Blood-1993-Melo-158-65

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1993 81: 158-165

JV Melo, DE Gordon, NC Cross and JM GoldmanThe ABL-BCR fusion gene is expressed in chronic myeloid leukemia

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Copyright 2011 by The American Society of Hematology; all rights reserved.Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.

Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American

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The ABL BCR Fusion Gene Is Expressed in Chronic Myeloid LeukemiaBy Junia V. Melo, D.E. Gordon, N.C.P. Cross, and J.M. Goldman

Although the BCR-ABL hybrid gene on chromosome 22q-plays a pivotal role in th e pathogenesis of chronic myeloidleukemia CML), lit tle is known of the reciprocal chimericgene, ABL-BCR, formed on chromosome 9q+. By reversetranscription/polymerase chain reaction amplifi cation RT/PCR) w e have detected ABL-BCR mRNA in cells from 31of 44 BCR-ABL positive CML patients and 3 of 5 CML celllines. Of the 34 posit ive samples, 31 had classical t 9;22)q3 4; ql l) translocations; in 3 samples there was no Phil-adelphia Ph) and/or 9q+ chromosomes. ABL-BCR expres-sion consisted of ABL lb)-BCR mRNA in 26 patients and ofboth ABL lb)-BCR and ABL la)-BCR mRNA species n 6 pa-tients. The ABL-BCR transcripts encoded one or, more

HRONIC MYELOID leukemia (CML) is characterizedC cytogenetically by the presence of the Philadelphia (Ph)chromosome, which originates from the reciprocal translo-cation t(9;22) (q34;qll). In the formation of the Ph chro-mosome, the bulk of the ABL protooncogene is translocatedfrom chromosome 9 onto the BCR gene in chromosome 22.This gives rise to a novel chimeric BCR-ABL gene, whichencodes a 210-Kd (P210) fusion protein with prominent ty-rosine kinase activity and transforming ability.

The product of the normal BCR gene is a 160-Kd (P160BCR)cytosolic phosphoprotein, whose physiological role is notclearly defined. It was shown to form cytoplasmic complexeswith P2 in Ph-positive CML cells, as well as with a53-Kd protein of unknown function in both Ph-positive andPh-negative cell lines. Sequences encoded by the first exonof BCR are responsible for the P160BCRerine/threonine ki-nase a~tivity.~hese sequences overlap the src-homology 2(SH2)-binding regions of the BCR gene that are essentialforthe activation of the ABL tyrosine kinase and the transform-ing potential of the chimeric BCR-ABL In itscentral segment, BCR has some homology to the dbl oncogeneand the yeast CDC24 gene.6 The product of the latter is in-volved in the control of cell division after DNA replication.The C-terminus of BCR has recently been shown to have aGTPase-activating protein (GAP) activity for p21, a mem-ber of the RAS family of small GTP-binding proteins.*

In the t(9;22) translocation, the p2 1 GAP domain ofBCR is absent from the BCR-ABL chimeric protein. The 3end of the BCR gene containing the coding sequence for thisdomain is, in turn, fused to the 5end of ABL on chromosome9. The fate of the 9q+ chromosome, the other partner in

From the MR C/ LR F Leukaemia Unit, Department of Haematol-ogy, Royal Postgraduate M edical School, London, UK.Submitted July I , 1992;accepted September 3, 1992.Address reprint requests to Junia V.Melo, MD , PhD, Departmentof Haematology, Royal Postgraduate Medical School, Ducane Rd,London WI2 O N N , UK.Th e publication costs of this article were defiayed in part b y pagecharge payment. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. section I734 solely toindicate this fact.993 by The American Society of Hematology.0006-4971/93/8101 0015 3.00/0

rarely, both of the two potential unctions, designated ABL-b3 and ABL-b4. which differed in size by 75 bp. In 2 pa-tients, t he BCR exon b 3 was not present in either the BCR-ABL or the cor respondingABL-BCR transcript, whereas in5 patients exon b3 w as present in both t ranscripts. Directsequencingof PCR fragments representing the full-lengthcoding sequence of ABL-BCR cDNAs type lb-b3,la-b3, Ib-b4, and la-b4 showed an open reading frame p redicted toencode fusion proteins of 3 70 to 41 4 amino-acids. If anABL-BCR gene product s produced n CML cells, it may berelevant as a mechanism for deregulating the GTPase ac-tivating protein GAP) funct ion of BCR.

993 by The American Society of Hematology.the reciprocal t(9;22), is not known. Whereas most of thepresently available data suggest that the P2 10 fusion proteinencoded by the BCR-ABL hybrid gene is involved in thepathogenesis of CML;-l2 it is likely that other genetic changesare also necessary for defining the full leukemic phenotype.The expression of the 5ABL-3BCR hybrid gene in this trans-location has not so far been studied, but it may have func-tional consequences if it leads to abnormal activation of BCR-GAP.

We show here that the reciprocal ABL-BCR gene is tran-scriptionally active in over two thirds of Ph-positive CMLpatients, and that translation of its cognate chimeric mRNAinto an ABL-BCR fusion protein is compatible with its se-quence.

MATERIALS AND METHODSPatients and cell lines. Cells from a total of 44 CML patientswere studied: 20 in chronic phase CP) and 24 in blast crisis BC).Among the latter, 15 were myeloid BC, 4 lymphoid, 1 mixed myeloid/lymphoid, and 4 of unknown phenotype. Thirty-eight patients showedcharacteristic Ph and 9q+ chromosomes, 1 had an atypical 22q-without a 9q+, and 5 were Ph-negative. All patients had clonal BCRgene rearrangement by Southern blot analysis.Five BCR-ABL-positive cell lines were also investigated. Thesewere well characterized lines from patients with CML in blast crisis:K562, KCL-22, KYO-1, BV173,I3and LAMA-84.I4The HL60 pro-

myelocytic cell line was used as a negative control for BCR-ABLexpression in all tests. Amplificationsof specific sequences on the ABL, BCR, BCR-ABL, and ABL-BCRgenes were performed by reverse transcription RT) of cDNA, fol-lowed by PCR RT/PCR) by standard methods. Briefly, PB whiteblood cells WBC) were obtained by dextran sedimentation or by redcell lysis of centrifuged buffy coat ~reparati0ns.l~he patients WBCand freshly explanted cells from lines in culture were washed twicein phosphate buffered saline and processed for RNA extraction bythe guanidinium thiocyanate/CsCl gradient method.16The RNA wasreverse transcribed into cDNA with Mo-MuLV reverse transcriptaseGIBCO-BRL, Gaithersburg, MD) using random hexamers and, insome samples, oligo-dT primers. PCR amplification of cDNA wasperformed as described elsewhere. Precautions towards eliminatingthe possibility of false PCR results were based on the recommenda-tions by Kwok and Higuchi. In brief: 1) cells, RNA, and cDNApreparations were always handled in a room separate from that spe-cifically dedicated to the analysisof PCR products; (2) different setsof pipettes were dedicated to sample preparation and PCR product

Polymerase chain reaction PC R) amplijication.

158 Blood, Vol 81, No 1 (January 1). 1993: pp 158-165

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ABL-BCR FUSION GENE IN CML 159

handling, and plugged tips (aerosol resistant) were used in all steps;(3) all reagents were prepare d under sterile conditions in a lam inar-flow cabinet, and stored as single-use aliquots; an d 4) each PCRexperiment included 6 to 8 test (CML) cDNA sample s plus 2 knownnegative controls: BCR-ABL- and ABL-BCR-negative cDNA fromHL60 cells, and a H20-blank (ie, no cDNA). In no instance wereABL-BCR products detected in either negative control. Furthermore,test samples showing no ABL-BCR amplification alongside samplesthat did yield an ABL-BCR PCR product were always found in an ygiven exper iment, reinforcing the validity of the positive results. Fivemicroliters from each PCR was electrophoresed through ethidiumbromide stained 1 to 2% agarose mini-gels, visualized, and pho-tographed under UV light. Samples that showed no ABLB CR prod-uct were submitted to a second ro und of am plification with nestedprimers and 1 pL of the original PCR products as template. Thesequences of the sy nthetic oligonucleotide primers used i n this in-vestigation are shown on T able 1. Some of the primers were designedto conta in natural o r forced restriction enzym e sites at their 5 endsto facilitate future cloning of the PCR fragments into phage or plasmidvectors. The size of PCR fragm ents amplified with each primer pairis shown in Table 2.Southern hybridization. Electrophoresed PCR products fromsome samples were transferred from agarose gels to nylon membranesby Southe rn blotting, and tested for h ybridization to a synthe tic oli-gonucleotideprobe (primer BS+) -labeled with Y -~ ~P -A TP .ybrid-ization and high-stringency washings were ca med out at the app ro-priate discriminating temperature s or the oligonucleotideas describedelsewhere. Direct seque ncing of PCR products was performedby the linear amplifica tion sequencing method and/o r by the Taqcycle sequencing m ethod [USB, Cleveland, OH]. PCR p roducts wereused directly as empla tes on an e stimated basis of 1 fmol ss-DNAper base per primer. Dena turing sequencing gels were prepared, elec-trophoresed , and autoradiographed by convention al techniques.

RESULTS

Primers for P CR , Southern hybridization, and sequencing.

Sequencing.

BCR-ABL gene. BCR-ABL amplificat ion of cD NA withprimers B2+ a n d CA3- yielded fragments 385 and 465 bp

Table 1. Synthet i c Ol igonuc leot ide Pr imers Used in This S tudyPrimer Sequence 5 . GeneL o c a t i o P Z 6Aa+Ab+PAa+PAb+Jc-CA3-81B2+B3+B4+B7+C48 4 -c5-G-PB-

ec

TTGGAGATCTCCCTGAAGCTTCTGGAAAGGGGTACCTATTAtacggaattcATGTTGGAGATCTCCCTGAACGCTGAgaaTTCTGGAAGATCTTGAAGGAGTGTTTCTCCAGACTGTTGTGTTGACTGGCGTGATGTAGTTGCTTGGGAGCGTGCAGAGTGGGGAGGGAGAACATCCGGTTCAGAAGCTTCTCCCTGACATTCTGAATGTCATCGTCCACTCAGCCAttcaaATCTGTACTGCACCCTGGAGCTGGTCgAaTTCAACAGCAGGGAGTTCAattagatCTGAAGCTGTACTTCCGTGAacgtgaaTTCCAGTTTGGCTCAGCTGTtcaaggaTCCACGCACTGGCGCACGATataggaTCCTTTGCAACCGGGTCTGAAgatgggaTCCAGCTGCAGGAGTATCAGGAAGGaTCCCGCTCTACGGAT

ABL la)ABL Ib)ABL la)ABL Ib)ABL I l l )ABL I l l )BCR b l )BCR b2)BCR b3)BCR b4)BCR b7)BCR c4)BCR b4)BCR c3)BCR c5)BCR c7)BCR ( 3 end)

Lower case indicates nucleotides that are not homologous to thecDNA sequence, but inco rporate restrict ion enzyme r ecognit ion sites.

Table 2. PCR Amp l i f icat ions of AB L, BCR, BCR-ABL.and ABL-BCR cDNA

PCR Produc tPrimers Gene length n bp)Aa+- c- ABL f rom la) 3 7 4Ab+ - c- ABL f rom Ib) 46 2B l +- - BCR 1,208385 b2a2)456 b3a2 )PAa+- - ABL la)-BCR 1,0 72 la-b4)

1,147 la-b3)

B2 ++ CA3- BCR-ABL

Ab+ G- ABL Ib)-BCR 1,1 48 lb-b 4)1,223 lb-b3)

PAa+ PB- ABL la)-BCR 1,2 08 la-b4)1,283 la-b3)

PAb+- B- ABL Ib)-BCR 1.31 6 lb-b4)1,391 lb-b3)

PAa+- 4 - * ABL la)-BCR 18 9 la-b4)264 la-b3)

PAb+ 84- ABL b-BCR 29 7 lb-b4)372 lb-b3)84 Bc- BCR in ABL-BCR) 4 5 6

B 7+- 5- BCR in ABL-BCR) 3 9 8c4+ PB- * BCR in ABL-BCR) 5 2 7

These were primers used for direct sequencing of overlapping seg-ments of the tem pla te ABL-BCR ampli fied cDNAs as PCRs PAa+- B -and PAb+- B-.long, representing the b2a2 and b3a2 type transcripts re-spectively (Fig 1A).The types of BCR-ABL transcript expressed in the 44 pa-t ients and th e five CM L cell l ines were as follows. In t he CPgroup, 45% of patients expressed only b2a2 and 50% onlyb3a2 B CR-ABL transcripts. A relatively similar distribu tionfor b2a2 an d b3a2 pat ients was observed in the BC group,which includes th e five cell lines (59% a nd 38%, respectively).One C P and o ne BC pat ient expressed bo th b2a2 an d b3a2transcripts.

Expression o f the2 m ajor al ternat ive t ranscripts from t he n orm al ABL al lelewas observed i n al l the 44 pat ients . This was shown by R T/PC R am plificat ion of the sequence spanning exon Ia to exon111(PCR PAa+ c- ) and , l ikewise, t he sequence f rom e xonIb to exon I11 ( P C R Ab c- Jc-). O f the five cell lines studied,only LAMA-84 a nd B V113 did not express ei ther ABL tran-script, which agrees with the absence of a norm al chrom osom e9 i n theseTh e normal BCR al lele was also found to be expressed inall patients an d cell lines, as assessed by PC R a mplificationof a 1,208-bp long fragment spa nning the m ajor breakpointcluster region (M-bcr), and extending downstream of exon~ 7 . ~ ~ 3 ~ ~his fragment includes the BCR -GAP coding regionin the normal Th e formation of an ABL-BCR fusiongene, the reciprocal pro duct o f th e BCR-ABL translocation,should theoretically yield different transcripts, dep endin g onthe posi t ions of the breakpoints in both ABL a nd BC R (Fig2). If the breakpoint in ABL occurs upstream o f exon Ib , no

N o r m a l A B L and normal BCR genes .

ABL-BCR gene.


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160

M 2 4 5 6 7 8 9 1 0 1 1 MMELO ET AL

ABL-BCR transcript is formed. If the breakpoint is betweenexons Ib and la, only transcripts originating from the exonIb promoter are possible (Ib-BCR); and if between exons laand 11, two R N A species can be transcribed from the inde-pendent promoters in exons Ib and la (Ib-BCR and la-BCR).

The 49 samples were tested for expression of ABL-BCRtranscripts by RT/PCR amplification using sense primers onABL exons Ib (Ah ) and la (PAa ). and anti-sense primerson BCR 3' end G- r P B - ) . A total of 34 out of 49 patients(69 ). ncluding2 Ph-negative patients and the patient with-out a 9q+ chromosome, showed ABL-BCR amplification ofthe Ib-BCR type and 6 of these 34 also expressed la-BCR.No case expressed la-BCR alone (Table 3). Likewise, abnor-mally large ABL-BCR fragments containing both exons Iband la were never found. In 7 of the 34 samples the ABL-BCR products were only detected by nested PCR. In all theothers, the level of ABL-BCR transcripts seemed comparableto that of ABL, BCR, and BCR-ABL amplified products, asestimated within the limitations of a standard, nonquanti-tative PCR assay. Among the I5ABL-BCR negative samples,4 (3 patients and the cell line K562) were Ph-negative, al-though BCR-rearranged and BCR-ABL positive.

A

+ 3a2+ 2a2

Beb-b3b-b4Fig 1. (A) BCR-ABL and B)

ABL-BCR PCR products in 11representative samples, as de-tected on ethidium bromide-stained agarose gels. BCR-ABLbands represent fragments of385 bp (b2a2) andlor 465 bp(b3a2). corresponding o ampli-fications with primers 82 andCA . ABL-BCR products spansequences between primersAb(in ABLexon Ib) andG (in BCR),representing fragments 1,223bp (lb-b3) and/or 1,1 48 bp (Ib-b4) long. Lanes 1 to 10 are CMLpatient samples; lane 1 1 is theHL60 cell l ine (negativecontrol).M, DNA molecular weightmarker (pEMBL digested withw I .

Like RCR-ABL. the ABL-BCR transcripts are predictedto vary in length, depending on whether BCR exons b3 orb4 are joined to the S'ABL. This was confirmed by Ib-BCRproducts 1,148 bp and/or 1,223 bp long when primersAh - G - were used for the PCR amplification (Fig IB).The same 75 bp difference in the length of the PCR prod-ucts was observed in la-BCR amplifications with primersPAa - .

Therefore, the ABL-BCR expressing cases fell into 3 cat-egories (Table 4): 12 showing b b 4 unction, 19 with b b 3junction, and 3 cases with double transcripts ( I b b 4 and Ibb3). In the 6 patients who also showed la-BCR transcription,the junction was of Ia-b4 type in 3 and Ia-b3 in 3. The pres-ence or absence of BCR exon b3 in each transcript was con-firmed by Southem hybridization of the ABL-BCR fragmentswith a 26-mer oligonucleotide (primer B3 ) spanning a sequence specific for exon b3 (Fig 3) and, in some cases, bydirect sequencing of the junction region in each PCR frag-ment.

Overall, the proportion of ABLBCR-expressing patientswho showed only b2a2 (56 ), only b3a2 (38 ), and bothb2a2 and b3a2 (6 )BCR-ABL transcripts matched the pro-


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Fig 2. Schematic represen-tation of the ABL, BCR, BCR-ABL, and ABL-BCR genes. Ar-rows indicate the most frequentregions for breakpoints in theABL and the BCR genes. Thepossible ABL-BCR transcriptsarisingfrom the different break-points in BCR are shown under-neath the corresponding BCR-ABL transcripts type b2a2 andb3a2, respectively.

BCR-ABLb2a2E2 1l b -b3 m I-- . ---=TI- ~I b WABL-BCR

l a -b3 l a Z E 2 1

X IWE l W NBCR-ABL I 1 IJLU 1b3a2E 2 1Ib gl b -b4ABL-BCR

l a X E 2 1l a - b 4 ~~ ~

portion of patients in each BCR-ABL category in the wholeseries. The frequency of ABL-BCR expression within eachBCR-ABL major group was 73% and 62%) n the b2a2 andthe b3a2 grou ps. respectively.When the expression o f the BCR-ABL and the ABL-BCRfusion genes was compa red in each individual sample (Table5). it was found that, in 27 patients (79% ). the junction typein ABL-BCR was exactly reciprocal to the junctio n in BCR-ABL. Thus, from the 19 b2a2-positive samples , 15 expressedan ABL-b3 type of junction, and 2 expressed both ABL-b3and ABL-b4 transcripts. Similarly, 8 out of I3 patients withsingle b3a2 and 2 patients with both b2a2 and b3a2 transcriptsfrom BCR-ABL showed ABL-BCR expression of the ABL-

b4 type, as expected. However. in 7 samples the junctiontype in the ABL-BCR transcripts did not correlate with thereciprocal BCR-ABL products: these were 2 b2a2 patientswho expressed only ABL-W transcripts, and 5 b3a2 patientsin whom ABL-b3 type o f transcripts were found either alone4 samples) or together with ABL-W transcripts ( 1 sample).Th e junction region of BCR-ABL and ABL-BCR products

Table3. ABL-BCR Transcripts Found in CML PatientsStage of Diseaseno. of cases) None Ib-BCR Ib-BCR and la-BCR

~

Chronic phase (20) 5 (25 ) 13 (65 ) 2 (10 )Blast crisis (29)' 10 (34 ) 15 (52 )t 4 (14 )TOTAL (49) 15 (31 ) 28 (57 ) 6 (12 )

Including the 5 CML cell lines.t Including the cell lines KCL-22. KYO-1, and BV173.

from this group of patients was sequenced (primers PAaR 4 - ) and results confirmed that, in 2 cases, BCR exon b3was not expressed in eith er BCR-ABL or ABL-BCR, whereas,in 5 cases. this exon was present in both fusion-gene produc ts.The same result was obtained by Southern hybridization o fBCR-ABL and ABL-BCR products with the oligonucleotideprobe R3 (Fig 3). In of th e 7 cases it was possible to repeatthe BCR-ABL and ABL-BCR amplifications in duplicateblood samples obtained at different times, and /or in duplicateRNA/cDNA preparations from the original blood samples.The initial results were always reproducible. In 2 patientsduplicate sa mples were not available for analysis.PCR amplifiedABL-BCR fragments from cDNA of 2 samples were sequenced in overlapping segmen ts. using 4 pairs of sense andantisense primers (Table 2). The full-length PCR productsrepresented ABL-BCR type I b b 3 and Ia-b3 from I patient.and Ibb4 and la-b4 from another. Each fragment started 60bp (Ib)o r IO bp (l a) upstream of ABL's initiation ATG. andended 86 bp downstream the stop codon for PI 60RCR.

Coding seqircvce qj clie ABL-BCR gene.

Table 4. Junction Type of the ABL(Ib)-BCR Transcriptsin the Positive Cases

Stage of Diseaseno. of cases) lb-b3 lb-b4 lb b3 and lbbChronic phase (15) 7 (47 ) 6 (40 ) 2 (13 )Blast crisis (19)' 12 (63 )' 6 (32 ) 1(5 )TOTAL (34) 19 (56 ) 12 (35 ) 3 (9 )

Including the cell lines KCL-22, KYO-1. and BV173.


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162 MELO ET AL

In the 4 products. the sequences matched exactly ABLexon or la. which joined in phase BCR exons b3 orb4, maintaining the open reading frame (ORF) up to thenormal BCR stopcodon. The ORFs ofthe ARL-BCR hybridcDNAs code for predicted fusion proteinsof 4 14 amino-acidsA A ) (Ihb3). 395 A A (la-b3). 389 A A lkb4). nd 370 A A(Ia-b4).

DISCUSSIONI n Ph-positive CML. the coding sequences of two genes,

ABL and BCR. are disrupted as a result of the reciprocalexchange between chromosomes 9 and 22. and two hybridgenes are formed. BCR-ABL ( on 22q-. Ph) and ABL-BCR(on 9q+). Expression of the BCR-ABL gene was shown inall cases of Ph-positive CML when the sensitive method

Table 5. BCR-ABL Transcriot Versus ABLfIbl-BCR TranscriDtABL(lb)-BCRBCR-ABL

(no. of cases) lb-b3 lb-b4 lbb 3 and lbb4b2a2 (19) 15 2 2b3a2 (13) 4 8 1b2a2 and b3a2 (2) 0 2 0

Cases in which the ABL-BCR transcripts are not reciprocal to theBCR-ABL transcripts.

of RT/PCR amplification was used. However, it wasnot known whether the reciprocal ABL-BCR hybrid geneis likewise functionally active i n this disease. This possi-bility was raised previously in a single report when a 3.5

M 2 4 5 6 7 8 9 1 0 1 1 M

.

2 3 4 5 6 7 8 9 10 11 M

b3a2

+lb-b3

Fig 3. Southern-blot hybrid-ization with oligonucleotide(primer)B3 of (A)BCR-ABL andthe same representative sam-ples shown in Fig 1. The oligo-nucleotide probe sequence isspecific for BCR exon b3. Notethat in samples 2 and 8 bothBCR-ABL and ABL-BCR tran-scripts hybridize to this probe,indicating the presence of BCRexon b3 in he twohybrid genes.Conversely, neither BCR-ABLnor ABL-BCR transcripts fromsample9 hybridize to he probe,which confirms the absence ofexon b3 in both genes.

(B) ABL-BCR PCR products Of


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kb transcript , presumed to be a SABL-3BCR fusion mes-sage, was detected in Northern blots of the BV173 cellline.

In the present study, we show that ABL-BCR expressioncan be detected by RT/PCR in WBC from approximately70 of CML patients. It is noteworthy that, among the ABL-BCR positive patients, 2 were Ph-negative and 1 had no 9q+chromosome, showing that, like BCR-ABL, the ABL-BCRgene rearrangement can also take place in the absence ofcytogenetic evidence of a t(9;22).

In one third of our cases, no ABL-BCR expression wasdetected, in spite of repeated PCR tests with several combi-nations of primers. This group contains 4 of the Ph-nega-tive, BCR-ABL positive samples that may represent com-plex and not reciprocal chromosome trans location^.^^-^'The remaining 14 samples (26 of the Ph-positive CML)presumably result from translocation breakpoints in chro-mosome 9 upstream of ABL exon Ib, or from deletionsin BCR sequences 3 to the chromosome 22 br e a k p ~ i n t . ~ ~In neither case would an ABL-BCR hybrid message bepresent.

The higher proportion (82%)of patients expressing onlyABL-BCR transcripts of the Ib-BCR type, compared withthose 18%) with transcripts initiated from both exons Iband Ia (Ib-BCR and Ia-BCR) is probably a reflection ofthe frequency of breakpoints between exons Ib and Ia, andIa and 11. Because the distance between exons Ib and Ia isgreater than 200 kb,24 pproximately I O times that betweenexons Ia and I1 ( 1 9 kb),34 he probability of a break oc-curring within the first large ABL intron might be 10-foldgreater than within the second i n t r ~ n . ~ ~ - ~ he fact that asingle transcript carrying both exons Ib and Ia was neverfound shows that the normal mechanism of alternativesplicing of these two exons3 is maintained in the ABL-BCR hybrid gene.

The overall similar distribution of ABL-BCR-positive pa-tients among the b2a2 and b3a2 BCR-ABL categories suggeststhat there is no simple correlation between the breakpointsites in ABL and in BCR. On the other hand, the structureof the BCR moiety of the BCR-ABL and ABL-BCR tran-scripts from 7 patients showed unexpected patterns. In 2 ofthese, neither BCR-ABL nor ABL-BCR transcripts includeBCR exon b3. This means that the breakpoint may be inexon b3 itself, or that this exon was included in deletions atthe breakpoint site.39Another possibility is that b3 was splicedout in the mature mRNA from whichever of the two chimericprimary transcripts that retained it. Precedents for alternativesplicing of exon b3 in BCR-ABL are well established in thosepatients who express both b2a2 and b3a2 transcript^^^,^^,^^and, in the present study, by the 3 patients with both Ib-b3and Ib-b4 ABL-BCR. More surprising are the 5 patients inthis study in whom both BCR-ABL and ABL-BCR transcriptscontain BCR exon b3. In 3 of these (3 BC), the karyotypeshowed single Ph and 9q+ chromosomes, in 1 (CP), the 22q-was longer than a typical Ph and there was no 9q+, and inthe fifth (CP),. additional i(Ph) chromosomes probably arosefrom duplication of the original Ph. Therefore in these cases,there is no evidence that the BCR-ABL and the ABL-BCR

hybrid genes are not part of the same clone, which impliesthat BCR exon b3 alone was duplicated in the t(9;22).Whether this b3 duplication took place before or during thetranslocation is not known. The breakpoint regions of bothBCR-ABL and ABL-BCR in these 5 cases are being clonedfor further studies.

The nucleotide sequence of the 4 different ABL-BCRhybrid cDNAs found in our series shows that in each casethe SABL-3BCR junc tion is in phase as expected. There-fore, ABL-BCR fusion proteins of about 390 AA could betranslated, the exact size of each varying according to theexon contribution of ABL and BCR to the transcript. Thefact that ABL-BCR amplifications were obtained fromoligo-dT primed cDNAs indicates the presence of poly-(A) on these transcripts and suggests that translatable RNAis produced. Investigations on the presence of such ABL-BCR proteins in cells from patients with CML are in pro-gress.

The significance of ABL-BCR expression in CML isstill unclear. It seems unlikely that the hybrid ABL-BCRgene has any primary oncogenic role, because it is notpresent in at least one third of the CML patients. Fur-thermore, the fact that ABL-BCR is expressed in CP aswell as in BC of CML argues against a role in causingdisease progression. On the other hand, ABL-BCRexpression could well be associated with specific clinicaland/or hematological features in subsets of CML patients,which may reflect prognosis and response to treatment.Data from our present series are still insufficient to addressthis question.

If a functional ABL-BCR fusion protein is indeed pro-duced, as predicted from the cDNA coding sequences, itwould contain a GAP-BCR domain in its C-terminus linkedto an N-terminal ABL sequence. Such an arrangement couldalter the rucGAP function of BCR leading to either con-stitutive activation or inactivation of the rucGAP activity.Moreover, the Ib-BCR-encoded fusion protein, like the ABLtype I1 P145,3,41,42 ould have a myristoylation site at itsN-terminus and, therefore, unlike p160BCR,t could becomeassociated with the cell membrane. Although mRNAexpression from the normal BCR allele can be detected inCML cells,43 t is not clear whether the level of P160BCRproduction is the same as in normal leukocytes. Coexpres-sion of an abnormal ABL-BCR gene product could resultin competition for the same target protein, ruc, and imbal-ance in the rate of ruc activation. The biologic effects of aderegulated BCR are unknown. However, because the rucproteins display relative myeloid specifi~ity~~nd are in-volved in activation of the NADPH oxidase system of neu-t r o p h i l ~ , ~ ~t is tempting to speculate that an ABL-BCR pro-tein with altered GAP activity might have a role ingranulocyte functional defects.

ACKNOWLEDGMENTWe thank Julie Bungey for reviewing the karyotypes of patientsand cell lines, and Dr Alan Hall Chester Beatty Institute for CancerResearch, London) for valuable discussion.


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164 MELO ET AL

REFERENCESI . Campbell M L, Li W, Arlinghaus RB: P210 BCR-ABL is com-plexed to PI60 BCR and ph-P53 proteins in K 562 cells. Oncogene5:773, 19902. Li W, Drea zen 0 Kloetzer W, Gale RP, Arlinghaus RB: Char-acterization of bcr gene products in hematopoietic cells. Oncogene

4:127, 19893. Maru Y Witte ON: The BCR gene encodes a novel serine/threonine k inase activity within a single exon. Cell 67:459, 19914. Muller AJ, Young JC, Pendergast AM, Pondel M, Landau N R,Littman DR, Witte O N BCR first exo n sequ ences specifically activatethe BCR/ABL tyrosine kinase oncogen e of Ph iladelphiachromosome-positive human leukemias. Mol Cell Biol 1 1:1785, I99 15. McW hirter JR, Wang JYJ: Activation of tyrosine kinase andmicrofilament-binding functions of c-ab1by bcr sequences in bcr/ablfusion proteins. Mol Cell Biol 11:1553, 19916. Ron D, Zannini M , Levis M, Wickner RB, Hun t LT, GrazianiG, Tronick SR, Aaronson SA, Eva A: A region of prot od bl essentialfor its transforming activity shows sequen ce similarity to a yeast cellcycle gene, CDC24, and the human breakpoint cluster region, bcr.

New Biol 3:372, 19917. Ohya Y Miyamoto S, Oshumi Y nraku Y Calcium sensitivec/s4 mutant of Saccharomyces cerevisiae with a defect in bud for-mation. J Bacteriol 165:28, 19868. Diekmann D, Brill S, Garrett MD, Totty N, Hsuan J, MonfriesC, Hall C, Lim L, H all A: BCR encodes a G TPase-activating proteinfor p21mc.Nature 351:400, 19919. Daley GQ, van Etten RA, Baltimore D: Induction of chronicmyelogenous leukemia in mice by the P210kr ab gene of the Phila-delphia chromosome. S cience 247:824, I9 90IO. Elefanty AG, Hariharan IK, Cory S bcr-ab/, he hallmark ofchronic myeloid leukaemia in man, induces multiple haemopoieticneoplasms in mice. EMBO J 9:1069, 19901 I. Daley GQ an Etten RA, Baltimore D: Blast crisis in a murin e

model of chronic my elogenous leukemia. Proc N atl Acad Sci USA88:11335, 199112. Gishizky M, Witte ON: Initiation of deregulated growth ofmultipotent progenitor cells by bcr/abl in vitro. Science 2563 36,199213. Keating A: Ph positive CML cell lines. Baillieres Clin Ha emato l1:1021, 198714. Seigneurin D, Champelovier P, Mouchiroud G, Berthier R,Leroux D, Prenant M, McGregor J, Starck J, Morle F, Micouin C,Pietrantuono A, Kolodie L: Human chronic myeloid leukemic cellline with positive Philadelphia chromosome exhibits megakaryocyticand erythroid characteristics. Exp Hematol 15322, 198715. Roos D, Loos JA: Changes in the carbohydrate metabolismof mitogenically stimulated huma n peripheral lymphocytes. I. Stim-ulation by phytohaemagglutinin. Biochem Biophys Acta 222565,197016. Chirgwin JM, Przybyla ARE, MacDon ald RJ, Rutter WJ: Isolation of biologically active ribonucleic acid from sources enrichedin ribonuclease. Biochemistry 18:5294 , 197917. Melo JV, Goldm an JM : Specific point-mutations that activatev-ab/ are not found in Philadelphia-negative chronic myeloid leu-kemia, Philadelphia-negative acute lymphoblastic leukemia or blasttransformation of chronic myeloid leukemia. Leukemia 6:786, 1992

18. Kwok S, Higuchi R Avoiding false positives with PCR . Na ture339:237, 198919. Cross NCP, de Franchis R, Sebastio G, Dazzo C, Tolan DR,Gregori C, Odievre M, Vidailhet M, Rom ano V, Mascali G, Roman oC, Musumeci S, Steinmann B, Gitzelmann R, Cox TM: Molecular

analysis of aldolase B genes in hereditary fructose intolerance. Lancet335:306, 1990

20. Craxton M: Linear amplification sequencing: A powerfulmethod for sequencing DNA. Methods: A companion to M ethodsin Enzymology 3:20, I9 9 1

2 1. Westbrook CA, Rubin C M, Carrino JJ, Le Beau MM, BernardsA, Rowley J D Long-range mapping of the Philadelphia chromosomeby pulsed-field gel electrophoresis. Blood 71:697, 1988

22. Heisterkamp N, Stam K, Groffen J, de Klein A, Grosveld G:Structural organization of the bcr gene and its role in the Ph' trans-location. Nature 315:758, 198523. Lifshitz B, Fainstei n E, Marc elle C, Shtivelm an E, Ams on R,Gale RP, Canaani E: bcr genes and transcripts. Oncogene 2:113,

198824. Bernards A, Rubin C M, Westbrook CA, Paskin d M, BaltimoreD The first intron in the human c-ab1 gene is at least 200 kilobaseslong and is a target for translocations in chronic myelogenous leu-kemia. Mol Cell Biol 7:3231, 198725. Fainstein E, Einat M, Gokkel E, Marcelle C, Croce CM, GaleRP, Canaani E: Nucleotide sequence analysis of hum an ab/ and bcr/

ab/ cDNA s. Oncogene 4: 1477, 198926. Morgan GJ, Hernandez A, Chan LC, Hughes T, Martiat P,Wiedemann LM: The role of alternative splicing patterns of BCR /ABL transcripts in the gene ration of the blast crisisof chronic myeloidleukaemia. Br J Haematol 76:33, 199027. Dobrovic A, Morley AA, Seshadri R, Januszewicz EH:Molecular diagnosis of Philadelphia-negative CML using thepolymerase chain reaction and DNA analysis: Clinical featuresand course of M-bcr negative and M -bcr positive CML. Leukem ia

5:187, 199128. Dhingra K, Talpaz M, Kantarjian H , Ku S Rothberg J, Gut-

terman JU, Kurzrock R: Appearance of acute leukemia-associatedPI 90BCR-ABLn chronic myelogenous leukemia may correlate withdisease progression. Leukemia 5: 19 199129. de Braekeleer M: Variant Philadelphia ranslocations in chronicmyeloid leukemia. Cytogenet Cell Genet 44:215, 198730. Mo ms CM , Heisterkamp N, K ennedy MA, Fitzgerald PH,Groffen J: Ph-negative chronic myeloid leukemia: Molecular analysisof ABL insertion into M-BCR on chromosome 22. Blood 76: 18 12,19903 1. Mo nis CM, K ennedy M, Heisterkamp N , Columbano-GreenL, Romeril K, Groffen J, Fitzgerald P A complex chromosome rear-rangement forms the BCR-ABL fusion gene in leukemic cells with

a normal karyotype. Genes Chrom Cancer 3:263, 199132. M om s CM, Heisterkamp N, Groffen J, Fitzgerald PH: EntireABL gene is joined with 5'BCR in som e patients with Ph iladelphia

positive leukemia. Blood 78: 1078, 199133. Popenoe DW, Schaefer-Rego K, M ean JG, Bank A, LeibowitzD: Frequent an d extensive deletion during the 9,22 translocation inCML. Blood 68:1123, 198634. Shtivelman E, Lifshitz B, Gale RP , Canaan i E: Fused transcrip tof ab1 and bcr genes in chronic myelogenou s leukaemia. Na ture 3 15:550, 198535. Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bar-tram CR, Grosveld G: Philadelphia chromosomal breakpoints areclustered within a limited region, bcr, on chromosome 22. Cell 36:93, 198436. Heisterkamp N, Stephenson JR, Groffen J, Hansen PF, deKlein A, Bartram CR, Grosveld G:Localization of the c-ab/ oncogeneadjacent to a translocation breakpoint in chronic my elocytic leukae-mia. Nature 306:239, 1983


8/12/2019 Blood-1993-Melo-158-65

9/9


37. Leibowitz D, Schaefer-RegoK, Popenoe DW, M ears JG, BankA: Variable breakpoints on the P hiladelphia chromosom e in chronicmyelogenous leukemia. Blood 66:243, 198538. Shtivelman E, Lifshitz B, Gale RP, Roe BA, Canaani E: Al-ternative splicing of RNA s transcribed from the hu man ab/ gene andfrom bcr-ab/ fused gene. Cell 47:277, 1 98639. Mills KI, Sprout AM, Leibowitz D, Bumett AK: M apping ofbreakpoints, and relationship to BCR -ABL RNA expression , in Phil-adelphia-chromosome-positive chronic myeloid leukaemia patientswith a breakpoint arou nd exon 14 (b3) of the BCR gene. Leukemia5:937, 199140. Lee MS, Le Maistre A, Kantarjian HM, Talpaz M, Freireich

ET Trujillo JM, Stass S A Detection of two alternativebcr/ab/mRNAjunctions and minimal residual disease in Philadelphia chromosom epositive chronic myelogenous leukemia by polymerase chain reaction.Blood 73:2165, 1989

41. Jackson P, Baltimore D N-terminal mutations activate theleukemogenic potential of the myristoylated form of c-ab/. EMB O J8:449, 198942. van Etten RA, Jackson P, Baltimore D Th e mouse type IVc-ab/ gene product is a nuclear protein, and activation of trans-forming ability is associated with cytoplasmic localization. Cell 58:669, 198943. Collins S, Coleman H , Groud ine M: Expression of BCR andBCR-ABL fusion transcripts in normal and leukemic cells. Mol CellBiol 7:2870, 198744. Didsbury J, Weber RF, Bokoch GM, Evans T, Snyderman Rrac, a novel vas-related family of proteins that are botu lin toxin sub-strates. J Biol Chem 264:16378, 198945. Abo A, Pick E, Hall A, Totty N, T eahan CG , Segal A W Ac-tivation of the NAD PH oxidase involves the small GTP-binding pro-tein p21 '. Nat ure 353:668, 1991


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