Introduction

An important determinant of the protein repertoire in human cells is alternative splicing, which can be defined as the differential identification and excision of intronic gene sequences during pre-mRNA processing. By connecting exons in different linear combinations, single genes can give rise to numerous protein products. Often, these differences contribute to distinct cellular phenotypes.^1,2 Recent studies of the human genome have suggested that 35–59% of human genes are alternatively spliced, indicating that alternative splicing contributes largely to the complexity of higher mammals.³

Splicing of pre-mRNA transcripts occurs co-transcriptionally and is mediated by large protein complexes, including but not limited to the spliceosome, that recognize distinct RNA sequences and coordinate the excision of introns and joining of exons.¹ Although these complexes and the elements they recognize have yet to be completely defined, it is thought that cell-type specific pre-mRNA splicing patterns in mammals are dependent on the relative expression levels of splicing machinery components.^2,4 Members of the serine–arginine (SR) protein and heterogeneous nuclear ribonucleoprotein (hnRNP) families play intricate roles in regulating alternative splicing.^1,5,6 These families of proteins are involved in splice site recognition and spliceosome assembly/disassembly. Altered phosphorylation and/or expression of these proteins affects their subcellular distribution and splice site recognition, thereby modifying the efficiency of alternative splicing at particular exon junctions.^4,7,8,9

Chronic myelogenous leukemia (CML) is a malignancy of the hematopoietic stem cell (HSC) characterized by the Philadelphia chromosome,¹⁰ which combines the N-terminus of the breakpoint cluster region (BCR) gene on chromosome 9, with the C-terminus of the Abelson kinase (ABL) gene on chromosome 22.^11,12 In contrast to p145^ABL kinase, the p210^BCR/ABL fusion protein has constitutively active tyrosine kinase activity.¹³ While p145^ABL is normally present in the nucleus, p210^BCR/ABL is located in the cytosol,^14,15 where it interacts with and phosphorylates numerous proteins.¹⁶ Although tyrosine kinase activity of p210^BCR/ABL is necessary and sufficient for malignant transformation,^17,18,19 the exact mechanism(s) underlying abnormal HSC behavior has yet to be determined.

Normal steady-state hematopoiesis occurs in the bone marrow (BM), where 1-integrins are, at least in part, responsible for adhesion to the extracellular matrix.^20,21,22 We and others have shown that the function of 1-integrins on CML CD34⁺ progenitors is abnormal, resulting in decreased adhesion, increased migration, and failure to inhibit proliferation upon integrin engagement.^23,24,25,26 How p210^BCR/ABL negatively influences 1-integrin function in CML is not known. Focal adhesion kinases are important mediators of 1-integrin signaling.²⁷ In contrast to fibroblasts and adherent cell types, Pyk2H is the predominantly expressed focal adhesion kinase in hematopoietic cells,^28,29,30 but whether signaling by Pyk2H and its full-length isoform, Pyk2, differs is not known.

We hypothesized that the presence of p210^BCR/ABL may cause changes in gene expression that contribute to the pathophysiology of BCR/ABL-mediated transformation. Other studies, using BCR/ABL-transduced cell lines, demonstrated that BCL-X_L,³¹ RAS-related KIR,³² melanoma-related antigen PRAME,³³ and Ian4³⁴ are differentially expressed in BCR/ABL⁺ cells. Since the cell lines used in these studies were transformed prior to the introduction of BCR/ABL, the exact role of p210^BCR/ABL in altered gene expression could not be accurately determined. Therefore, we transduced primary umbilical cord blood (UCB) CD34⁺ cells with the BCR/ABL cDNA and analyzed p210^BCR/ABL-kinase-dependent changes in gene expression by subtractive hybridization 48 h after transduction.

We demonstrate that expression of numerous proteins involved in pre-mRNA splicing is increased in p210^BCR/ABL-transduced CD34⁺ cells and CML CD34⁺ cells. Increased expression of these genes was correlated with alternative splicing of the focal adhesion kinase PYK2. Inhibition of p210^BCR/ABL with the Abelson tyrosine kinase inhibitor STI571 reduced the expression of pre-mRNA splicing genes and returned PYK2 mRNA splicing patterns to a more normal configuration, demonstrating the dependence of altered splicing on BCR/ABL kinase activity.

Materials and methods

Cell lines and materials

Mo7e cells transfected with p210^BCR/ABL (MBA4) or an empty vector (Mo7e) (kind gift from Dr J Dick, Hospital for Sick Children, Toronto, Canada) and HL-60 (CCL-240) and K562 cells (CCL-243), obtained from ATCC, were maintained in Iscove's modified Dulbecco's medium (IMDM; GibcoBRL, Grand Island, NY, USA) supplemented with 20% fetal calf serum (FCS; HyClone, Logan, UT, USA) and 1% L-glutamine (GibcoBRL). Mo7e cell medium also included 50 ng/ml IL-3 (R&D, Minneapolis, MN, USA).

Antibodies (Abs) used in this study were directed against phosphotyrosine (mouse monoclonal IgG₁; UBI, Lake Placid, NY, USA), pan-Pyk2 (UBI, Transduction Labs, Lexington, KY, USA), SRPK1 (Santa Cruz, Santa Cruz, CA, USA), RNA Helicase II/Gu (kind gift from Dr B Valdez, Baylor, Houston, TX, USA), and -actin (Sigma, St Louis, MO, USA). Unspliced Pyk2-specific rabbit polyclonal Abs (#639) raised against the excised amino-acid sequence of Pyk2H (ie exon 23) were kindly provided by Dr I Dikic (Ludwig Institute, Uppsala, Sweden). STI571 was a generous gift from Novartis. Rabbit serum and other bulk chemicals were obtained from Sigma.

Primary cells

Peripheral blood or BM from patients with chronic phase (CP) BCR/ABL⁺ CML or normal (NL) healthy volunteers and UCB samples were obtained following informed consent, using guidelines approved by the Committee on the Use of Human Subjects at the University of Minnesota. CD34⁺ cell-enriched populations were selected from mononuclear cells immunomagnetic column separation techniques (Miltenyi Biotec, Sunnyvale, CA, USA). Purity following two passes over the immunomagnetic columns was >95% CD34⁺.

Primary CD34⁺ cell culture and manipulation

UCB CD34⁺ cells were transduced with MSCV-p210^BCR/ABL-eGFP or control MSCV-eGFP vectors as described,³⁵ and CD34⁺eGFP⁺ cells were sorted on a FACSVantage SE™, maintained at the University of Minnesota Cancer Center Flow Core. Prior to lysis for RNA or protein extraction, FACS-sorted UCB CD34⁺ cells, CML or NL BM CD34⁺ cells were maintained for 16–24 h in BIT-9500 media (Stem Cell Technologies; Vancouver, Canada) supplemented with 50 M 2-mercaptoethanol, 40 g/ml low-density lipoprotein, 250 pg/ml G-CSF (Amgen, Thousand Oaks, CA, USA), 10 pg/ml GM-CSF (Immunex, Seattle, WA, USA), 1 ng/ml IL-6 (R&D), 50 pg/ml LIF (R&D), 200 pg/ml MIP-1 (R&D), and 200 pg/ml SCF (Amgen).

Quantitative and standard RT-PCR

Total RNA was isolated using the RNeasy procedure (Qiagen, Valencia, CA, USA), treated with DNAse I (GibcoBRL) for 15 min at room temperature, and mRNA was reverse transcribed for 50 min at 42°C using Oligo(dT) primers and Superscript™ II RT (GibcoBRL). Quantitative (q) RT-PCR was performed using the SYBR Green I dye (SYBR Green PCR Master Mix, Applied Biosystems). Sequence-specific primers were designed (Table 1) and, to prevent nonspecific amplification, primer concentration optimization was performed and verified by gel analysis. The amplification was performed with 40 cycles of two-step PCR (15 s at 95°C and 60 s at 60°C) after initial denaturation (95°C for 10 min) using an ABI Prism 7700 Sequence Detector System (Applied Biosystems). Amplification of -actin mRNA as an endogenous control was used to standardize the amount of sample added to the reactions. To compare the relative amount of target gene in the different CP CML samples, we designated one of the NL BM samples as a reference (ie calibrator) for all qRT-PCR experiments and expressed the averaged sample value as percentage of the calibrator value.

Table 1 - Primer sequences.

Full table

Standard RT-PCR was performed for 35 cycles. The relative amount of PYK2 mRNA isoforms was assessed using primers located 5' and 3' of the outermost alternatively spliced exons (Table 1). -actin was amplified as previously described.³⁶

Southern blots

cDNA was separated on 2% TAE-agarose gels, transferred to nylon membranes (Roche, Mannheim, Germany) and crosslinked with UV light. A pan-Pyk2-specific oligonucleotide probe was 3'-end labeled with digoxygenin (DIG), hybridized in DIG EasyHyb™ (Roche) overnight at 54°C, and visualized using alkaline phosphatase-conjugated DIG Abs (Roche) and chemiluminescence. Densitometric analysis was performed using a model GS-700 Imaging Densitometer™ and Molecular Analyst™ software (BioRad).

Immunoprecipitation and western blot

Cells were lysed in immunoprecipitation wash buffer (IPWB, 50 nM Tris (pH 7.4), 250 mM NaCl, 2 mM EDTA, 1% NP-40, 50 mM NaF, 2 mM NaVO₄, 1 mM NaPO₄, and Complete™ protease inhibitor cocktail (Roche)). For immunoprecipitation experiments, equal amounts of protein, as determined by the BCA protein assay (Pierce, Rockford, IL), were precleared with 35 l of recombinant protein-G agarose beads (Roche) for 1 h at 4°C, then centrifuged. Cleared supernatants were incubated with specific Abs for 3 h at 4°C, followed by addition of 35 l of recombinant Protein-G agarose beads for 75 min at 4°C. After three washes with PBS/1% NP-40, proteins were eluted with sample buffer (2% SDS, 10% glycerol, 0.96 M 2-mercaptoethanol, 0.3 M Tris (pH 6.8), and 0.02% bromophenol blue) by boiling for 5 min, separated by 8% SDS-PAGE, and then transferred to Immuno-Blot™ PVDF membrane (BioRad, Hercules, CA, USA). Membranes were blocked using 4% nonfat dry milk in PBS-T (pH 7.6, 0.1% Tween-20) and incubated for 75 min with specific Abs. Immunoreactive bands were visualized using secondary horseradish peroxidase-conjugated Abs and chemiluminescence (Amersham, Arlington Heights, IL, USA).

Results

Identification of pre-mRNA splicing genes overexpressed in BCR/ABL⁺CD34⁺ cells

To identify BCR/ABL-dependent changes in gene expression, we utilized a CML model system in which p210^BCR/ABL cDNA was retrovirally introduced into primary UCB CD34⁺ cells. We have previously shown that BCR/ABL-transduced UCB CD34⁺ cells exhibit similar defects in proliferation, apoptosis, adhesion, and migration as primary CML CD34⁺ cells.^35,37 We previously reported, using subtractive hybridization, the identification of differentially expressed genes in BCR/ABL vs control eGFP cDNA-transduced UCB CD34⁺ cells.³⁸ Some genes have previously been implicated in cellular processes thought to be disturbed in CML, while other genes may be involved in new cellular pathways responsible for the BCR/ABL-mediated leukemic process. Interestingly, five out of the 34 genes confirmed as overexpressed in BCR/ABL-transduced CD34⁺ cells corresponded to known genes directly implicated in pre-mRNA splicing: such as SRPK1, RNA Helicase II/Gu, hnRNPA2/B1, DDX10, and SF3b (ie SAP130).

To verify the changes in gene expression identified in the subtractive library, we evaluated mRNA levels of SRPK1, RNA Helicase II/Gu, hnRNPA2/B1, DDX10, and SF3b (ie SAP130) in four to seven individual BCR/ABL-transduced UCB CD34⁺ populations and six to eight CD34⁺ cell populations from CP CML patients by qRT-PCR (Figure 1a). A 2.1–4.6-fold increase in expression of pre-mRNA processing genes was observed in BCR/ABL- vs control eGFP-transduced UCB CD34⁺ cells. Despite patient-to-patient, and normal donor-to-donor variability, all genes with increased expression in BCR/ABL-transduced UCB CD34⁺ cells were also 1.5–5.9-fold overexpressed in CP CML (n=6–8) vs NL BM (n=3) CD34⁺ cells (Figure 1a).

Figure 1.

Increased expression of genes involved in pre-mRNA splicing in BCR/ABL⁺ UCB and CP CML CD34⁺ cells. qRT-PCR experiments were performed on cDNA from (a) eGFP and BCR/ABL-transduced UCB CD34⁺ cells (n4; P0.01), and (b) primary CD34⁺ cells from NL donors (n=3) or CML patients (n6; P0.05). (a) Results in eGFP⁺BCR/ABL⁺ samples are expressed relative to those in corresponding eGFP⁺CD34⁺ samples. Individual results in (b) are expressed as log % of the calibrator value. Results were normalized against -actin.

Full figure and legend (30K)

We next evaluated protein expression of SRPK1 and RNA Helicase II/Gu in BCR/ABL- vs control eGFP-transduced UCB CD34⁺ cells and CP CML vs NL CD34⁺ cells. Both SRPK1 and RNA Helicase II/Gu proteins were more abundant in p210^BCR/ABL-transduced CD34⁺ cells compared with untransduced or eGFP-transduced CD34⁺ cells (Figure 2a). In CD34⁺ cells from two NL donors, SRPK1 and RNA Helicase II/Gu protein were barely detectable, while SRPK1 and RNA Helicase II/Gu were readily observed in four CP CML patient samples (Figure 2b).

Figure 2.

SRPK1 and RNA Helicase II/Gu protein expression is increased in BCR/ABL⁺ UCB and CP CML CD34⁺ cells. Western blots were performed with cell lysate from 5 10⁴ (a) eGFP and BCR/ABL-transduced UCB CD34⁺ cells or (b) NL and CP CML CD34⁺ cells. Membranes were stripped and reprobed with a -actin Ab. Blots are representative of n=2 experiments.

Full figure and legend (96K)

Overexpression of genes implicated in pre-mRNA splicing is p210^BCR/ABL kinase dependent

To test whether increased expression of pre-mRNA processing proteins was p210^BCR/ABL-kinase dependent, CD34⁺ cells from BCR/ABL- or control eGFP-transduced UCB, NL donors or CML patients were cultured for 48 h in the presence or absence of 1 M STI571, and mRNA and protein levels were analyzed. STI571 (1 M) inhibited p210^BCR/ABL-kinase activity in CD34⁺ cells from CP CML patients, as demonstrated by decreased phosphorylation of p210^BCR/ABL itself, as well as CrkL.³⁸ Moreover, no significant changes were observed in the basal phosphorylation of CrkL in control GFP-transduced or NL CD34⁺ cells upon STI571 treatment.³⁸

Treatment with STI571 significantly decreased the mRNA levels of SRPK1, RNA Helicase II/Gu, hnRNPA2/B1, DDX10, and SF3b (ie SAP130) 2.4–5.1-fold in BCR/ABL-transduced UCB CD34⁺ cells (Figure 3a) and 1.3–6.3-fold in CP CML CD34⁺ cells (Figure 3b). Meanwhile, STI571 did not significantly alter gene expression in BCR/ABL-negative CD34⁺ cells (data not shown). Treatment with STI571 for 48 h resulted in reduced SRPK1 and RNA Helicase II/Gu protein levels (Figures 4a–d).

Figure 3.

p210^BCR/ABL-induced gene expression is kinase dependent. qRT-PCR experiments were performed on cDNA from (a) BCR/ABL- and eGFP-transduced UCB CD34⁺ cells (n3; P0.02), or (b) NL (n=3) and CP CML CD34⁺ cells, which were incubated without or with 1 M STI571 for 48 h. Results were normalized against -actin, and expressed relative to the corresponding untreated samples.

Full figure and legend (32K)

Figure 4.

SRPK1 and RNA Helicase II/Gu protein expression is p210^BCR/ABL-kinase dependent. Western blots were performed using cell lysate from 5 10⁴ (a, c) NL or CP CML CD34⁺ cells, and (b, d) UCB BCR/ABL⁺CD34⁺ cells incubated without or with 1 M STI571 for 48 h. Blots demonstrating (a, b) RNA Helicase II/Gu expression or (c, d) SRPK1 expression in CD34⁺ cells are shown and representative of n2 experiments.

Full figure and legend (82K)

PYK2 splicing and expression are altered in BCR/ABL⁺ cells

Experiments evaluating the expression of proteins involved in 1-integrin signaling resulted in the observation of a 'Pyk2H mobility shift' in Western blots using CD34⁺ cell lysates from CP CML patients vs NL donors (data not shown). Further analysis of CML vs NL CD34⁺ cells resulted in the conclusion that the slower migrating band in p210^BCR/ABL-positive samples did not result from increased Pyk2H phosphorylation. Meanwhile, RT-PCR experiments using primers located 5' and 3' of the excised exon of Pyk2H (ie exon 23), and subsequent Southern blots and DNA sequencing, confirmed that the slower migrating transcript in CML CD34⁺ cells represented full-length Pyk2 (Figures 5a and b). Serial immunoprecipitation (ie immunodepletion) experiments using Abs against exon 23 of PYK2 (ie Ab#639) clearly demonstrated that expression of full-length Pyk2 was increased in primary CML CD34⁺ cells (Figure 5c). A similar increase in full-length Pyk2 transcripts and protein was observed in BCR/ABL⁺ cell lines (Figure 5b) and in primary UCB CD34⁺ cells transduced with p210^BCR/ABL cDNA compared to mock- (data not shown) or control GFP-transduced cells (Figure 6a).

Figure 5.

PYK2 gene splicing and expression are altered in BCR/ABL⁺ cells. (a) The open reading frame of Pyk2H and Pyk2 are shown, including the location of the kinase domain (), proline-rich regions (), and focal adhesion targeting domain (FAT; ). The excised portion of Pyk2H is indicated. (b) RT-PCR was performed using cDNA from cell lines or NL and CP CML CD34⁺ cells, and Southern blots were done. (c) A measure of 300 g of cell lysate from NL or CP CML CD34⁺ cells was serially immunodepleted with rabbit serum (rIg), full-length Pyk2 specific Ab #639 (3 ), and pan-Pyk2 Ab (2 ), and Western blots were performed. Data are representative of n3 experiments.

Full figure and legend (92K)

Figure 6.

Alternative splicing and expression of PYK2 is p210^BCR/ABL kinase-dependent. (a) RT-PCR was performed using cDNA from BCR/ABL- or eGFP-transduced CD34⁺ cells, and NL or CP CML CD34⁺ cells, incubated without or with 1 M STI571 for 48 h. Data are representative of n3 experiments. (b) Densitometry was performed to determine the relative abundance of Pyk2 vs Pyk2H. ^*Significantly different from BCR/ABL-negative cells (n7; P0.039), or ^**untreated cells (n=3; P=0.014).

Full figure and legend (80K)

PYK2 alternative splicing is dependent on p210^BCR/ABL-kinase activity

To determine whether changes in PYK2 splicing were dependent on the kinase activity of p210^BCR/ABL, we treated CML and BCR/ABL-transduced CD34⁺ cells with STI571. The relative expression of Pyk2 vs Pyk2H mRNA was returned to baseline levels in CML and p210^BCR/ABL-transduced CD34⁺ cells treated with STI571 (Figures 6a and b) for 48 h, whereas no effect was observed in NL BM CD34⁺ cells or eGFP-transduced UCB CD34⁺ cells (Figure 6a). These studies therefore suggest a direct relationship between the kinase activity of p210^BCR/ABL and increased expression of the full-length Pyk2 isoform.

Discussion

It has been long recognized that alternative splicing can generate numerous protein isoforms, at times with variant function, which are encoded by the same pre-mRNA transcript.² Although it is well accepted that p210^BCR/ABL modifies various signaling cascades and gene expression, the effect of BCR/ABL on pre-mRNA processing has not been studied. We demonstrate here that expression of an uncharacteristically large proportion of genes involved in pre-mRNA processing (ie 15% of subtractive library) is increased in primary p210^BCR/ABL-positive CD34⁺ cells, which correlates with altered splicing patterns of the PYK2 gene whose product is an important mediator of integrin signaling; which is defective in p210^BCR/ABL-positive cells. Increased expression of pre-mRNA processing genes and altered splicing of gene products (eg PYK2) are not likely secondary to cellular transformation, as altered gene expression and associated alternative splicing are reversed by the Abl-specific tyrosine kinase inhibitor STI571.

SR protein kinase-1 (SRPK1) is highly specific for RS domain-containing splicing factors, and directly influences the functional activity of SR proteins that participate in spliceosome assembly and splice site selection.³⁹ SR protein phosphorylation is necessary for spliceosome assembly, but dephosphorylation is critical for subsequent steps of the splicing process.⁷ Furthermore, SRPK1 affects the subcellular localization of splicing factors.⁸ Overexpression of SRPK1 results in constitutive phosphorylation of SR proteins, thus inhibiting their dephosphorylation, and possibly subcellular localization, which is required for efficient catalysis of the splicing reaction.⁸ hnRNPs are abundant nuclear RNA-binding proteins also involved in the processing of pre-mRNA.⁴⁰ hnRNPA/B proteins influence splice site selection in vitro and in vivo, and antagonize the effects of SR proteins in regulating alternative splicing.^9,41 Like SR proteins, cellular distribution of hnRNPA/B is controlled by phosphorylation and affects splice site selection.⁴ In addition, the ratio between SR proteins and hnRNPA/B in the nucleus is a critical factor in the control of alternative splicing.⁴ RNA Helicase II/Gu and putative RNA Helicase DDX10 are DEAD-box superfamily members potentially involved in the modification of RNA secondary structure, important for pre-mRNA splicing, translation initiation, ribosome/spliceosome assembly, and mRNA stability.^42,43,44 Finally, SAP130, together with SAP49, -145, and -155 constitute the SF3b U2 snRNP-associated protein complex essential for spliceosome assembly, and may play a critical role at, or near, the catalytic core of the spliceosome.⁴⁵

Overexpression of these pre-mRNA processing genes may be responsible for the altered splicing patterns of the PYK2 gene observed in CML cells, of which SRPK1, RNA Helicase II/Gu, and hnRNPA2/B1 may be particularly important. By modulating the expression of one, or a subset, of these pre-mRNA processing genes in NL or BCR/ABL⁺ cells, it may be possible to elucidate the mechanism by which splicing of the PYK2 gene is modified. However, because an intricate balance of splicing factors is required for specific pre-mRNA splicing patterns on a gene-by-gene basis,^1,2 simply manipulating the expression of one, or a few, pre-mRNA processing genes may not significantly influence alternative splicing in a manner analogous to p210^BCR/ABL.

In addition to increasing the expression of proteins involved in spliceosome assembly and splice site recognition, it is possible that p210^BCR/ABL also alters the expression of proteins that interact with various splicing enhancer or silencing elements that are located within both introns and exons.^1,46 These elements play an intricate role in mediating tissue- and cell-type-specific splicing patterns.^1,4 Identification of the DNA elements and proteins that bind them, which together act to promote or impede exon inclusion, may provide insight into the pathogenesis of CML and numerous other disorders.

Integrin signaling is disrupted in p210^BCR/ABL-positive CD34⁺ cells. We show here that primary CD34⁺ cells from CP CML patients, or p210^BCR/ABL-transduced UCB CD34⁺ cells, have BCR/ABL kinase-dependent alterations in PYK2 splicing, resulting in increased inclusion of exon 23 in the mRNA transcript. Further shifts were observed in PYK2 splicing in CD34⁺ cells from CML patients in blast crisis (data not shown), wherein the ratio of Pyk2 to Pyk2H was further skewed in favor of full-length Pyk2, much like that observed in the erythroid blast crisis-derived cell line K562 (Figure 5B). We have demonstrated elsewhere that Pyk2H is phosphorylated upon integrin engagement in NL CD34⁺ cells, and both Pyk2 and Pyk2H are phosphorylated in primary CML CD34⁺ cells.⁴⁷ Although the two PYK2 isoforms appear functionally redundant when singly overexpressed in primary NL CD34⁺ cells, simple overexpression of either Pyk2 or Pyk2H may not recapitulate the expression profile of Pyk2[H] effecter proteins that cooperate to modulate signaling in BCR/ABL⁺ cells.⁴⁷ In support of this possibility, expression of the Pyk2[H]-interacting proteins Graf, p130^Cas, and Hck are each altered in BCR/ABL⁺ vs BCR/ABL^- Mo7e cells (data not shown), suggesting that modification of Pyk2[H] signaling may, in fact, occur because effecter molecules (eg Graf) differentially associate with the two PYK2 isoforms.⁴⁸ Further studies regarding the effects of Pyk2[H] signaling via its effecter molecules in NL and BCR/ABL⁺ cells are needed to ascertain whether such a hypothesis might be correct.

Changes in the expression of proteins involved in alternative splicing, and concomitant processing of pre-mRNA, suggest that the pathogenic effects of p210^BCR/ABL involve both altered gene expression and RNA editing. BCR/ABL-induced expression of uncharacteristic, or even novel, isoforms of genes that are intimately involved in the regulation of hematopoietic progenitor behavior may, in part, be responsible for the altered HSC proliferation, differentiation, and/or migration inherent to BCR/ABL⁺ cells.

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Acknowledgements

We thank Dr M Garcia-Blanco for valuable comments and Janet Peller, Brad Anderson, and members of the Stem Cell Lab for technical assistance. Additional thanks to Drs B Valdez, B Turner, I Dikic, and Novartis for reagents critical to these studies. This work was supported by grants from the National Institute of Health (RO1 HL-49930 and RO1 DK-53673), Leukemia and Lymphoma Society of America H6377-97; the Tulloch Family Foundation and the McKnight Foundation to CMV, and the Bone Marrow Transplant Research Fund and NCI-awarded Cancer Biology Training Grant (CA09138) to SJD.