3. Growth of BCR/ABL-expressing CD34+ cells in long-term bone marrow culture. CD34+green fluorescent protein+ cells were plated in triplicate in long-term bone marrow culture medium on M2-10B4 murine fibroblast feeders subcultured in 24-well plates. Cultures were maintained at 37°C in a humidified atmosphere with 5% CO2 and fed at weekly intervals by removal of half the medium from the wells and replacement with fresh medium. After culture for 2, 4, and 6 weeks, all of the nonadherent and adherent cells were harvested, pooled, and plated in colony forming cell (CFC) culture and the number of colonies evaluated. Results of data obtained from six separate experiments are reported as the mean; bars, ±SE. Significance levels determined by Student’s t test analysis are shown: *, P < 0.05; **, P = 0.01, MIG versus MIG210 vector-transduced cells; {dagger}, P < 0.05, MIG210 versus MIG210KI vector-transduced cells; LTC, long-term culture.

 
The above studies indicate that increased proliferation and survival of BCR/ABL-transformed committed and primitive progenitors are completely reversed by inactivation of the BCR/ABL tyrosine kinase.

Adhesion and Migration of BCR/ABL and Kinase-Inactive BCR/ABL-Expressing CD34+ Cells.
Adhesion assays were performed to evaluate the effect of BCR/ABL expression on CD34+ progenitor adhesion to the extracellular matrix protein fibronectin. MIG210-transduced cells demonstrated significantly reduced adhesion to fibronectin compared with MIG-transduced controls (Fig. 4A)Citation . In contrast, adhesion to fibronectin was significantly greater for MIG210KI-transduced cells compared with MIG210-transduced cells. Adhesion of MIG210KI-transduced cells was also less than for control MIG-transduced cells, although the difference did not reach statistical significance (P = 0.07).



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  		Fig. 4. Adhesion and migration of BCR/ABL-expressing progenitors. A, progenitor adhesion was measured by incubating CD34+green fluorescent protein+ cells on fibronectin CH-296 (8 µg/cm2) or control BSA-adsorbed plates for 2 h. Nonadherent and adherent fractions were collected, and the percentage of adherent colony forming cell (CFC) calculated {% adherent CFC = [adherent CFC ÷ (adherent CFC + nonadherent CFC)] x100%}. B, progenitor migration was measured in a transwell assay by evaluating the movement of CD34+green fluorescent protein+ progenitors from the upper to the lower chamber of transwells with filters of 5-µm diameter pore size. Transwell membranes were adsorbed with fibronectin CH-296 (8 µg/cm2). Stromal cell-derived factor 1 (SDF-1{alpha}; 100 ng/ml) was added to the lower chamber. The percentage of CFC that migrated to the lower chamber over 6 h is shown. Results represent data obtained from five separate experiments and are reported as the mean; bars, ±1 SE. Significance levels determined by Student’s t test analysis are shown: *, P < 0.05; **, P < 0.005, versus MIG-transduced cells; {dagger} P < 0.05, MIG210 versus MIG210KI vector-transduced cells.

 
Progenitor migration was measured in a transwell assay. Spontaneous migration through fibronectin-coated transwells appeared to be higher for MIG210-transduced CD34+ cells compared with MIG-transduced cells, but the difference did not reach statistical significance (3.6 ± 0.8% for MIG versus 6.6 ± 2.0% for MIG210-transduced cells; P = 0.08). Spontaneous migration was also not significantly different between wild-type and kinase-inactive BCR/ABL-expressing cells (6.6 ± 2.0% for MIG210 versus 8.0 ± 2.7% for MIG210KI-transduced cells). In contrast, directed migration to a gradient of the chemokine SDF-1{alpha} was significantly reduced in MIG210-transduced cells compared with controls (Fig. 4B)Citation . Chemotaxis to SDF-1{alpha} was increased significantly in cells expressing kinase-inactive BCR/ABL compared with MIG210-transduced cells. However, migration of kinase-inactive BCR/ABL-expressing cells remained significantly less than for control cells.

Contact with fibronectin results in integrin-dependent inhibition of proliferation of normal progenitors when studied in low, physiological concentrations of GFs. This mechanism has been shown to be perturbed in CML progenitors. Using a thymidine suicide assay to measure the percentage of proliferating CFC, we observed that contact with fibronectin resulted in significant inhibition of proliferation of MIG-transduced cells (26.2 ± 3.3% reduction in S-phase cells on fibronectin compared with BSA controls; P < 0.001 for fibronectin compared with BSA controls; n = 5) but did not inhibit proliferation of MIG210-transduced cells (3.2 ± 1.3% reduction in S-phase cells on fibronectin compared with BSA controls; P < 0.005 compared with MIG-transduced cells). In contrast, proliferation of MIG210KI-transduced cells was inhibited by fibronectin (19.3 ± 3.6% reduction in S-phase cells on fibronectin compared with BSA controls; P < 0.01 compared with MIG210-transduced cells; P = 0.08 compared with MIG-transduced cells).

The above results indicate that abnormalities in adhesion and migration in p210BCR/ABL-transduced CD34+ cells are significantly reduced but are not completely reversed after kinase inactivation.

Effect of Imatinib Mesylate on BCR/ABL-Expressing Progenitors.
We investigated the effect of the BCR/ABL kinase inhibitor imatinib mesylate on BCR/ABL-induced proliferation, adhesion, and migration abnormalities in MIG210-transduced CD34+ cells. The concentrations of imatinib used correspond to levels achieved in patients being treated with imatinib (34) . Exposure to imatinib mesylate markedly reduced the number of CFC generated from BCR/ABL-expressing CD34+ cells and the numbers of cells generated after GF culture (Fig. 5, A and B)Citation . However, imatinib also significantly inhibited the growth of cells transduced with MIG vectors. Inhibition of MIG-transduced cells was significantly less than for MIG210-transduced cells (P < 0.05 for MIG210 compared with MIG-transduced cells treated with 0.2 and 1 µM imatinib). In contrast, imatinib exposure did not alter adhesion of MIG210-transduced progenitors to fibronectin (Fig. 5C)Citation . However, imatinib significantly reduced adhesion of MIG-transduced progenitors to fibronectin. Imatinib exposure resulted in significantly increased chemotaxis of MIG210-transduced progenitors to an SDF-1{alpha} gradient (Fig. 5D)Citation , but migration remained significantly less than that of MIG-transduced cells (P < 0.05 for MIG210-transduced cells treated with 0.2–5 µM imatinib compared with MIG-transduced cells). Imatinib did not affect chemotaxis of MIG-transduced cells. These results indicate that imatinib treatment, although profoundly reducing abnormally increased proliferation of BCR/ABL-expressing progenitors, does not completely correct the defect in migration and does not alter progenitor adhesion. However, imatinib also significantly reduces proliferation and adhesion of control progenitors expressing GFP alone, which must be considered while interpreting these results.



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  		Fig. 5. Effect of imatinib mesylate on BCR/ABL-expressing progenitors. The effect of increasing concentrations of imatinib on MIG210 and control MIG vector-transduced CD34+ cells was evaluated. A, CD34+green fluorescent protein+ cells were plated in methylcellulose progenitor culture with or without addition of imatinib to the culture medium for 14 days and assessed for colony forming cell (CFC) growth. B, CD34 green fluorescent protein+ cells were cultured in growth factor-containing medium as described for Fig. 2DCitation , with or without addition of imatinib, and assessed for the number of cells generated after 14 days. C, adhesion of progenitors to fibronectin CH-296 (8 µg/cm2) was evaluated after prior exposure of cells to imatinib for 24 h in serum-free medium containing low concentrations of growth factors. The total number of CFC obtained from 1000 CD34+ cells after 24-h exposure to imatinib for MIG210-transduced cells was 157 ± 16 without imatinib, 107 ± 16 with 0.2 µM, 94 ± 14 with 1 µM, and 80 ± 11 with 5 µM imatinib; and for MIG transduced cells was 127 ± 23 without imatinib, 131 ± 21 with 0.2 µM, 132 ± 24 with 1 µM, and 135 ± 24 with 5 µM imatinib. D, migration of progenitors toward an stromal cell-derived factor 1 gradient (100 ng/ml) through fibronectin CH-296-coated transwells was evaluated after prior exposure of cells to imatinib for 24 h in serum-free medium containing low concentrations of growth factors. Results represent mean of four separate experiments; bars, ±1 SE. Significance levels were determined by Student’s t test analysis: *, P < 0.05; **, P < 0.005; ***, P < 0.0005, versus cells not exposed to imatinib.

 
Proliferation, Adhesion, and Migration of Progenitors Expressing Actin-Binding Domain and Proline-Rich Domain-Deleted BCR/ABL Gene Mutants.
To additionally investigate potential mechanisms contributing to abnormal adhesion and migration of BCR/ABL-expressing progenitors, vectors containing BCR/ABL genes with deletion of the COOH-terminal actin-binding domain (MIG210dA) or the proline-rich domain (MIG210dP1P2) were generated and used to transduce CD34+ cells. These mutants have been well characterized in cell lines, but their role in transformation of primary human progenitors has not been studied. We confirmed that these mutants were expressed at similar levels as wild-type BCR/ABL-transduced cells by Western blotting using an anti-BCR antibody (Fig. 6A)Citation . Phosphotyrosine blotting indicated that the deletion mutants retained tyrosine kinase activity (Fig. 6B)Citation . CD34+ cells expressing either of the mutant genes proliferated in CFC (data not shown) and liquid GF culture (Fig. 6C)Citation to a similar extent as cells expressing wild-type BCR/ABL. In contrast, expression of these mutants resulted in enhanced progenitor adhesion to fibronectin compared with wild-type BCR/ABL (Fig. 6D)Citation . In addition, progenitors expressing either mutant demonstrated enhanced migration toward an SDF-1{alpha} gradient compared with cells expressing wild-type BCR/ABL (Fig. 6E)Citation . These mutants did not affect spontaneous migration on fibronectin (data not shown). These results suggest that the COOH-terminal actin binding and proline-rich domains of BCR/ABL contribute to abnormal adhesion and migration of BCR/ABL expressing human progenitors but do not contribute significantly to abnormal proliferation.



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  		Fig. 6. Proliferation, adhesion, and migration of progenitors expressing ABD and proline-rich domain-deleted BCR/ABL genes. CD34+ cells were transduced with vectors expressing BCR/ABL genes with deletion of the actin binding (MIG210dA) or proline-rich (MIG210dP1P2) domains, as well as wild-type BCR/ABL (MIG210) and control vectors expressing the green fluorescent protein (GFP) alone (MIG), and CD34+GFP+ cells were selected by flow cytometry. A, lysates were made from CD34+GFP+ cells expanded in liquid growth factor culture, and BCR/ABL expression was assessed by Western blotting (WB) using an anti-BCR antibody. To confirm equal sample loading blots were reprobed with an antiactin antibody. B, protein tyrosine phosphorylation was assessed by probing blots with antiphosphotyrosine (anti-PY) antibodies. C, CD34+GFP+ cells were cultured in growth factor containing medium as described for Fig. 2DCitation , with or without addition of imatinib. The number of cells generated per 1000 CD34+ cells is shown (n = 7). D, adhesion of progenitors to fibronectin CH-296 (8 µg/cm2) -coated plates was evaluated (n = 6–7). E, migration of progenitors toward an stromal cell-derived factor 1 (SDF-1{alpha}) gradient (100 ng/ml) through fibronectin CH-296-coated transwells was evaluated (n = 6). Significance levels were determined by Student’s t test analysis: *, P < 0.05; **, P < 0.005 versus MIG-transduced cells, {dagger}, P < 0.05 versus MIG210-transduced cells.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have established a human model of CML based on retrovirus-mediated transfer of the BCR/ABL gene into CD34+ progenitor cells and have used this model to evaluate the role of specific BCR/ABL signaling mechanisms in abnormal cellular regulation in CML progenitors.

Ectopic expression of p210BCR/ABL in human hematopoietic progenitors resulted in significantly increased progenitor proliferation. Although CFC frequency was unchanged, very large colonies with significantly increased numbers of cells per colony were seen indicating increased clonal proliferation of individual progenitor cells. Increased GF-independent survival of BCR/ABL-transduced progenitors was also observed. While these studies were in progress, two additional studies of human progenitor transduction with BCR/ABL have been reported (45 , 46) . Our results are consistent with those of Zhao et al. (45) , who also observed increased proliferation and survival of BCR/ABL-transduced committed progenitors. Here we additionally demonstrate that abnormal proliferation extends to more primitive progenitors capable of generating hematopoiesis in long-term bone marrow culture. In addition, BCR/ABL expressing human CD34+ cells had significantly reduced adhesion to fibronectin and significantly impaired inhibition of proliferation after coculture with fibronectin. Zhao et al. (45) also observed reduced adhesion to fibronectin in BCR/ABL-transduced human progenitors. We also demonstrate that migration in response to the chemokine SDF-1{alpha} was impaired significantly in BCR/ABL-expressing progenitors compared with controls. In contrast to our results and those of Zhao et al., Chalandon et al. (46) reported that BCR/ABL expression in human CD34+ cells was associated with increased erythroid differentiation response, including granulocytic lineage programmed cells. Increased erythroid differentiation response is not a common feature of CML, and the observed abnormalities in erythroid differentiation may be explained by much higher levels of BCR/ABL expression in transduced CD34+ cells in the study by Chalandon et al. (46) .

The abnormalities resulting from ectopic expression of BCR/ABL in CD34+ cells are similar to those observed in committed and primitive progenitors from CML patients. CML CD34+ progenitors also demonstrate increased proliferation in response to GF stimulation and resistance to apoptosis after GF withdrawal (8, 9, 10, 11, 12) , reduced ß1 integrin-mediated adhesion and signaling but increased mobility on fibronectin (19 , 20 , 22) , and impaired migration in response to SDF-1{alpha} (23, 24, 25, 26 , 43) . CD34+ progenitors ectopically expressing BCR/ABL appeared to be more proliferative in progenitor culture and more resistant to apoptosis on GF withdrawal than we have observed previously for primary CML progenitors. The use of the murine stem cell virus promoter results in enhanced BCR/ABL expression in transduced CD34+ cells that is not subject to normal regulatory influences, which may result in enhanced cellular proliferation during in vitro culture (47) . It is also possible that cord blood CD34+ progenitors, used for their ease of transduction, may be more proliferative than their adult counterparts in their response to BCR/ABL expression. Despite these limitations, the model described here closely recapitulates abnormalities of cell growth and adhesion characteristic of CML progenitors and has a major advantage of allowing investigation of molecular mechanisms underlying BCR/ABL transformation of primary human progenitors using a mutational approach not possible with patient-derived cells.

Experimental and clinical evidence support a critical role for BCR/ABL kinase activity in cellular transformation by BCR/ABL (34 , 48) . Abnormal kinase activity results in activation of downstream signaling pathways implicated in mitogenic signaling and survival as well as regulation of adhesion and migration (7 , 27) . Although previous studies have shown that constitutively activated tyrosine kinase activity plays an important role for in abnormal proliferation and antiapoptotic signaling, these studies have not clearly defined its role in abnormal adhesion and migration of BCR/ABL-expressing cells. We have shown that the kinase inhibitor tyrphostin AG957 resulted in significant improvement in abnormal adhesion and adhesion-mediated signaling in CML progenitors (49) . In contrast, Wertheim et al. (44) found that abnormalities in adhesion of BCR/ABL-transformed 32D cells were not affected by expression of a kinase-inactive BCR/ABL mutant or by exposure to imatinib. It is possible that variances between these studies are related to differences in effects of BCR/ABL expression and kinase inactivation in cell lines compared with primary progenitor cells. Unlike in primary progenitors, BCR/ABL transformation resulted in increased adhesion in the 32D cell line. Differences in cell culture and assay conditions could also play a role, because GF stimulation up-regulates integrin-mediated adhesion in normal but not BCR/ABL-transformed cells. BCR/ABL-expressing cells tested in the absence of GFs may demonstrate relatively increased adhesion (43 , 50) . The present study demonstrates that BCR/ABL-induced enhancement of proliferation and survival was completely reversed in cells expressing a kinase-inactive BCR/ABL gene and confirms that abnormal proliferation of CML progenitors and abnormal survival in GF-deprived conditions is absolutely kinase dependent. Our observations that BCR/ABL-induced defects in progenitor adhesion to fibronectin and chemotaxis to SDF-1{alpha} were significantly corrected in cells after kinase inactivation indicate an important role for kinase-dependent signaling mechanisms in adhesion and migration defects. However, lack of complete correction of adhesion and chemotactic defects in cells expressing the kinase-inactive BCR/ABL gene suggests that kinase-independent mechanisms may also contribute to these abnormalities.

The role of tyrosine kinase activity in functional abnormalities in BCR/ABL-expressing cells was additionally evaluated by exposing cells to the kinase inhibitor imatinib mesylate. Imatinib resulted in a profound reduction in abnormally increased proliferation of BCR/ABL-expressing progenitors. However, proliferation of control GFP-expressing cells was also inhibited, indicating that mechanisms other than BCR/ABL kinase inhibition can contribute to inhibition of progenitor growth. Consistent with results obtained using kinase-inactive BCR/ABL, imatinib exposure improved but did not fully correct the chemotactic defect in BCR/ABL-expressing progenitors. In contrast to results obtained with kinase-inactive mutants, no improvement in adhesion of BCR/ABL-expressing progenitors was observed after imatinib exposure. However, imatinib significantly reduced adhesion of control progenitors expressing GFP alone. These additional non-BCR/ABL-mediated effects of imatinib may account for differences in results obtained using kinase-inactive mutants compared with those obtained after imatinib treatment. The lack of increase in adhesion of BCR/ABL-expressing progenitors with imatinib may represent a balance between adhesion-enhancing effects of BCR/ABL kinase inhibition and "nonspecific" adhesion-inhibitory effects.

Our studies support a role for the BCR/ABL COOH-terminal actin-binding and proline-rich domains in abnormal adhesion and migration of BCR/ABL-expressing progenitors. Deletion of these domains did not reduce BCR/ABL-induced increased progenitor proliferation. The actin-binding domain is necessary for colocalization of BCR/ABL with actin cytoskeleton (51 , 52) . Actin-binding domain deletions reduced Rat-1 fibroblast transformation and reversed abnormal adhesion of hematopoietic cell lines (32 , 53) . However, actin-binding domain deletions have had varying effects in murine CML models with reduced oncogenicity or no effect seen in different studies (32 , 54) . The proline-rich region of BCR/ABL can mediate interactions with SH3 domains in other proteins including CRKL and the ABL interactor protein (38 , 55) . CRKL is a major phosphoprotein in primary CML cells (56) . Signaling through CRKL could potentially affect adhesion and migration via interactions with paxillin, CAS, and CBL, and formation of complexes with focal adhesion proteins (57) . The ABL interactor protein has been shown to regulate cytoskeletal function in cell lines (58) . Combined SH3 and proline-rich region deletions abrogate spontaneous cell migration on fibronectin and impair the ability to induce CML-like disease in mice (55) . The full range of adhesion and migration defects in CML progenitors may reflect a combination of signaling abnormalities, including cytoskeletal alteration mediated through the actin-binding and proline-rich domains and/or other kinase-independent interactions, and alteration in signaling mechanisms regulating cytoskeletal and focal complex assembly through kinase-dependent mechanisms. The model developed here will allow exploration of the role of these and other candidate signaling mechanisms in abnormal adhesion of CML progenitor cells.

Clinical studies with imatinib indicate that BCR/ABL kinase activity is required to sustain the proliferative advantage of CML cells. However, it is not clear whether kinase inhibition is sufficient to eradicate leukemia cells. There is considerable evidence that residual leukemia cells may persist and that malignant hematopoietic progenitors can be detected in CML patients responsive to imatinib treatment (59 , 60) . The mechanisms underlying incomplete elimination of malignant progenitors are not clear. One possibility is that complete inhibition of kinase activity is not achieved in all of the progenitor cells after imatinib exposure. However, in the current study, CD34+ cells expressing kinase-inactive BCR/ABL retained capacity for normal proliferation and survival. These results indicate that kinase inactivation in BCR/ABL-expressing cells does not by itself abrogate responsiveness to normal proliferation and survival signals and, therefore, may not lead to elimination of BCR/ABL-expressing cells under physiological conditions. The failure of kinase inhibition to fully correct adhesion and migration defects in BCR/ABL-expressing progenitors could also play a role in incomplete elimination of progenitor cells in the context of the marrow microenvironment, and this possibility warrants investigation in future studies.

In conclusion, we show that mutational inactivation and pharmacological inhibition of BCR/ABL kinase activity completely reverses abnormal progenitor proliferation but does not completely ameliorate defects in progenitor adhesion and migration. We additionally demonstrate that the BCR/ABL actin-binding and proline-rich domains significantly contribute to defects in progenitor adhesion and migration. These results indicate that both kinase-dependent and kinase-independent mechanisms contribute to abnormal adhesion and migration. These studies demonstrate the effectiveness and utility of this approach for direct investigation of the role of specific BCR/ABL signaling activities in human progenitor cell transformation.


   ACKNOWLEDGMENTS
 
We thank Khristine van Heijzen and Tinisha McDonald for assistance with progenitor assays and Lucy Brown and Claudio Spalla of the Analytical Cytometry Core. We also thank Dr. Warren Pear, University of Pennsylvania (Philadelphia, PA), Dr. Brian Druker, Oregon Health Sciences University (Portland, OR), Dr. Ruibao Ren, Brandeis University (Waltham, MA), and Dr. Martin Haas, University of California, San Diego (San Diego, CA) for plasmid constructs and Priscilla Yam (Department of Virology Research) for assistance with the virus production protocol.


   FOOTNOTES
 
Grant support: Leukemia and Lymphoma Society Translational Research Grant 6468 and the American Cancer Society Grant RPG-99-202-01-LBC (R. Bhatia). R. Bhatia is a Clinical Scholar of the Leukemia and Lymphoma Society.

Note: P. Ramaraj, H. Singh, and N. Niu contributed equally to this work. P. Ramaraj is currently at the Division of Endocrinology, Metabolism & Molecular Medicine, Charles R. Drew University, Los Angeles, CA.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Ravi Bhatia, Division of Hematology and Bone Marrow Transplantation, City of Hope National Medical Center, Duarte, CA 91010. Phone: (626) 359-8111, extension 62683; Fax: (626) 301-8973; E-mail: rbhatia{at}coh.org

Received 11/21/03. Revised 4/13/04. Accepted 5/26/04.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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