[CANCER RESEARCH 60, 2049–2055, April 1, 2000]

BCR-ABL Tyrosine Kinase Activity Regulates the Expression of Multiple GenesImplicated in the Pathogenesis of Chronic Myeloid Leukemia1

Michael W. N. Deininger,2 Sara Vieira, Rezzeline Mendiola, Beate Schultheis, John M. Goldman, and Junia V. Melo3

Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, United Kingdom

ABSTRACT

The BCR-ABL chimeric protein is thought to play a central role in thepathogenesis of Philadelphia (Ph) chromosome-positive leukemias, nota-bly chronic myeloid leukemia (CML). There is compelling evidence thatmalignant transformation by BCR-ABL is critically dependent on itsprotein tyrosine kinase (PTK) activity. As a result, multiple signalingpathways are activated in a kinase-dependent manner, and thus theactivation of such pathways may affect the expression of genes that conferthe malignant phenotype. In this study, we used differential display toinvestigate the alterations of gene expression in BV173, a CML cell linederived from lymphoid blast crisis, after exposure to STI571, whichselectively inhibits ABL PTK activity. We show that the expression of a setof 12 genes is correlated with the kinase activity and that the profile ofthese genes reflects mechanisms implicated in the pathogenesis of CML.Several of the genes show a consistent pattern of altered regulation in allPh-positive lymphoid cell lines, whereas others appear to be unique toBV173 cells. We conclude that BCR-ABL PTK activity drives the expres-sion of specific target genes that contribute to the malignant transforma-tion of Ph-positive cells. The identification of downstream molecules witha consistent regulation pattern may provide suitable targets for therapeu-tic intervention in the future.

INTRODUCTION

Over 90% of cases of CML4 (1) and 10–25% of cases of ALL (2)are characterized by a reciprocal translocation between chromosomes9 and 22. As a result, aBCR-ABLhybrid gene is formed on thederivative Ph chromosome. Depending on the location of the break-point inBCR, three types of fusion protein can be formed, all of whichexhibit deregulated PTK activity compared to normal ABL (2–4). Asa result, there is excessive tyrosine phosphorylation of many intracel-lular proteins including the BCR-ABL protein itself (5, 6). Althoughnot all interactions of BCR-ABL with other proteins are phosphoty-rosine dependent, it is clear from mutational analysis that the PTKactivity is an absolute requirement for malignant transformation, andthat it cannot be complemented by any downstream effector (7, 8). Incontrast, it is, at present, less clear which of the various signalingpathways activated by BCR-ABL (such as RAS (9), Janus-activatedkinase-signal transducers and activators of transcription (10, 11), andphosphatidylinositol 39-kinase (12) is essential for transformation,and, indeed, redundancy is likely.

Basic mechanisms that have been attributed toBCR-ABL-positivecells, particularly in CML, are increased proliferation, increased re-sistance to apoptosis (13–15), and an alteration of their adhesionproperties (16, 17). Given the fact that most of the pathways activatedby BCR-ABL transmit signals to the transcriptional machinery ofthe transformed cell, we reasoned that the phenotype ofBCR-ABL-positive cells should at least partially be explicable on the grounds ofaltered gene expression. In this scenario, BCR-ABL PTK activitywould maintain the activation or suppression of a set of target genesthat confer the malignant phenotype on a Ph-positive cell. Until now,only a few such downstream targets have been identified. Moreover,the data are mostly derived from cell lines transformed to growthfactor independence by ectopic expression ofBCR-ABLrather thanfrom cells that acquired the translocation “naturally.”

To define downstream targets ofBCR-ABL, we studied alterationsin the profile of gene expression in BV173 cells (derived from a CMLpatient in lymphoid blast crisis; Ref. 18) exposed to STI571 (formerlyknown as CGP57148B), a specific inhibitor of BCR-ABL PTK (19–21). DD was used to compare mRNA expression in the cells afterinhibition of BCR-ABLPTK for 24 h with untreated control cells.

We identified 12 differentially regulated mRNAs, 7 of which cor-respond to known genes, and 5 of which correspond to unknowngenes. All known genes are implicated in cellular processes that arethought to be disturbed in Ph-positive leukemias.

MATERIALS AND METHODS

Cell Culture. All cell lines were grown in RPMI 1640 supplemented with10% FCS, penicillin, streptomycin, andL-glutamine. Table 1 provides a list ofthe cell lines used. All experiments were performed on exponentially growingcells with viability of.90% as assessed by trypan blue exclusion. The tyrosinekinase inhibitor STI571 (kindly provided by Dr. Elisabeth Buchdunger; NO-VARTIS, Basel, Switzerland) was added to the culture media at the requiredconcentration. In one experiment, BV173 cells were cultured in the presence ofa blocking antibody to the PDGFRb [M4T9.22 (22); 10mg/ml; a generous giftof Dr. Nick Landolfi (Protein Design Laboratories, Fremont, CA)].

Apoptosis Assay.Analysis of apoptosis in BV173 cells exposed to STI571was done by assessment of DNA fragmentation on agarose gels as describedpreviously (21).

DD. Total RNA was extracted from BV173 cells treated with 1.0mM

STI571 for 24 h using the acid guanidinium thiocyanate method (23). Con-taminating DNA was removed by digestion of 100mg of RNA with RNase-free DNase (Boehringer Mannheim, Mannheim, Germany) in a 200ml reactioncontaining 10 mM Tris (pH 8.3), 5 mM KCl, 1.5 mM MgCl2, and 18ml ofDNase, 2ml of RNase inhibitor (Promega, Southampton, United Kingdom).Single-use aliquots (1mg/ml) of DNA-free RNA were stored at280°C. DD(24) was performed with the RNAimage kit (Gene Hunter, Nashville, TN),following the instructions of the manufacturer, with a few modifications. ThecDNA synthesis was done with Superscript reverse transcriptase (Life Tech-nologies, Inc., Paisley, United Kingdom), and 1ml of RNase inhibitor (Pro-mega) was regularly added to the 40ml reaction. PCR was performed with Taqpolymerase from Boehringer Mannheim in 10ml reactions. Thermocyclingconditions were as recommended by Gene Hunter, except that the extensiontime was increased to 90 s. In several experiments, “long” 21-mer or 18-merprimers (a gift from Dr. Feilan Wang; Dana-Farber Cancer Institute, Boston,MA) were used, and the annealing temperature of the PCR was increased to50°C. Reamplified PCR products were cloned into the TOPO-TA vector

Received 10/29/99; accepted 1/31/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the Leukaemia Research Fund of Great Britain, the Dr.Ernst und Anita Bauer Stiftung, Nurnberg, Germany, the Dr. Mildred Scheel-Stiftung fu¨rKrebsforschung, Germany, and the Fundac¸ao para a Ciencia e Tecnologia (PRAXIS XXIBD/15769/98 Program), Portugal.

2 Present address: Hamatologisches Speziallabor, Medizinische Klinik II, Universita¨tLeipzig, Germany.

3 To whom requests for reprints should be addressed, at Department of Haematology,Imperial College School of Medicine, Hammersmith Hospital, Ducane Road, LondonW12 0NN, United Kingdom. Phone: 44-20-8383-2167; Fax: 44-20-8742-9335; E-mail:j.melo@ic.ac.uk.

4 The abbreviations used are: CML, chronic myeloid leukemia; ALL, acute lympho-blastic leukemia; Ph, Philadelphia; PTK, protein tyrosine kinase; DD, differential display;R-PTP-k, receptor-protein tyrosine phosphatasek; CSCP, chondroitin sulfate core pro-tein; OSM, oncostatin M; OSMRb, OSM receptorb; RT-PCR, reverse transcription-PCR;PDGFRb, platelet-derived growth factor receptor; poly(A), polyadenylic acid.

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(Invitrogen, NV Leek, the Netherlands), according to the instructions of themanufacturer.

Northern Blotting. Fifteen mg of total RNA or 2 mg of poly(A) RNA(isolated directly from cells with the MACS mRNA isolation kit; MiltenyiBiotech, Bergisch Gladbach, Germany) were separated on a 0.8% agarose geland blotted onto nylon membrane (Hybond N; Amersham, Amersham, UnitedKingdom). Hybridizations were carried out at 42°C in 50% formamide, 10%dextran sulfate (Pharmacia, Uppsala, Sweden), 1M NaCl, 1% SDS, and 0.2mg/ml sonicated salmon sperm DNA (Sigma, Paisley, United Kingdom).

Probes were prepared by PCR amplification of the vector inserts fromflanking primers (M13 standard primers), restriction enzyme digestion of theplasmid, or PCR amplification of the target gene with specific primers (se-quences are available on request). The purified probes were labeled with32P(Megaprime; Amersham). Specific activities of more than 53 108 cpm/mgDNA were routinely achieved.

As a control for RNA loading, the blots were stripped and reprobed withb-actin cDNA (kindly provided by Dr. Philip Mason, Imperial College Schoolof Medicine, London, United Kingdom). Densitometric analysis of the auto-radiographs was performed using GELBLOTPRO software (Ultra Violet Prod-ucts, Cambridge, United Kingdom).

Semiquantitative RT-PCR. In several instances, no signal could be ob-tained even on Northern blots prepared from poly(A) RNA after exposure ofthe filters for up to 7 days. In these cases, the cloned PCR products weresequenced, and sequence-specific primers were designed (sequences are avail-able on request). PCR conditions were optimized for each individual fragment.

Amplification of glucose-6 phosphate dehydrogenase (25) served as a controlfor equal amounts of cDNA. The number of cycles was chosen so that thereaction was in its logarithmic phase. PCR products were resolved on anethidium bromide-stained 2% agarose gel and transferred to a nylon membrane(Hybond N; Amersham), as described previously (26). The filter was thenprobed with an internal oligonucleotide.

Rapid Amplification of cDNA Ends-PCR. In one case (oncostatinbreceptor), the 59rapid amplification of cDNA ends-PCR kit (version 2.0; LifeTechnologies, Inc.) was used to extend the initially unknown fragment derivedfrom DD into the published sequence.

Western Blotting for ABL and Phosphotyrosine. Lysates from 2.53 106

cells were prepared as described by Kabarowskiet al. (27). The proteinconcentration was determined with the Dc protein assay (Bio-Rad, Hercules,CA). One hundredmg of protein were separated on 6% SDS-PAGE gels,blotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA),and probed with an anti-phosphotyrosine antibody (PY99; Santa Cruz Bio-technology, Santa Cruz, CA). Bands were visualized by enhanced chemilumi-nescence (Amersham) and subsequently quantified by densitometry. To con-trol for equal protein loading, the blots were stained with Coomassie Blueand/or stripped and reprobed with anti-ABL antibody (Abl-3; Calbiochem,Nottingham, United Kingdom). All experiments were done at least twice, withsimilar results.

RESULTS

Establishment of Experimental Conditions. We have previouslyshown that STI571 inhibits the proliferation of BV173 cells in adose-dependent manner (21). Analysis of phosphotyrosine blotsshowed that STI571 reduced BCR-ABL autophosphorylation withIC50 and IC90 values of approximately 0.25 and 1.0mM, respectively(Fig. 1). Little DNA laddering above background levels was detect-able in BV173 cells incubated with STI571 for 24 h, whereas exten-sive laddering was seen after 48 h (Fig. 2). The cultures could berescued after up to 36 h of exposure if STI571 was thoroughly washedout, whereas longer exposure times invariably led to cell death. Basedon these observations, we chose an exposure time of 24 h to avoiddetection of changes of gene expression associated with the generalmetabolic breakdown that accompanies the final stages of apoptosis.To establish the optimal dose of STI571, a series of pilot experimentswas performed that showed no appreciable difference in the pattern ofbands obtained by DD between cells incubated with 0.25 and 1.0mM

Fig. 1.Top panels, tyrosine phosphorylation of BCR-ABL (arrow) at graded concentrations of STI571.Middle panels, To control for equal protein loading, the blots were strippedand reprobed with an anti-ABL antibody or stained with Coomassie Blue.Bottom panels, densitometric analysis of the BCR-ABL phosphotyrosine bands, and IC50. Analogousexperiments were performed in TOM1 and ALL/MIK cells (Table 3). Attempts to quantify the phosphorylation of P180BCR-ABL in MY cells were not successful.

Table 1 Phenotype and expression of BCR-ABL in the cell lines used in this study

Cell linea Phenotype PBCR-ABLReference

no.

BV173 Pre-B 210 18NALM1 Pre-B 210 57TOM1 Pre-B 190 58ALL/MIK Pre-B 190 59MY Biphenotypic (myeloid/B-lymphoid) 180b 60SD1 B-lymphoblastoid 190 30CML-T1 T-lymphoid 210 61697 Pre-B 62REH Pre-B 63Jurkat T 64a These cell lines were purchased from cell repository banks (American Type Culture

Collection, Rockville, Maryland; European Collection of Cell Cultures, Winchester,United Kingdom; or German Collection of Microorganisms and Cell Cultures, Braun-schweig, Germany) or kindly donated by the originators.

b MY cells have BCR-ABL transcripts with an atypical e1a3 junction, encoding aslightly smaller fusion protein ofMr 180,000.

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STI571, respectively. Therefore, 1.0mM (corresponding to the IC90

for inhibition of BCR-ABL tyrosine phosphorylation in BV173 cells)was chosen for the experiments. This is also the concentration thatdiscriminates maximally between CML and normal progenitor cells inclonogenic assays (21).

Differentially Expressed Genes.An estimated 8000 bands weredisplayed from RT-PCR amplifications with 114 primer combina-tions. The regulation pattern seen on the DD gels was confirmed for12 cDNA fragments. In nine cases, this was done by Northern blotanalysis (Fig. 3). Three fragments gave no signal at all, even afterhybridization to poly(A)-selected RNA and prolonged exposure of theautoradiographs. For these fragments, sequence-specific primers weredesigned, and the expression was analyzed by semiquantitative RT-PCR. In all three cases, the regulation pattern expected from theoriginal DD gel (data not shown) was confirmed (Fig. 4). Of the 12differentially expressed mRNAs, 7 represent known genes, and 5 arenovel sequences (Table 2). Known genes found to be up-regulated oninhibition of BCR-ABLPTK wereMPP1 (28), BCL-6, andR-PTPk(29). Genes whose expression decreased wereintegrin a6, cyclin D2,CSCP, andOSMRb. Four of the five novel sequences correspond toexpressed sequence tags, whereas one (DD-W) does not match anysequence in public databases. These genes are currently under inves-tigation.

Time Course Experiments.Time course experiments showed thatthe up- or down-regulation of the differentially expressed genesoccurred after various times of treatment with STI571 (Fig. 5). CyclinD2, integrin a6, and OSMRb were strongly down-regulated within3 h, and BCL-6 was strongly up-regulated within 6 h, and their levelof expression did not change significantly thereafter. In the case of theother genes, the up- or down-regulation followed a more protractedcourse. However, a change in the level of expression was usuallyevident after 3 h. Given the limitations of semiquantitative PCR, no

attempt was made to quantify the expression of the respective genesover time.

Comparison with other Cell Lines. We then asked whether thepattern of regulation seen in BV173 cells would be reproducible inother Ph-positive cell lines and, conversely, be absent in Ph-negativelines. Generally, the regulation pattern could not be reproduced inPh-positive myeloid cell lines (Fig. 6). The expression of the genes inquestion was either below the threshold of sensitivity of total RNANorthern blots or unaffected by STI571 (data not shown). In contrast,in many cases, the pattern observed in BV173 cells was reproduciblein other Ph-positive lymphoblastic cell lines (Table 1), with thenotable exception of SD1. This cell line is unique because it isEBV-transformed (30). In particular, cyclin D2 was consistentlydown-regulated and the unknown gene DD221 was consistently up-regulated in all Ph-positive lymphoblastic cell lines treated withSTI571. Similarly, there was a consistent pattern of up-regulation forBCL-6, although the extent was variable, and the gene is not ex-pressed in all of the cell lines. Integrina6 was down-regulated in allEBV-negative B-cell lines, whereas its expression seemed to increasein CML-T1 cells on treatment with STI571. The level of expressionwas below the threshold of detection in TOM1 cells. Up-regulation ofMPP1 and DDI was observed in the two B-cell lines (BV173 andNALM1) that originated from CML in blast crisis rather than fromALL. In some cases (OSMRb, CSCP, and DDQ), the expression ofthe mRNA in question appeared to be restricted to BV173 cells at the

Fig. 2. Analysis of high molecular weight DNA from BV173 cells treated with STI571for the indicated times.

Fig. 3. Northern blot analysis of BV173 cells treated with STI571 for 24 h. The blots were probed with (a) MPP1, (b) CSCP, (c) integrina6, (d) cyclin D2, (e) BCL-6, (f) DDI,(g) DD221, (h) DDQ, and (i) OSMRb. The b-actin control hybridization is shown on thebottom panelof each blot.

Fig. 4. Semiquantitative RT-PCR was performed on (a) untreated BV173 cells and (b)cells incubated with 1mM STI571 for 24 h. The cDNAs for R-PTPk, DDM, DDW, andglucose-6 phosphate dehydrogenase (G-6PD; control gene) were PCR-amplified withincreasing numbers of cycles (Lanes 1, lowest number of cycles;Lanes 2, medium numberof cycles;Lanes 3, highest number of cycles). The PCR products were resolved on anagarose gel and transferred to a nylon membrane. The autoradiographs shown wereobtained after the filters were probed with an internal oligonucleotide.

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given level of sensitivity. Importantly, none of the changes wasdetectable in any of the Ph-negative cell lines exposed to STI571.

R-PTPk, HRF-5, and DD-W showed highly variable levels ofexpression in cell lines other then BV173, although they were detect-able in all cases under optimal conditions for RT-PCR amplification.Thus, given the limitations of semiquantitative PCR, no attempt wasmade at quantifying their expression in cell lines other than BV173.

To exclude the possibility that insufficient inhibition of BCR-ABLtyrosine kinase activity was responsible for the differences betweenthe lymphoid cell lines, we prepared phosphotyrosine blots and de-termined the respective IC50 values (Fig. 1; Table 3). With theexception of CML-T1 cells, tyrosine phosphorylation of BCR-ABLwas reduced in a dose-dependent manner in all cell lines, although theIC50 values differed slightly from cell line to cell line.

DISCUSSION

Intensive research during the past decade has elucidated many ofthe signal transduction pathways activated by BCR-ABL. In contrast,few data are available that addressBCR-ABL-dependent gene expres-sion. Genes reported to be overexpressed in v-abl- or BCR-ABL-positive cells includeMYC (31), BCL-2 (32), the melanoma-related

antigenPRAME(33), and the RAS-like geneKIR (34). A more recentstudy compared the gene expression profile in Mo7 cells transfectedwith a P210BCR-ABL expression vector using DNA arrays and sug-gested differential expression of multiple genes (35). Confirmation byNorthern analysis has not yet been presented.

We show that inhibition of BCR-ABL tyrosine kinase by a specificinhibitor (STI571) alters the expression of multiple genes in BV173and in other lymphoid cell lines. Hence, these genes can be consideredas downstream targets ofBCR-ABL. We chose BV173 cells for theinitial screening for several reasons: (a) they do not express normalABL, which excludes effects related to ABL PTK (36); (b) theyexhibit a dose-dependent growth inhibition on exposure to STI571that is correlated to inhibition of BCR-ABL autophosphorylation; and(c) unlike other CML cell lines, they readily engraft in SCID-NODmice (37), suggesting that they may be closer to the clinical disease.

We found 12 genes (7 previously known genes and 5 unknowngenes) whose expression is dependent on BCR-ABL PTK activity inBV173 cells. All known genes reflect pathogenetic principles thoughtto be relevant in CML.

Integrin a6 (VLA-6), CSCP, andMPP1 are genes involved in theorganization of the cell membrane and its adhesion properties. It istherefore conceivable that their aberrant regulation contributes to thewell-described adhesion defect of CML cells (38). Abnormal function

Fig. 5. Northern blot analysis of BV173 cells treated with 1mM STI571 for theindicated times. The blots were probed with (a) MPP1, (b) CSCP, (c) integrina6, (d)cyclin D2, (e) BCL-6, (f) DDI, (g) DD221, (h) DDQ, (i) OSMRb, and (j)b-actin (control).

Fig. 6. Northern blot analysis of lymphoid cell lines treated with 1mM STI571 for 24 h.The blots were probed with (a) MPP1, (b) CSCP, (c) integrina6, (d) cyclin D2, (e) BCL-6,(f) DDI, (g) DD221, (h) DDQ, (i) OSMRb, and (j) b-actin (control).

Table 2 Genes differentially expressed in BV173 cells after a 24-h exposure to 1mM

ST1571

Gene Pattern of regulationa Fold regulation

MPP1 1 4–8Integrin a6 2 .20CSCP 2 10–15Cyclin D2 2 .20BCL-6 1 .20OSMRb 2 .20R-PTPk 1 RT-PCRDDM 1 RT-PCRDD221 1 5–10DDI 1 4–6DDQ 2 .20DDW 1 RT-PCR

a1, up-regulated;2, down-regulated.

Table 3 IC50 values for inhibition of BCR-ABL tyrosine phosphorylation by STI571 inthe Ph-positive cell lines

Cell line IC50 (mM STI571)

BV173 0.25NALM1 0.5TOM1 0.5MIK/ALL 0.3SD1 0.5CML-T1 .10

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and expression ofa integrins inBCR-ABL-positive cells have beenreported (16, 35, 39). Similarly, adhesion of hematopoietic progenitorcells to fibronectin appears to require the coordinated function ofchondroitin sulfate proteoglycans, CD44 anda4b integrins (40, 41). Itis thus conceivable thatBCR-ABLperturbs the ordered expression ofCSCP with resulting alterations in the composition of proteoglycans,which, in turn, may impact on homing and adhesion to stroma.Surprisingly, we failed to detect a CSCP message of identical size tothat in BV173 cells in any of the other cell lines tested, and we arecurrently investigating whether this message represents a splice var-iant that is peculiar to BV173 cells. The interaction of MPP1 withprotein 4.1 and glycophorin C is vital for the stability of the erythro-cyte membrane, but the wide expression of glycophorin C in noner-ythroid cells indicates that the system may have additional functions.With respect to CML, it is noteworthy that talin, a cytoskeletal proteinrelated to protein 4.1, is tyrosine-phosphorylated by BCR-ABL (42)and that abnormal processing of glycophorin C has been demonstratedin leukemic cells (43). Moreover, loss of MPP1 exon 5 due to exonskipping has recently been described in a patient with CML in blastcrisis (44). Thus, repression of MPP1 may be yet another way bywhich BCR-ABL PTK activity alters the properties of the cell mem-brane.

BCL-6 functions as a transcriptional repressor (45, 46) and isessential for germinal center formation (47). It is absent in B cellsbefore and after germinal center formation (45). The up-regulationobserved on inhibition of BCR-ABL PTK could indicate that BCR-ABL inhibits the expression of BCL-6, which, in turn, prevents theexecution of a differentiation program at a stage prior to germinalcenter formation.

Cyclin D2andD3 mediate the G1-S-phase transition in hematopoi-etic cells and, if overexpressed, allow for G1-S-phase progressionunder conditions of growth-factor deprivation (48). Thus, it is likelythat BCR-ABL provides a mitogenic signal that results in overexpres-sion of cyclin D2 and facilitates the G1-S-phase transition. Our ob-servation is in line with a recent report that showed cooperationbetween cyclin D1 and BCR-ABL in the transformation of fibroblastsand murine B cells (49).

OSMRb. The BCR-ABL-dependent expression of the OSMRbappears to be limited to BV173 cells, at least at the given level ofsensitivity. OSMRb is the high-affinity receptor for OSM, a cytokinewith pleiotropic biological activities. The biological effects of OSMdepend on the cellular context: it inhibits the proliferation of manycancer cell lines (50) but can also act as a potent growth factor,e.g.,for the spindle cells in AIDS-related Kaposi’s sarcoma (51). BecauseOSM mRNA is detectable in BV173 cells by RT-PCR (data notshown), it is possible that an OSM autocrine loop exists in BV173cells that is maintained by the BCR-ABL-induced expression ofOSMRb.

R-PTPk. The role of protein tyrosine phosphatases such as PTP1B(52), SYP (53), SHP-2 (54), and SHPTP1 (55) in the transformationby BCR-ABL is complex. For example, PTB1B expression is inducedby BCR-ABL PTK activity, and it has been suggested that this mayrepresent a compensatory mechanism by which the cell attempts tolimit the effects of BCR-ABL. Down-regulation of R-PTPk in BV173cells would be expected to increase rather than decrease the impact ofBCR-ABL by reducing the activity of a potential antagonist. We arecurrently investigating this possibility.

It is interesting to note that not all genes show a consistent patternof up- or down-regulation within the lymphoid cell lines used in thisstudy. Even phenotypically very similar cell lines such as BV173 andNALM1 show significant differences that cannot be explained byvariations in IC50 values among the individual cell lines. In somecases (OSMRb, DDQ, and CSCP), the expression of the gene in

question appears to be restricted to BV173 cells, at least at the givenlevel of sensitivity. Thus, the set of genes regulated by BCR-ABL ispartly dependent on the cell line studied. This is most strikinglyillustrated by the EBV-transformed SD1 cell line that expresses alargely different set of genes and is very resistant to STI571. More-over, the results were generally not reproducible in Ph-positive my-eloid cell lines. It should be noted that the difference in BCR-ABL-regulated gene expression profile appears to be dependent on theactual cell lineage (lymphoidversusmyeloid) rather than the type ofBCR-ABL fusion protein (i.e., p210versusp190) because both p210-and p190-producing cell lines were included in the lymphoid collec-tion examined. Taken together, our data underline the importance ofthe cellular context in the pathogenesis of Ph-positive leukemias. Onemight speculate that this is also reflected in the diverse clinicalbehavior of individual cases of CML and Ph-positive ALL.

The issue of specificity is of great importance in any study thatinvestigates biological changes induced by a chemical agent. STI571is known to inhibit the tyrosine kinases of KIT and PDGFb receptorsat concentrations comparable to ABL PTK. In our system, effectsrelated to inhibition of ABL can be excluded because it is notexpressed in the BV173 cell line. Effects of STI571 related to inhi-bition of PDGFRb PTK can be ruled out because blockage of thePDGFRb with a specific antibody (22) did not induce any of thechanges seen on incubation with STI571 (Fig. 7). In contrast, wecannot exclude effects due to inhibition of KIT because the latter maybe activated by BCR-ABL in a tyrosine kinase-dependent manner, atleast in myeloid cells (56). Similarly, potential effects of STI571 onother kinases downstream of BCR-ABL cannot be excluded withcertainty because not all possible candidates have been tested (19, 20).On the other hand, nonspecific effects not related to the expression ofBCR-ABL are unlikely because none of the effects was seen inPh-negative cell lines of similar phenotype.

Bearing these caveats in mind, our approach has two advantagesover systems that use ectopic expression of BCR-ABL in factor-dependent cell lines: (a)BCR-ABL is expressed from its naturalpromoter at physiological levels; and (b) cell transformation to growthfactor independence does not reflect all aspects ofBCR-ABL-mediatedmalignant transformation. It will be important to investigate whetherthe gene expression changes identified in this study are also observedin primary CML or Ph-positive ALL. Preliminary results of compet-itive RT-PCR tests suggest that expression of theMPP1 gene, forexample, follows an up-regulation pattern on BCR-ABL inhibition inCML CD341 progenitors similar to that found in BV173 cells (datanot shown). Detailed investigations on the functional significance ofthe changes detected in each gene are currently in progress.

Fig. 7. Northern blot analysis of untreated BV173 cells (2) and cells incubated with10 mg/ml of a blocking antibody to the PDGFRb for 24 h. The blots were probed with (a)MPP1, (b) CSCP, (c) integrina6, (d) cyclin D2, (e) OSMRb, (f) DD221, (g) DDQ, and(h) b-actin (control). BCL-6 and DDI were also tested, and no signal was detected intreated or untreated cells (data not shown).

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In summary, our results demonstrate that BCR-ABL PTK activityregulates the expression of specific genes that partly explain theabnormal biological behavior of Ph-positive cells. They further indi-cate that genes hitherto not implicated in the pathogenesis of Ph-positive leukemias could be important in the transformation processand may constitute targets for future therapeutic intervention.

REFERENCES

1. Cortes, J. E., Talpaz, M., Beran, M., O’Brien, S. M., Rios, M. B., Stass, S., andKantarjian, H. M. Philadelphia chromosome-negative chronic myelogenous leukemiawith rearrangement of the breakpoint cluster region. Long-term follow-up results.Cancer,75: 464–470, 1995.

2. Clark, S. S., McLaughlin, J., Timmons, M., Pendergast, A. M., Ben Neriah, Y., Dow,L. W., Crist, W., Rovera, G., Smith, S. D., and Witte, O. N. Expression of a distinctiveBCR-ABL oncogene in Ph1-positive acute lymphocytic leukemia (ALL). Science(Washington DC),239: 775–777, 1988.

3. Konopka, J. B., Watanabe, S. M., and Witte, O. N. An alteration of the human c-ablprotein in K562 leukemia cells unmasks associated tyrosine kinase activity. Cell,37:1035–1042, 1984.

4. Quackenbush, R. C., Reuther, G. W., and Pendergast, A. M. Cloning and character-ization of the chronic neutrophilic leukemia-associated P230 BCR/ABL oncogene.Blood, 90 (Suppl.1):247a, 1997.

5. Laneuville, P. Abl tyrosine protein kinase. Semin. Immunol.,7: 255–266, 1995.6. Liu, J., Campbell, M., Guo, J. Q., Lu, D., Xian, Y. M., Andersson, B. S., and

Arlinghaus, R. B. BCR-ABL tyrosine kinase is autophosphorylated or transphospho-rylates P160 BCR on tyrosine predominantly within the first BCR exon. Oncogene,8: 101–109, 1993.

7. Lugo, T. G., Pendergast, A. M., Muller, A. J., and Witte, O. N. Tyrosine kinaseactivity and transformation potency of bcr-abl oncogene products. Science (Wash-ington DC),247: 1079–1082, 1990.

8. Cortez, D., Kadlec, L., and Pendergast, A. M. Structural and signaling requirementsfor BCR-ABL-mediated transformation and inhibition of apoptosis. Mol. Cell. Biol.,15: 5531–5541, 1995.

9. Pendergast, A. M., Quilliam, L. A., Cripe, L. D., Bassing, C. H., Dai, Z., Li, N.,Batzer, A., Rabun, K. M., Der, C. J., Schlessinger, J., and Gishizky, M. L. BCR-ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain ofthe GRB-2 adaptor protein. Cell,75: 175–185, 1993.

10. Ilaria, R. L., Jr., and Van Etten, R. A. P210 and P190(BCR/ABL) induce the tyrosinephosphorylation and DNA binding activity of multiple specific STAT family mem-bers. J. Biol. Chem.,271: 31704–31710, 1996.

11. Carlesso, N., Frank, D. A., and Griffin, J. D. Tyrosyl phosphorylation and DNAbinding activity of signal transducers and activators of transcription (STAT) proteinsin hematopoietic cell lines transformed by Bcr/Abl. J. Exp. Med.,183: 811–820,1996.

12. Skorski, T., Kanakaraj, P., Nieborowska Skorska, M., Ratajczak, M. Z., Wen, S. C.,Zon, G., Gewirtz, A. M., Perussia, B., and Calabretta, B. Phosphatidylinositol-3kinase activity is regulated by BCR/ABL and is required for the growth of Philadel-phia chromosome-positive cells. Blood,86: 726–736, 1995.

13. Bedi, A., Zehnbauer, B. A., Barber, J. P., Sharkis, S. J., and Jones, R. J. Inhibition ofapoptosis by BCR-ABL in chronic myeloid leukemia. Blood,83: 2038–2044, 1994.

14. Cambier, N., Chopra, R., Strasser, A., Metcalf, D., and Elefanty, A. G. BCR-ABLactivates pathways mediating cytokine independence and protection against apoptosisin murine hematopoietic cells in a dose-dependent manner. Oncogene,16: 335–348,1998.

15. Cortez, D., Stoica, G., Pierce, J. H., and Pendergast, A. M. The BCR-ABL tyrosinekinase inhibits apoptosis by activating a Ras-dependent signaling pathway. Oncogene,13: 2589–2594, 1996.

16. Bazzoni, G., Carlesso, N., Griffin, J. D., and Hemler, M. E. Bcr/Abl expressionstimulates integrin function in hematopoietic cell lines. J. Clin. Investig.,98: 521–528, 1996.

17. Zhao, R. C., Tarone, G., and Verfaillie, C. M. Presence of the adhesion inhibitoryb1Bintegrin isoform on CML but not normal progenitors is at least in part responsible forthe decreased CML progenitor adhesion. Blood,90 (Suppl.1):393a, 1997.

18. Pegoraro, L., Matera, L., Ritz, J., Levis, A., Palumbo, A., and Biagini, G. Establish-ment of a Ph1-positive human cell line (BV173). J. Natl. Cancer Inst.,70: 447–453,1983.

19. Buchdunger, E., Zimmermann, J., Mett, H., Meyer, T., Muller, M., Druker, B. J., andLydon, N. B. Inhibition of the Abl protein-tyrosine kinasein vitro and in vivo by a2-phenylaminopyrimidine derivative. Cancer Res.,56: 100–104, 1996.

20. Druker, B. J., Tamura, S., Buchdunger, E., Ohno, S., Segal, G. M., Fanning, S.,Zimmermann, J., and Lydon, N. B. Effects of a selective inhibitor of the Abl tyrosinekinase on the growth of Bcr-Abl positive cells. Nat. Med.,2: 561–566, 1996.

21. Deininger, M., Goldman, J. M., Lydon, N. B., and Melo, J. V. The tyrosine kinaseinhibitor CGP57148B selectively inhibits the growth of BCR-ABL positive cells.Blood, 90: 3691–3698, 1997.

22. Shulman, T., Sauer, F. G., Jackman, R. M., Chang, C. N., and Landolfi, N. F. Anantibody reactive with domain 4 of the platelet-derived growth factorb receptorallows BB binding while inhibiting proliferation by impairing receptor dimerization.J. Biol. Chem.,272: 17400–17404, 1997.

23. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem.,162: 156–159, 1987.

24. Liang, P., and Pardee, A. B. Differential display of eukaryotic messenger RNA bymeans of the polymerase chain reaction. Science (Washington DC),257: 967–971,1992.

25. Hochhaus, A., Lin, F., Reiter, A., Skladny, H., Mason, P. J., van Rhee, F., Shepherd,P. C., Allan, N. C., Hehlmann, R., Goldman, J. M., and Cross, N. C. Quantificationof residual disease in chronic myelogenous leukemia patients on interferon-a therapyby competitive polymerase chain reaction. Blood,87: 1549–1555, 1996.

26. Hochhaus, A., Yan, X. H., Willer, A., Hehlmann, R., Gordon, M. Y., Goldman, J. M.,and Melo, J. V. Expression of interferon regulatory factor (IRF) genes and responseto interferon-a in chronic myeloid leukaemia. Leukemia,11: 933–939, 1997.

27. Kabarowski, J. H., Allen, P. B., and Wiedemann, L. M. A temperature sensitive p210BCR-ABL mutant defines the primary consequences of BCR-ABL tyrosine kinaseexpression in growth factor dependent cells. EMBO J.,13: 5887–5895, 1994.

28. Kim, A. C., Metzenberg, A. B., Sahr, K. E., Marfatia, S. M., and Chishti, A. H.Complete genomic organization of the human erythroid p55 gene (MPP1), a mem-brane-associated guanylate kinase homologue. Genomics,31: 223–229, 1996.

29. Yang, Y., Gil, M. C., Choi, E. Y., Park, S. H., Pyun, K. H., and Ha, H. Molecularcloning and chromosomal localization of a human gene homologous to the murineR-PTP-k, a receptor-type protein tyrosine phosphatase. Gene (Amst.),186: 77–82,1997.

30. Dhut, S., Gibbons, B., Chaplin, T., and Young, B. D. Establishment of a lympho-blastoid cell line, SD-1, expressing the p190 bcr-abl chimeric protein. Leukemia,5:49–55, 1991.

31. Cleveland, J. L., Dean, M., Rosenberg, N., Wang, J. Y., and Rapp, U. R. Tyrosinekinase oncogenes abrogate interleukin-3 dependence of murine myeloid cells throughsignaling pathways involving c-myc: conditional regulation of c-myc transcription bytemperature-sensitive v-abl. Mol. Cell. Biol.,9: 5685–5695, 1989.

32. Sanchez Garcia, I., and Martin Zanca, D. Regulation of Bcl-2 gene expression byBCR-ABL is mediated by Ras. J. Mol. Biol.,267: 225–228, 1997.

33. Watari, K., Tojo, A., Nagamura-Inoue, T., Tsujimura, H., Nagamura, F., Tani, K., andAsano, S. A new melanoma antigen, PRAME, is identified as a BCR/ABL-induciblegene in TF-1 cells. Blood,90 (Suppl.1):197a, 1997.

34. Cohen, L., Mohr, R., Chen, Y. Y., Huang, M., Kato, R., Dorin, D., Tamanoi, F., Goga,A., Afar, D., Rosenberg, N., and Witte, O. Transcriptional activation of a ras-like gene(kir) by oncogenic tyrosine kinases. Proc. Natl. Acad. Sci. USA,91: 12448–12452,1994.

35. Xie, Z., Chang, S., McQueen, T., and Andreeff, M. Effect of BCR/ABL transfectionon the gene expression profile of human hematopoietic MO7e cells: analysis by DNAexpression array. Blood,92 (Suppl.1):91a, 1998.

36. Melo, J. V., Gordon, D. E., Cross, N. C., and Goldman, J. M. The ABL-BCR fusiongene is expressed in chronic myeloid leukemia. Blood,81: 158–165, 1993.

37. Dazzi, F., Capelli, D., Hasserjian, R., Cotter, F., Corbo, M., Poletti, A., Chinswang-watanakul, W., Goldman, J. M., and Gordon, M. Y. The kinetics and extent ofengraftment of chronic myelogenous leukemia cells in non-obese diabetic/severecombined immunodeficiency mice reflect the phase of the donor’s disease: anin vivomodel of chronic myelogenous leukemia biology. Blood,92: 1390–1396, 1998.

38. Bhatia, R., Wayner, E. A., McGlave, P. B., and Verfaillie, C. M. Interferon-a restoresnormal adhesion of chronic myelogenous leukemia hematopoietic progenitors to bonemarrow stroma by correcting impairedb1 integrin receptor function. J. Clin. Inves-tig., 94: 384–391, 1994.

39. Verfaillie, C. M., McCarthy, J. B., and McGlave, P. B. Mechanisms underlyingabnormal trafficking of malignant progenitors in chronic myelogenous leukemia.Decreased adhesion to stroma and fibronectin but increased adhesion to the basementmembrane components laminin and collagen type IV. J. Clin. Investig.,90: 1232–1241, 1992.

40. Verfaillie, C. M., Benis, A., Iida, J., McGlave, P. B., and McCarthy, J. B. Adhesionof committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain of fibronectin: cooperation between the integrina4b1 and the CD44 adhesion receptor. Blood,84: 1802–1811, 1994.

41. Lundell, B. I., McCarthy, J. B., Kovach, N. L., and Verfaillie, C. M. Activation ofb1

integrins on CML progenitors reveals cooperation betweenb1 integrins and CD44 inthe regulation of adhesion and proliferation. Leukemia,11: 822–829, 1997.

42. Salgia, R., Brunkhorst, B., Pisick, E., Li, J. L., Lo, S. H., Chen, L. B., and Griffin,J. D. Increased tyrosine phosphorylation of focal adhesion proteins in myeloid celllines expressing p210BCR/ABL. Oncogene,11: 1149–1155, 1995.

43. Villeval, J. L., Le Van Kim, C., Bettaieb, A., Debili, N., Colin, Y., el Maliki, B.,Blanchard, D., Vainchenker, W., and Cartron, J. P. Early expression of glycophorinC during normal and leukemic human erythroid differentiation. Cancer Res.,49:2626–2632, 1989.

44. Ruff, P., Chishti, A. H., Grimm, E., Bischoff, D., and Kim, A. C. Exon skippingtruncates the PDZ domain of human erythroid p55 in a patient with chronic myeloidleukemia in acute megakaryoblastic blast crisis. Leuk. Res.,23: 247–250, 1999.

45. Chang, C. C., Ye, B. H., Chaganti, R. S., and Dalla, F. R. BCL-6, a POZ/zinc-fingerprotein, is a sequence-specific transcriptional repressor. Proc. Natl. Acad. Sci. USA,93: 6947–6952, 1996.

46. Seyfert, V. L., Allman, D., He, Y., and Staudt, L. M. Transcriptional repression by theproto-oncogene BCL-6. Oncogene,12: 2331–2342, 1996.

47. Ye, B. H., Cattoretti, G., Shen, Q., Zhang, J., Hawe, N., de, W. R., Leung, C., Nouri,S. M., Orazi, A., Chaganti, R. S., Rothman, P., Stall, A. M., Pandolfi, P. P., and Dalla,F. R. The BCL-6 proto-oncogene controls germinal-centre formation and Th2-typeinflammation. Nat. Genet.,16: 161–170, 1997.

48. Ando, K., Ajchenbaum Cymbalista, F., and Griffin, J. D. Regulation of G1/S transi-tion by cyclins D2 and D3 in hematopoietic cells. Proc. Natl. Acad. Sci. USA,90:9571–9575, 1993.

49. Afar, D. E., McLaughlin, J., Sherr, C. J., Witte, O. N., and Roussel, M. F. Signaling byABL oncogenes through cyclin D1. Proc. Natl. Acad. Sci. USA,92: 9540–9544, 1995.

2054

BCR-ABL-DEPENDENT GENE EXPRESSION

Research. on February 9, 2021. © 2000 American Association for Cancercancerres.aacrjournals.org Downloaded from

50. Horn, D., Fitzpatrick, W. C., Gompper, P. T., Ochs, V., Bolton, H. M., Zarling, J.,Malik, N., Todaro, G. J., and Linsley, P. S. Regulation of cell growth by recombinantoncostatin M. Growth Factors,2: 157–165, 1990.

51. Miles, S. A., Martinez, M. O., Rezai, A., Magpantay, L., Kishimoto, T.,Nakamura, S., Radka, S. F., and Linsley, P. S. Oncostatin M as a potent mitogenfor AIDS-Kaposi’s sarcoma-derived cells. Science (Washington DC),255: 1432–1434, 1992.

52. LaMontagne, K. R., Jr., Flint, A. J., Franza, B. R., Jr., Pandergast, A. M., and Tonks,N. K. Protein tyrosine phosphatase 1B antagonizes signalling by oncoprotein tyrosinekinase p210 bcr-ablin vivo. Mol. Cell. Biol.,18: 2965–2975, 1998.

53. Tauchi, T., Feng, G. S., Shen, R., Song, H. Y., Donner, D., Pawson, T., andBroxmeyer, H. E. SH2-containing phosphotyrosine phosphatase Syp is a target ofp210bcr-abl tyrosine kinase. J. Biol. Chem.,269: 15381–15387, 1994.

54. Sattler, M., Salgia, R., Shrikhande, G., Verma, S., Choi, J-L., Rohrschneider, L. R.,and Griffin, J. D. The phosphatidylinositol polyphosphate 5-phosphatase SHIP andthe protein tyrosine phosphatase SHP-2 form a complex in hematopoietic cells whichcan be regulated by BCR/ABL and growth factors. Oncogene,15: 2379–2384, 1995.

55. Liedtke, M., Pandey, P., Kumar, S., Kharbanda, S., and Kufe, D. Regulation ofBCR-ABL-induced SAP kinase activity and transformation by the SHPTP1 proteintyrosine kinase. Oncogene,17: 1889–1892, 1998.

56. Hallek, M., Danhauser Riedl, S., Herbst, R., Warmuth, M., Winkler, A., Kolb, H. J.,Druker, B., Griffin, J. D., Emmerich, B., and Ullrich, A. Interaction of the receptortyrosine kinase p145c-kit with the p210bcr/abl kinase in myeloid cells. Br. J. Haema-tol., 94: 5–16, 1996.

57. Minowada, J., Tsubota, T., Greaves, M. F., and Walters, T. R. A non-T, non-B humanleukemia cell line (NALM-1): establishment of the cell line and presence of leukemia-associated antigens. J. Natl. Cancer Inst.,59: 83–87, 1977.

58. Okabe, M., Matsushima, S., Morioka, M., Kobayashi, M., Abe, S., Sakurada, K.,Kakinuma, M., and Miyazaki, T. Establishment and characterization of a cell line,TOM-1, derived from a patient with Philadelphia chromosome-positive acute lym-phocytic leukemia. Blood,69: 990–998, 1987.

59. Higa, T., Okabe, M., Kunieda, Y., Kodama, S., Itaya, T., Kurosawa, M., Sakurada, K.,Maekawa, I., Shoji, M., Kasai, M., and Miyasaki, T. Establishment and character-ization of a new Ph1-positive ALL cell line (ALL/MIK) presentingbcr gene rear-rangement on bcr-2 and ALL-type bcr/abl transcript: suggestion ofin vitro differen-tiation to monocytoid lineage. Leuk. Lymphoma,12: 287–296, 1994.

60. Inokuchi, K., Shinohara, T., Futaki, M., Hanawa, H., Tanosaki, S., Yamaguchi,H., Nomura, T., and Dan, K. Establishment of a cell line with variant BCR/ABLbreakpoint expressing P180BCR/ABL from late-appearing Philadelphia-positiveacute biphenotypic leukemia. Genes Chromosomes Cancer,23: 227–238, 1998.

61. Kuriyama, K., Gale, R. P., Tomonaga, M., Ikeda, S., Yao, E., Klisak, I., Whelan, K.,Yakir, H., Ichimaru, M., Sparkes, R. S., and Dreazer, O. CML-T1: a cell line derivedfrom T-lymphocyte acute phase of chronic myelogenous leukemia. Blood,74: 1381–1387, 1989.

62. Findley, H. W., Jr., Cooper, M. D., Kim, T. H., Alvarado, C., and Ragab, A. H. Twonew acute lymphoblastic leukemia cell lines with early B-cell phenotypes. Blood,60:1305–1309, 1982.

63. Rosenfeld, C., Goutner, A., Choquet, C., Venuat, A. M., Kayibanda, B., Pico, J. L.,and Greaves, M. F. Phenotypic characterisation of a unique non-T, non-B acutelymphoblastic leukaemia cell line. Nature,267: 841–843, 1977.

64. Schneider, U., and Schwenk, H. U. Characterization of “T” and “non-T” cell linesestablished from children with acute lymphoblastic leukemia and non-Hodgkinlymphoma after leukemic transformation. Hamatol. Bluttransfus.,20: 265–269,1977.

2055

BCR-ABL-DEPENDENT GENE EXPRESSION

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2000;60:2049-2055. Cancer Res   Michael W. N. Deininger, Sara Vieira, Rezzeline Mendiola, et al.   Myeloid Leukemia of Multiple Genes Implicated in the Pathogenesis of Chronic

Tyrosine Kinase Activity Regulates the ExpressionBCR-ABL

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