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NEOPLASIA
From the Department of Medicine, Stem Cell Institute,
and Cancer Center, University of Minnesota, Minneapolis.
Most insights into the molecular mechanisms underlying
transformation by the p210BCR/ABL oncoprotein are derived
from studies in which BCR/ABL cDNA was introduced into hematopoietic or
fibroblast cell lines. However, such cell line models may not represent
all the features of chronic myelogenous leukemia (CML) caused by
additional genetic abnormalities and differences in the biology of cell
lines compared with primary hematopoietic progenitor and stem cells. A
primary human hematopoietic progenitor cell model for CML was developed
by the transduction of b3a2 BCR/ABL cDNA in normal CD34+
cells. Adhesion of BCR/ABL-transduced CD34+ cells to
fibronectin was decreased, but migration over fibronectin was enhanced
compared with that of mock-transduced CD34+ cells. Adhesion
to fibronectin did not decrease the proliferation of BCR/ABL-transduced
CD34+ cells but decreased the proliferation of
mock-transduced CD34+ cells. This was associated with
elevated levels of p27Kip in
p210BCR/ABL-expressing CD34+ cells. In
addition, the presence of p210BCR/ABL
delayed apoptosis after the withdrawal of cytokines and serum. Finally,
significantly more and larger myeloid colony-forming units grew from
BCR/ABL than from mock-transduced CD34+ cells. Thus, the
transduction of CD34+ cells with the b3a2-BCR/ABL cDNA
recreates most, if not all, phenotypic abnormalities seen in primary
CML CD34+ cells. This model should prove useful for the
study of molecular mechanisms associated with the presence of
p210BCR/ABL in CML.
(Blood. 2001;97:2406-2412) Chronic myelogenous leukemia (CML) is a
malignant disease of the human hematopoietic stem cell, characterized
by the Philadelphia chromosome (Ph) and a rearrangement between the
breakpoint-cluster region (BCR) gene on chromosome 22 and
the Abelson leukemia (ABL) gene on chromosome
9.1 The presence of p210BCR/ABL tyrosine
kinase is essential and sufficient for the malignant transformation of hematopoietic cells.2-7 Studies have
shown p210BCR/ABL binding or activation of molecules for a
number of intracellular signaling pathways, including cytoskeletal
proteins,8,9 RAS,10,11 phosphatidylinositol
3'-kinase,12 and Janus kinase-signal transducer and
activator of transcription (Jak/Stat).13 However, how
p210BCR/ABL causes the clinical CML syndrome is
still unclear.
CML is characterized by the abnormal accumulation of immature myeloid
progenitors and precursors in blood and marrow.14 At the
cellular level, CML progenitors show predominant myeloid differentiation, decreased adhesion, enhanced migration, uncontrolled proliferation, and delayed apoptosis,11,15-21 all of which
contribute to the clinical phenotype.14 Cell lines derived
from patients with CML or generated by the transfection of the BCR/ABL
cDNA in existing cell lines have been used extensively to correlate molecular effects of BCR/ABL with the CML
phenotype.3,22,23 Even though hematopoietic cell lines
have been used to evaluate the effect of p210BCR/ABL on
cell function, these cells do not represent normal hematopoietic stem
and progenitor cells, where clinical CML originates. Additional genetic
abnormalities may occur in the cell lines, either as a result of the
underlying transformed cell type or of the long-term presence of
p210BCR/ABL, and may interfere with the interpretation of
changes thought to be the result of p210BCR/ABL. Finally,
the use of primary hemopoietic cells from patients with chronic-phase
CML, though informative, may be complicated by the fact that additional
genetic changes may already have occurred that influence the
interpretation of p210BCR/ABL- mediated effects. To
avoid such complicating factors, we here developed a primary
CD34+ CML model by transducing normal CD34+
cells with b3a2 BCR/ABL cDNA, and we demonstrate that this recreates the phenotype of CML.
Preparation of primary progenitors
p210-eGFP retroviral vector
Transduction of primary CD34+ cells CD34+ cells obtained from umbilical cord blood or bone marrow (n = 2) were cultured in serum-free medium (Stem Cell Technologies, Vancouver, Canada) with 50 ng/mL stem cell factor (SCF; Amgen, Thousand Oaks, CA), 50 ng/mL fetal liver-tyrosine kinase-3 ligand (Flt3-L; Immunex, Seattle, WA), and 50 ng/mL thrombopoietin (TPO; Amgen) for 2 days. 105 CD34+ cells were then resuspended in 200µL retroviral supernatant supplemented with SCF, Flt3-L, and TPO and were placed in collagen-coated transwells (Transwell-COL; Corning Costar, Cambridge, MA) of 24-well plates that had previously been incubated with 50µg/mL recombinant CH296 fibronectin (Takara Shuzo, Otsu, Japan). Once the viral medium was drained through the transwell, the filtrate was removed from the bottom compartment, and 200 µL fresh viral supernatant with cytokines was added to the transwell until 1 mL p210-eGFP or eGFP containing retroviral supernatant was filtered.26 When the last 200 µL viral supernatant was added, serum-free medium with cytokines was added to the bottom compartment, and cells were cultured with virus for 8 hours at 32°C in 5% CO2.21 Afterward, all viral supernatant was drained, and fresh serum-free medium with cytokines was added to the transwell and bottom compartment for 16 hours. This was repeated once or twice. Twenty-four hours after the last transduction, cells were labeled with anti-CD34-phycoerythrin (Becton Dickinson, Palo Alto, CA), and eGFP+ CD34+ cells were selected by FACS (FACStar Plus with Consort 32 computer) (Becton Dickinson) using isotype control-labeled cells.Adhesion assays For adhesion assays, 104 p210-eGFP- or eGFP-transduced GFP+ CD34+ cells suspended in 150 µL serum-free medium were plated in fibronectin-coated (50 µg/mL) or bovine serum albumin (BSA)-coated (5 mg/mL) (both from Sigma) wells of 48-well plates for 2 hours. Nonadherent and adherent cells were recovered and plated in short-term methylcellulose assay as described.18 The percentage of adherent colony-forming cells (CFCs) was calculated as: [no. adherent CFCs/(no. adherent CFCs + no. nonadherent CFCs)] × 100.Migration assay For migration assays, 104 p210-eGFP- or GFP-transduced eGFP+ CD34+ cells, or normal or CML marrow, were plated in fibronectin-coated (50 µg/mL) or BSA-coated (5 mg/mL) dishes and tilted to 80° for 1 hour, which allowed cells to accumulate in the lower rim. Dishes were lowered to 20° for 12 hours, and migration was assessed after enumerating the number of cells that had migrated beyond the middle line. In some experiments a blocking anti- 1-integrin antibody (P4C10) or a
control IgG was added (both from Gibco-BRL).
Proliferation inhibition assay For proliferation inhibition assays, eGFP- or p210-eGFP-transduced GFP+ CD34+ cells were treated with 5 µg/mL1-integrin-activating antibody 8A2 (a kind
gift from Dr N. Kovach, University of Washington, Seattle), as
previously described.27 Then 104
CD34+ cells were plated in fibronectin-coated (50 µg/mL)
or poly-L-lysine-coated (Sigma) (5 mg/mL) dishes for 12 hours.
Nonadherent and adherent cells were collected as described. Cell cycle
status was assessed using propidium iodide as
described.28
Determination of p27Kip levels in transduced CD34+ cells The levels of p27Kip in eGFP- and p210-eGFP-transduced CD34+ cells were determined by FACS 24 hours after transduction.28 Transduced cells were fixed and permeabilized with 1% paraformaldehyde, 80 µg/mL lysolecithin (all from Sigma) in phosphate-buffered saline (PBS; Gibco) for 5 minutes at 4°C. Cells were labeled with anti-CD34-antigen-presenting cells (Becton Dickinson) and then incubated with anti-p27Kip antibodies (Pharmingen), washed, and incubated with phycoerythrin-conjugated goat antimouse immunoglobulin (Pharmingen) for 30 minutes. CD34+ eGFP+ cells and CD34+ eGFP cells were gated, and
p27Kip levels were compared.
Progenitor survival assay CD34+ cells were plated in serum-free medium with or without 500 pg/mL recombinant human interleukin-3 (rhIL-3) for 2 days. The absolute number of viable CFCs was determined by plating culture progeny in methylcellulose assay. Survival of CFCs was determined by comparing the number of CFCs in culture progeny with those in fresh, uncultured cells.Colony-forming assay CD34+ cells were plated in methylcellulose containing Iscoves modified Dulbecco medium supplemented with 30% fetal calf serum, 3 IU erythropoietin (Amgen), 10 ng/mL each rhIL-3 (R&D Systems, Minneapolis, MN), SCF, granulocyte-macrophage-colony-stimulating factor (GM-CSF; Immunex), and 3 IU erythropoietin (Amgen), as previously described.18Western blot analysis for p210BCR/ABL Western blot analysis was performed as previously described.29 Briefly, 5 × 105 p210-eGFP- or eGFP-transduced GFP+ CD34+ cells were lysed directly in SDS sample buffer (50 mM/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 5%-mercaptoethanol; all from Sigma). After boiling,
the lysates were separated by SDS-PAGE and transferred onto
polyvinylidene difluoride. Blots were incubated with Blocker Blotto in
TBS (Pierce, Rockford, IL) and 0.1 µg/mL anti-ABL-mouse IgG1
antibody (clone 24-11; Santa Cruz Biotechnology, Santa Cruz, CA),
washed, and incubated for 1 hour with a goat antimouse horseradish
peroxidase-conjugated antibody (1:10 000 dilution; Jackson
ImmunoResearch Laboratories, West Grove, PA). Protein bands were
visualized using an ECL detection system (du Pont de Nemours,
Boston, MA).
Immunohistochemistry For immunochemistry eGFP- or p210-eGFP-transduced GFP+ CD34+ cells were cytospun on glass slides and incubated in 100% ethanol (Sigma) for 20 minutes. Endogenous peroxidase was blocked with 0.3% H2O2 in 50% methanol for 5 minutes (Sigma). Slides were stained using an established avidin-biotin complex (ABC) protocol (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA) according to the manufacturer's recommendation. Slides were incubated with 20% horse serum (Stem Cell Technologies) for 60 minutes, then incubated with 4 µg/mL anti-BCR mouse IgG1 (clone 7C6; Santa Cruz Biotechnology) at 4°C overnight. Slides were rinsed in PBS, and goat antimouse secondary antibody (Vector Laboratories) was added at room temperature for 30 minutes. After another PBS rinse, slides were incubated in freshly prepared ABC solution, rinsed again, and developed with diaminobenzidine. All slides were lightly counterstained with hematoxylin (Sigma).Statistical analysis Results of experimental points obtained from multiple experiments were reported as mean ± SEM. Significance levels for differences between different samples were determined using the 2-tailed Student t test.
We constructed the p210-eGFP retroviral vector by cloning the b3a2
BCR/ABL cDNA in the multicloning site upstream from the IRES sequence
of the MSCV-IRES-eGFP (eGFP) vector. Because p210BCR/ABL
is toxic to fibroblast,23 we performed transient
cotransfection of the p210-eGFP or eGFP plasmid with the pCL plasmid in
293kj cells to generate retroviral vector-containing
supernatants.25 Umbilical cord blood CD34+
cells were transduced with p210-eGFP or eGFP supernatant on 2 or 3 consecutive days using a transwell transduction procedure as described
previously.26 Twenty-four hours after the last transduction, eGFP+ CD34+ cells were selected
by FACS, and adhesion, migration, proliferation, and apoptosis were
evaluated (Figure 1). Between 40% and
65% of all cells recovered from transduction cultures were
CD34+. Thirty percent to 40% of CD34+ cells
transduced with the eGFP vector expressed eGFP, whereas 5% to 10% of
CD34+ cells transduced with the p210-eGFP vector were
eGFP+. We assessed the expression of p210-eGFP transduced
CD34+ cells by immunohistochemistry and Western blot
analysis. As shown in Figure 2A, the
cytoplasm of p210-eGFP-transduced CD34+ cells stained
considerably stronger with an anti-BCR antibody than did control
eGFP-transduced CD34+ cells, indicating the expression of
p210BCR/ABL in the cytoplasm of transduced cells.
Similarly, Western blot analysis showed the presence of
p210BCR/ABL at levels similar to those for endogenous
p145ABL in p210-eGFP-transduced, but not in
eGFP-transduced, CD34+ cells (Figure 2B). We then examined
whether the transduction of normal CD34+ cells with BCR/ABL
would recreate the cellular phenotype of CML. Specifically, we examined
the effect on cell adhesion, cell migration, cell proliferation and
differentiation, and cell survival.
We performed adhesion assays of p210-eGFP- or eGFP-transduced
GFP+ CD34+ cells to fibronectin or
BSA.18 We found that 24.4% ± 4% of eGFP-transduced
CFCs adhered to fibronectin, whereas only 3.4% ± 1% of
p210-eGFP-transduced CFCs adhered to fibronectin (n = 4) (Figure
3). Thus, the presence of
p210BCR/ABL in normal CD34+ cells caused
decreased adhesion. This is similar to what we have previously
described for primary, chronic-phase CML (10% ± 4% adhesion)
CD34+ cells versus normal bone marrow (22% ± 5%
adhesion) CD34+ cells.30
We next tested the effect of p210BCR/ABL on
CD34+ cell migration. We plated normal bone marrow
CD34+ cells (n = 4), CML bone marrow CD34+
cells (n = 4), p210-eGFP- or eGFP-transduced GFP+
CD34+ cells in fibronectin- or BSA-coated wells. Wells were
then tilted to 80° for 1 hour and lowered to 20°. After 12 hours,
we enumerated the fraction of cells that had migrated at least halfway
across the well. When 104 primary CML CD34+
cells were allowed to migrate on fibronectin-coated tilted dishes, 758 ± 79 cells migrated beyond the middle line, and this
could be inhibited by pretreating the cells with a blocking
anti-
Engagement of
Considerable evidence shows that p210BCR/ABL has
antiapoptotic effects.11,21,32-34 Therefore, we assessed
whether the transduction of CD34+ cells with
p210BCR/ABL would render cells resistant to apoptosis
induced by serum and cytokine withdrawal. p210-eGFP- or
eGFP-transduced GFP+ CD34+ cells were cultured
in serum-free medium with or without 500 pg/mL IL-3 for 2 days, and
CFCs were enumerated in fresh and cultured cells. We found that
78% ± 5% of eGFP-transduced CFCs remained viable after 48 hours in
culture with IL-3, whereas only 15% ± 2% of eGFP-transduced CFCs
were recovered after culture for 48 hours without IL-3 (Figure
7) (n = 3). In contrast, 81% ± 6%
of p210-eGFP-transduced CFCs were maintained after 2 days of culture without IL-3, and 77% ± 5% were maintained when cultured with IL-3.
Finally, CML is characterized by extensive expansion of the myeloid
cell pool and significantly smaller expansion of the erythroid cell
pool.15,16,35 To determine whether transduction with BCR/ABL would recreate this last characteristic, we assessed the number
of granulocyte-macrophage colony-forming units (GM-CFU) and
burst-forming units-erythroid (BFU-E) generated per 1000 eGFP- or
p210-eGFP-transduced GFP+ CD34+ cells cultured
in methylcellulose progenitor assay supplemented with SCF, GM-CSF, and
IL-3, each at a final concentration of 5 ng/mL and erythropoietin 3 IU/mL. In cultures initiated with 1000 p210-eGFP-transduced
CD34+ cells, we detected 493 ± 58 GM-CFU and
8 ± 2 BFU-E (n = 4) (Figure 8A,B). In contrast, in cultures initiated
with 1000 eGFP-transduced CD34+ cells, we detected
89 ± 10 GM-CFU and 19 ± 4 BFU-E (n = 4).
We have developed a human model of p210BCR/ABL+ CML by
transducing normal, human umbilical cord blood CD34+ cells
with a retroviral vector containing the b3a2 BCR/ABL cDNA. The model
recreates most, if not all, features of CML. Like chronic-phase CML
CD34+ cells, BCR/ABL-transduced CD34+ cells
adhere less well to,18,30 but migrate better over,
fibronectin than normal or mock-transduced CD34+
cells.8,36 BCR/ABL-transduced CD34+ cells, but
not untransduced or mock-transduced CD34+ cells, continue
to proliferate after the engagement of We show that BCR/ABL transduced CD34+ cells differentiate chiefly to the myeloid lineage, which is consistent with findings demonstrated by Clarkson et al16 and Marley et al.15,35 In addition, the size of GM-CFU in cultures with BCR/ABL-transduced CD34+ cells was greater than that of mock-transduced GM-CFU. The increase in the number of GM-CFU generated may be due to the increased sensitivity of BCR/ABL-containing progenitors to cytokines, resulting in the recruitment of otherwise quiescent progenitors in cycle.33 In addition, we found that p210-eGFP-transduced CD34+ cell survival under cytokine-depleted conditions is significantly better than that of mock-transduced cells, consistent with a series of studies indicating that p210BCR/ABL has antiapoptotic activity.11,21,32,33 However, survival and number of GM-CFU was not different when mock- or p210BCR/ABL-transduced cells were cultured in the presence of cytokines for 2 days. Nevertheless, the increased number and size of GM-CFU in cultures of BCR/ABL-transduced CD34+ cells may also be due to increased proliferation or decreased apoptosis of progenitors, which becomes obvious only after a longer period in culture under cytokine-replete conditions. Single-cell assays will be needed to discriminate between these possibilities. Compared with what we have observed for primary CML CD34+ cells,27,30,37,38 the excess GM-CFU growth is more pronounced in the model described here. Similarly, the increased size of GM-CFU in cultures of BCR/ABL-transduced CD34+ cells is more pronounced than what we have commonly seen in cultures of primary CML CD34+HLA-DR+ cells. Finally, the degree of resistance against apoptosis, when cultured in serum-free and cytokine-free cultures, was greater in the model system than in primary CML. It is possible that differences seen between our CML model and primary chronic-phase CML may be related to the level of p210BCR/ABL in transduced cord blood cells. In the p210-eGFP model, p210BCR/ABL expression is regulated from the MSCV-LTR promoter and not the endogenous BCR promoter, as in chronic-phase CML. Levels of p210BCR/ABL in the p210-eGFP model are similar to those of endogenous p145ABL, which is 3- to 5-fold higher than p210BCR/ABL levels in primary CML CD34+ cells. Our finding that the effects of BCR/ABL are exaggerated is similar to what has been reported by a number of investigators who generated a murine model of CML-like disease by transducing mouse stem cells with a BCR/ABL-containing retroviral vector.2,6,7 They, too, speculated that this was the result of high levels of p210BCR/ABL oncoprotein in the hematopoietic stem and progenitor cells. This was elegantly illustrated by Cambier et al,33 who found that effects on cell survival and in vivo tumorigenicity of cell lines varies significantly with different levels of p210BCR/ABL expression. Use of an inducible vector will be needed to determine whether the higher expression of p210BCR/ABL in the model system is responsible for these differences. An alternative explanation may be that we introduced the BCR/ABL cDNA in cord blood CD34+ cells and not in adult marrow cells. We chose umbilical cord blood rather than marrow because the transduction of primary normal marrow-derived CD34+ cells with the p210-eGFP construct was usually less than 1%, which precludes the studies described here. We have previously shown that cord blood CD34+ cells adhere to fibronectin, migrate over fibronectin, and are regulated by adhesion to fibronectin in a manner similar to that of marrow-derived CD34+ cells.39 Whether differences in proliferative potential between cord blood and adult bone marrow CD34+ cells40,41 contribute to the "exaggerated" proliferation and resistance to apoptosis seen between p210-eGFP-transduced cord blood CD34+ cells and primary CML CD34+ cells is unknown. Despite these differences, we believe that the model in which normal CD34+ cells are transduced with p210-eGFP recreates the cardinal clinical features of CML and should be invaluable in studies aimed at further characterizing the molecular mechanisms through which BCR/ABL causes functional defects in CML CD34+ cells. Several groups have shown that the transplantation of murine progenitors transduced with a BCR/ABL cDNA-containing retroviral vector creates a myeloproliferative disease akin to chronic-phase CML in 100% of recipient mice, with a latency of 4 to 6 weeks.2,6,7 Whether functional abnormalities resulting from the transduction of human progenitors with p210-eGFP at the cellular level identified in the human model described in this article are also seen in the murine models is yet to be evaluated. Transplantation of human chronic-phase CML marrow or blood in immunodeficient mice leads to the development of a CML-like disease in the marrow and spleen of engrafted mice.42-44 However, the frequency and level of engraftment are variable when different patient samples are examined, somewhat compromising the usefulness of the model. This variability may be due to the fact that some, but not all, primary chronic-phase CML samples have already acquired additional genetic abnormalities that contribute to the final phenotype seen for each patient.45 This would be circumvented in the model developed here. Finally, our study confirms that the p210BCR/ABL oncoprotein is necessary and sufficient for the abnormal functional characteristics seen in CML. To prove that functional abnormalities in CML are caused by the p210BCR/ABL oncoprotein, several studies used antisense29,46,47 or ribozyme48 strategies to suppress p210BCR/ABL expression, or they used tyrosine kinase inhibitors such as STI57149,50 or AG95751 to inhibit the p210BCR/ABL kinase activity. The fact that we can recreate all cellular functional defects ascribed to CML by transferring the BCR/ABL gene in normal primary hematopoietic cells further confirms the causal relation between p210BCR/ABL and the CML phenotype. In conclusion, this model recreates most characteristics of chronic-phase CML in vitro. It should, therefore, prove a useful tool for investigators interested in determining the molecular mechanisms underlying p210BCR/ABL-mediated abnormalities in progenitor function.
We thank Valerie McCullar, Erjin Fan, and the members of the Stem Cell Laboratory for their excellent technical assistance.
Submitted August 18, 2000; accepted December 1, 2000.
Supported by National Institutes of Health grants RO1-HL-49930, RO1-DK-53673, and RO1-CA 74887; the Leukemia and Lymphoma Society of America; University of Minnesota Bone Marrow Transplant Research Fund; Minnesota Medical Foundation; University of Minnesota Graduate School; and the Institute of Hematology at the Chinese Academy of Medical Science.
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: Catherine M. Verfaillie, Stem Cell Institute, University of Minnesota, Mayo Mail Code 806, 420 Delaware St SE, Minneapolis, MN 55455; e-mail: verfa001{at}tc.umn.edu.
1. Rowley J. The Philadelphia chromosome translocation: a paradigm for understanding leukemia. Cancer. 1990;65:2178-2184[CrossRef][Medline] [Order article via Infotrieve].
2.
Daley GQ, Van Etten RA, Baltimore D.
Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome.
Science.
1990;247:824-830
3.
Gishizky mL, Witte ON.
Initiation of deregulated growth of multipotent progenitor cells by bcr-abl in vitro.
Science.
1992;256:836-839 4. Elefanty AG, Hariharan IK, Cory S. bcr-abl, the hallmark of chronic myeloid leukaemia in man, induces multiple haemopoietic neoplasms in mice. EMBO J. 1990;9:10691078.
5.
Li S, Ilaria RLJ, Million RP, Daley GQ, Van Etten RA.
The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity.
J Exp Med.
1999;189:1399-1412
6.
Xiaowu Z, Ruibao R.
Bcr-Abl efficiently induces a myeloproliferative disease and production of excess interleukin-3 and granulocyte-macrophage colony-stimulating factor in mice: a novel model for chronic myelogenous leukemia.
Blood.
1998;92:3829-3840
7.
Pear WS, Miller JP, Xu L, et al.
Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow.
Blood.
1998;92:3780-3792 8. Salgia R, Li JL, Ewaniuk DS, et al. BCR/ABL induces multiple abnormalities of cytoskeletal function. J Clin Invest. 1997;100:46-55[Medline] [Order article via Infotrieve].
9.
Salgia R, Avraham S, Pisick E, et al.
The related adhesion focal tyrosine kinase forms a complex with paxillin in hematopoietic cells.
J Biol Chem.
1996;271:31222-31226
10.
Lugo TG, Pendergast AM, Muller AJ, Witte ON.
Tyrosine kinase activity and transformation potency of bcr-abl oncogene products.
Science.
1990;247:1079-1082 11. Cortez D, Stoica G, Pierce JH, Pendergast AM. The BCR-ABL tyrosine kinase inhibits apoptosis by activating a Ras-dependent signaling pathway. Oncogene. 1996;13:2589-2594[Medline] [Order article via Infotrieve].
12.
Jain SK, Susa M, Keeler ML, Carlesso N, Druker B, Varticovski L.
PI 3-kinase activation in BCR/abl-transformed hematopoietic cells does not require interaction of p85 SH2 domains with p210 BCR/abl.
Blood.
1996;88:1542-1550 13. Shuai K, Halpern J, ten Hoeve J, Rao X, Sawyers CL. Constitutive activation of STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia. Oncogene. 1996;13:247-254[Medline] [Order article via Infotrieve].
14.
Kantarjian HM, Smith TL, McCredie KB, et al.
Chronic myelogenous leukemia: a multivariate analysis of the associations of patient characteristics and therapy with survival.
Blood.
1985;66:1326-1335 15. Marley SB, Lewis JL, Scott MA, Goldman JM, Gordon MY. Evaluation of "discordant maturation" in chronic myeloid leukaemia using cultures of primitive progenitor cells and their production of clonogenic progeny (GM-CFU). Br J Haematol. 1996;95:299-305[CrossRef][Medline] [Order article via Infotrieve]. 16. Clarkson BD, Strife A, Wisniewski D, Lambek C, Carpino N. New understanding of the pathogenesis of CML: a prototype of early neoplasia. Leukemia. 1997;11:404-428. 17. Gordon M, Dowding C, Riley G, Goldman J, Greaves M. Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia. Nature. 1987;328:342-345[CrossRef][Medline] [Order article via Infotrieve]. 18. 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.
19.
Bhatia R, McCarthy JB, Verfaillie CM.
Interferon-alpha restores normal beta 1 integrin-mediated inhibition of hematopoietic progenitor proliferation by the marrow microenvironment in chronic myelogenous leukemia.
Blood.
1996;87:3883-3891
20.
Jiang Y, Zhao RCH, Verfaillie CM.
Inactivation of the cyclin-dependent kinase inhibitor, p27, is responsible for overriding the integrin-mediated inhibition of CML CD34+ cells.
Proc Natl Acad Sci U S A.
2000;97:10538-10543
21.
Bedi A, Zehnbauer BA, Barber JP, Sharkis SJ, Jones RJ.
Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia.
Blood.
1994;83:2038-2044 22. Matulonis U, Salgia R, Okuda K, Druker B, Griffin JD. Interleukin-3 and p210 BCR/ABL activate both unique and overlapping pathways of signal transduction in a factor-dependent myeloid cell line. Exp Hematol. 1993;21:1460-1466[Medline] [Order article via Infotrieve]. 23. Renshaw MW, McWhirter JR, Wang JYJ. The human leukemia oncogene bcr-abl abrogates the anchorage requirement but not the growth factor requirement for proliferation. Mol Cell Biol. 1995;15:1286-1293[Abstract]. 24. Pui JC, Allman D, Xu L, et al. Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity. 1999;11:299-308[CrossRef][Medline] [Order article via Infotrieve].
25.
Naviaux RK, Costanzi E, Haas M, Verma IM.
The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses.
J Virol.
1996;70:5701-5705 26. Liu HJ, Hung Y, Wissink SD, Verfaillie CM. Improved retroviral transduction of hematopoietic progenitors by combining methods to enhance virus-cell interaction. Leukemia. 2000;14:307-311[CrossRef][Medline] [Order article via Infotrieve].
27.
Lundell BI, McCarthy JB, Kovach NL, Verfaillie CM.
Activation-dependent 28. Jiang Y, Zhao RCH, Verfaillie CM. Inactivation of the cyclin-dependent kinase inhibitor, p27, is responsible for overriding the integrin-mediated inhibition of CML CD34+ cells. Proc Natl Acad Sci U S A. 2000;97:10538-10543.
29.
Zhao R, McIvor R, Griffin J, Verfaillie C.
Gene therapy for chronic myelogenous leukemia (CML): a retroviral vector that renders hematopoietic progenitors methotrexate-resistant and CML progenitors functionally normal and nontumorigenic in vivo.
Blood.
1997;90:4687-4698 30. Bhatia R, Wayner EA, McGlave PB, Verfaillie CM. Interferon-alpha restores normal adhesion of chronic myelogenous leukemia hematopoietic progenitors to bone marrow stroma by correcting impaired beta 1 integrin receptor function. J Clin Invest. 1994;94:384-391. 31. Hurley RW, McCarthy JB, Verfaillie C. Direct adhesion to bone marrow stroma via fibronectin receptors inhibits hematopoietic progenitor proliferation. J Clin Invest. 1995;96:511-512.
32.
McGahon A, Bissonnette R, Schmitt M, Cotter KM, Green DR, Cotter TG.
BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death.
Blood.
1994;83:1179-1187 33. Cambier N, Chopra R, Strasser A, Metcalf D, Elefanty AG. BCR-ABL activates pathways mediating cytokine independence and protection against apoptosis in murine hematopoietic cells in a dose-dependent manner. Oncogene. 1998;16:335-348[CrossRef][Medline] [Order article via Infotrieve]. 34. Maguer-Satta V, Burl S, Liu L, et al. BCR-ABL accelerates C2-ceramide-induced apoptosis. Oncogene. 1998;16:237-248 |
Top
Abstract
Introduction
) or eGFP-transduced (
) GFP+
CD34+ cells (104) from 4 umbilical cord blood
samples were plated in 24-well plates coated with 100 µg/mL
fibronectin (FN, left) or 5% BSA (right) for 2 hours. Nonadherent
cells and adherent cells were collected separately and plated in
methylcellulose progenitor assay. The percentage of adherent CFCs was
calculated as [no. adherent CFCs/(no. adherent CFCs + no.
nonadherent CFCs)] × 100. The number of CFCs in the
nonadherent + adherent population was 66 ± 6/1000 cells for
eGFP-transduced GFP+ CD34+ cells and
268 ± 25/1000 cells for p210-eGFP-transduced GFP+
CD34+ cells. Comparison between p210-eGFP- and
eGFP-transduced GFP+ CD34+ cells
(P = .001).
, eGFP/+IL-3; 
, p210-eGFP/no IL-3; 
,
p210-eGFP/+IL-3.
5