Leukemia Research 23 (1999) 1055–1060

ABL-BCR expression in BCR-ABL-positive human leukemia celllines

Cord C. Uphoff a,*, Silke Habig a, Severine Fombonne a, Yoshinobu Matsuo b,Hans G. Drexler a

a DSMZ-German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Mascheroder Weg 1 B,D-38124 Braunschweig, Germany

b Fujisaki Cell Center, Hayashibara Biomedical Labs, Okayama, Japan

Received 8 April 1999; accepted 4 June 1999

Abstract

Expression of normal ABL and BCR and of reciprocal fusion genes BCR-ABL and ABL-BCR was examined in a panel of 53BCR-ABL-positive cell lines by RT-PCR to determine the influence of the various transcripts on leukemogenesis. Seventeen outof 18 lymphoid cell lines expressed ABL1a and/or ABL1b, whereas only 16 out of 35 myeloid cell lines expressed one or bothnormal ABL transcripts. Normal BCR was expressed in seven lymphoid cell lines; all cell lines from the m-bcr group (n=9) wereBCR-negative. Among the myeloid cell lines, 77% expressed the BCR gene. The M-bcr and m-bcr translocations were equallydistributed among cell lines with lymphoid phenotype. The m-bcr translocation was not found in myeloid cell lines. b3-a2constitutes the predominant form of fusion gene in myeloid cell lines with an incidence of about 68%. One myeloid cell lineexhibited the m-bcr variant. An ABL-BCR transcript of the 1a splice variant was not detected in any of the cell lines. ABL1b-BCRwas expressed in all varieties of cell types and translocation forms: 56 and 66% in the lymphoid and myeloid cell lines, respectively;similar distributions were found for the fusion gene types: 67% among e1-a2, 73% among b2-a2, and 61% among b3-a2translocations. Except for the lack of expression of normal BCR in m-bcr cell lines and of ABL1a-BCR expression in all cell lines,no consistent correlation of expression or lack of expression of BCR and ABL or of ABL-BCR reciprocal fusion genes could befound with cell lineages and translocation types. Further work is required to determine the exact role of the reciprocal fusion genetranscripts on the pathophysiological mechanisms of leukemogenesis. © 1999 Published by Elsevier Science Ltd. All rightsreserved.

Keywords: Philadelphia chromosome; ABL; m-bcr; M-bcr; m-bcr; Cell lines

www.elsevier.com/locate/leukres

1. Introduction

Chronic myeloid leukemia (CML) is characterised bythe occurrence of the Philadelphia (Ph) chromosome inabout 95% of CML patients (for review see [1]). The Phchromosome originates from the reciprocal transloca-tion t(9;22)(q34;q11) by which the downstream portionof the ABL protooncogene from chromosome 9 isjuxtaposed with the upstream portion of the BCR geneon chromosome 22 to give rise to the chimeric BCR-ABL gene. The formation of the BCR-ABL gene is

thought to be one of the key events in the pathogenesisof the leukemia, but other thus far unidentified geneticchanges are probably additionally involved in the pro-gression of the disease, in particular for the transitionto blast crisis [2].

The positions of the breakpoints in the genes arevariable. In the ABL gene, the breakpoints are scat-tered between the 5%-end of the ABL gene and exon a2,resulting in unique transcripts all displaying ABL exona2 in the BCR-ABL fusion mRNA [3]. Concerning theBCR gene, the majority of the breakpoints (M-bcr)found in Ph chromosomes lies within a 5.8 kb regionspanning BCR exons 12 to 16 (also designated as b1 tob5). The bulk of breakpoints in the M-bcr results in oneof two possible transcripts which differ by 75 bp and

* Corresponding author. Tel.: +49-531-2616156; fax: +49-531-2616150.

E-mail address: cup@dsmz.de (C.C. Uphoff)

0145-2126/99/$ - see front matter © 1999 Published by Elsevier Science Ltd. All rights reserved.

PII: S 0 1 4 5 -2126 (99 )00131 -9

C.C. Uphoff et al. / Leukemia Research 23 (1999) 1055–10601056

which either includes or deletes BCR exon b3. Theexpression of the M-bcr fusion gene leads to 210 kDproteins with enhanced tyrosine kinase activity(p210BCR-ABL). A second breakpoint cluster region,rarely found in CML patients but in two thirds ofPh+ acute lymphoblastic leukemia (ALL) patients(designated m-bcr), lies within a long intron betweenBCR exons 1 and 2 (termed e1 and e2). This m-bcrchimeric gene produces a 190 kD protein (p190BCR-ABL)[3]. Recently, a third breakpoint cluster region (desig-nated m-bcr) downstream of BCR exon 19 was discov-ered leading to the large p230BCR-ABL fusion proteinwhich is found very rarely in CML patients [4].

The BCR-ABL fusion protein is thought to be in-volved in a defective b1-integrin function which mightlead to the abnormal circulation and proliferation of thePh+ progenitor cells. BCR-ABL is also involved in anumber of signal transduction pathways including theactivation of the RAS oncogene. The ABL part of thefusion protein appears to be responsible for the latter twofunctions as the normal ABL protein was described tohave similar activities [5]. The normal ABL gene product(p145ABL) is an ubiquitously expressed non-receptorprotein tyrosine kinase which fluctuates between thenucleus and the cytoplasm. The function of the normalBCR gene product is not yet entirely understood. Therole of the reciprocal fusion gene, namely ABL-BCR, intumorigenesis of CML is currently unknown. Melo et al.[6] and MacKenzie et al. [7] have shown that theABL-BCR gene is transcriptionally active in a portion ofCML patients and may have functional consequencesdue to the dysregulated GTPase activating proteins(GAP) activity. It is not yet clear whether the expressionor absence of normal BCR and ABL transcripts as wellas the chimeric break-region transcripts exert any influ-ence on the phenotype of the leukemic cells. It should benoted that in primary CML cells, normal ABL and BCRtranscripts were always found [8].

The aim of the present study was to investigate theincidence and distribution of the expression of theABL-BCR transcripts in a panel of human BCR-ABLpositive cell lines in relation to the occurrence of M-, m-,or m-bcr breakpoint cluster regions as well as the detec-tion of the alternative ABL splicing variants from exons1b or 1a in normal and aberrant ABL genes (ABL1b,ABL1a, ABL1b-BCR and ABL1a-BCR).

2. Materials and methods

2.1. Culture of cell lines

The continuous cell lines were taken from the stock ofthe DSMZ cell bank or were provided for researchpurposes by the original investigators [9,10]. Cell lineswere grown at 37°C in a humidified atmosphere of air

containing 5% CO2. The basic growth media (LifeTechnologies, Eggenstein, Germany) were supplementedwith 10–20% foetal bovine serum (Sigma, Deisenhofen,Germany). For factor-dependent cell lines, specificgrowth factors or conditioned media containing growthfactors were added. Cells were harvested in their logarith-mic growth phase with viabilities exceeding 90% asdetermined by trypan blue dye exclusion using standardhematocytometers. Cell pellets were stored at −20°C orprocessed immediately.

2.2. RNA isolation and re6ersetranscriptase-polymerase chain reaction (RT-PCR)

Total cellular RNA was isolated using the guani-dinium isothiocyanate method followed by cesium chlo-ride gradient centrifugation for purification. Fivemicrograms of the total RNA were used for the synthesisof first strand cDNA applying a RT preamplification kit(SuperScript; Life Technologies). The RT was carried outwith 50 ng of random hexamers in a final volume of 20ml RT buffer (containing 20 mM Tris–HCl, pH 8.4, 50mM KCl, 1.5 mM MgCl2, and 0.1 mg/ml bovine serumalbumin). The mixture was incubated at 70°C for 10 minand 10 min at room temperature; 200 U of Moloneymurine leukemia virus RT and 1 ml of 10 mM dNTP mixwere added to the reaction and incubated at 42°C for 50min. The reaction was stopped by heating to 70°C for 5min and then the tube was quickly chilled on ice. Afterbrief centrifugation, 2 U of RNase H were added to thereaction mixture and incubated for 20 min at 37°C. Onemicrolitre of the first strand cDNA (corresponding to0.25 mg of RNA) was diluted with PCR buffer (10× : 500mM KCl, 15 mM MgCl2, 100 mM Tris–HCl, pH 8.8,0.8% Nonidet P40) containing 10 pmol of each senseF-ACTIN and antisense R-ACTIN primer, 5 nmol ofdNTP mix, 10 ml of Q-solution and 1.25 U of Taq DNApolymerase (Qiagen, Hilden, Germany) for the PCRreaction to determine the quality of the RNA, the RTreaction and the PCR amplification. The PCR reactionwas performed with a DNA thermal cycler (Perkin ElmerCetus, Heidelberg, Germany) under the following condi-tions: denaturation for 7 min at 94°C; 3 min at 72°C andaddition of the Taq polymerase to perform a hot startPCR; 2 min at 60°C and 10 min at 72°C for one cycle.The amplification was carried out for 35 cycles of 30 sat 94°C, 30 s at 60°C and 60 s at 72°C with 2 s of extensiontime per cycle. Nine microlitres of the reaction mix wereelectrophoresed on an ethidium bromide-stained 1.3%agarose-TAE-gel and observed under UV-light. For theamplification of BCR-ABL and ABL-BCR fragments,the same conditions were used except for the amount ofcDNA (3 ml corresponding to 0.75 mg RNA), no Q-solu-tion was added and different annealing temperatureswere applied (see Table 1). Primers were purchased fromEurogentec (Seraing, Belgium).

C.C. Uphoff et al. / Leukemia Research 23 (1999) 1055–1060 1057

3. Results and discussion

3.1. Expression of fusion gene mRNAs

The expression of the various gene products of BCRand ABL in t(9;22) positive leukemia cell lines, whichinclude the normal BCR and ABL1b and ABL1a mR-NAs, the transcripts of the fusion gene of the Phchromosome (BCR-ABL) and the mRNAs of the recip-rocal fusion gene (ABL-BCR), was investigated. Thecell lines used for this study contain all described formsof translocations and were established from patientswith ALL, acute myeloid leukemia (AML) and CML inblast crisis (CML-bc). Except for SD-1, a B-lymphoblastoid cell line which was immortalised byEpstein–Barr virus (EBV), all cell lines were establishedfrom the leukemic cells.

Since the BCR-ABL translocation and the expressionof the BCR-ABL fusion gene do not appear to repre-sent the sole causative event in leukemogenesis, theexpression of the reciprocal fusion gene ABL-BCRmight as well be involved in the transformation of thecells [6]. In reciprocal translocations, the identificationof the derivative chromosome containing the criticaljunction is essential. This analysis is not difficult in

cases where only one transcript is realised. However,balanced translocations may lead to the expression oftranscripts from both fusion genes; both might be es-sential for the resulting phenotype, in particular whenseveral breakpoints in one gene or a chromosomalregion including several genes are found. For severalfusion genes generated by translocations it was shownthat both transcripts of the fusion products are indeedexpressed and are considered to be oncoproteins drivingthe malignant leukemic transformation: AF1P, AF4,AF6, AF9, AF17, ENL and AFX1 are some of thenumerous fusion partners of the MLL gene on chromo-some 11 commonly found in lymphoblastic andmyeloid (monoblastic) acute leukemia [11]. Likewiseboth transcripts are found in the fusion genes of ERGand TLS (t(16;21)) as well as AML1 and TEL (t(12;21))[12,13].

3.2. Expression of normal BCR and ABL transcripts

The translocation of chromosomes 9 and 22 usuallyaffects only one of the two alleles. To demonstrate thatthe normal genes are still expressed in the BCR-ABLpositive cell lines and that their integrity is preserved,we employed RT-PCR for the amplification of the

Table 1Primer sequences and annealing temperatures applied for RT-PCR and PCR fragment lengths

Annealing temperaturePrimer SequenceGene and fu- Fragment length°Csion type (bp)

b-Actin 6011285%-ATG-GAT-GAT-GAT-ATC-GCC-GCG-3%F-ACTINR-ACTIN 5%-CTA-GAA-GCA-TTT-GCG-GTG-GAC-3%

65F-BCR-E1A 5%-CCC-CCG-GAG-TTT-TGA-GGA-TTG-C-3%BCR-ABL 577(e1-a2)

R-ABL-A3 5%-CCA-TTT-TTG-GTT-TGG-GCT-TCA-CAC-CAT-TCC-3%5%-CAT-GGC-CTT-CAG-GGT-GCA-CAG-3%BCR-ABL 467F-BCR-E12A 65

(b2-a2, b3-a2) R-ABL-A3 See above 53855ABL-BCR F-ABL-1A2 5%-GAG-ATC-TGC-CTG-AAG-CTG-GT-3% 440

(1a-e17)3655%-ATC-CCA-TTC-ATG-GCG-ATG-AC-3%R-BCR-E17585 55ABL-BCR 5%-ACG-TAT-ATG-CCA-TTT-CCC-TC-3%F-ABL-1B2

(1b-e17) R-BCR-E17 see above 510ABL-BCR 335see aboveF-ABL-1A2 55

R-BCR-E2(1a-e2) 5%-AGG-TAA-GTC-TCC-TCG-CTA-GCC-AGG-ATT-3%ABL-BCR F-ABL-1B2 See above 480 55

R-BCR-E2(1b-e2) See above55F-ABL-1A2ABL See above 320

(1a)R-ABL-A3 See above

55F-ABL-1B2ABL See above 466(1b)

R-ABL-A3 See aboveSee aboveF-BCR-E12A 582BCR 55

R-BCR-E17 See aboveF-BCR-E1ABCR See above 591 55R-BCR-E2 See above

C.C. Uphoff et al. / Leukemia Research 23 (1999) 1055–10601058

normal BCR and ABL genes. The primer combinationswere chosen depending on the type of BCR-ABL translo-cation (Table 1). A region that spans the chromosome22 BCR breakpoint was amplified to avoid the amplifi-cation of the BCR region in the mutated gene.

Except for BV-173, all lymphoid cell lines expressedone (n=1 cell line) of the two possible ABL transcripts(ABL exon 1a or ABL exon 1b) or both (n=16 cell lines)(Table 2). The lack of ABL expression in BV-173 mightbe due to the absence of the normal chromosome 9,which could not be detected by G-banding and FISH [9].Seven out of 18 (39%) lymphoid cell lines expressed thenormal BCR gene. In the group of m-bcr cell lines, allnine cell lines are BCR-negative. On the other hand,CML-T1, NALM-24, and SD-1 were the only cell linescarrying a b2-a2 or b3-a2 M-bcr translocation which didnot express a normal BCR.

Concerning the myeloid lineage, only 45% (16/35)expressed the normal ABL gene whereas 77% (27/35) ofthe myeloid cell lines expressed the BCR gene mRNA.Except for two cell lines (expressing only ABL1a), themyeloid cell lines were either positive (n=14 cell lines)or negative (n=19 cell lines) for both ABL1a and ABL1btranscripts. The present findings are at variance withprevious results which showed that normal ABL isubiquitously expressed and that both ABL gene tran-scripts can be detected in all patients [6]. This might bedue to secondary alterations in the cell lines or to uniqueaberrations, as seen in the cell line KBM-7 which exhibitstwo Ph chromosomes [14].

3.3. Expression of BCR-ABL gene transcripts

Two forward primers for the BCR gene (from exon 1and from exon 12) and one oligonucleotide for the ABLgene (exon 3) were used to amplify the different formsof BCR-ABL fusion cDNA (Table 1). All known typesof BCR-ABL translocations can be identified using theseprimer combinations (Fig. 1).

The types of molecular translocation in the cell linesinvestigated correspond to those found in primary mate-rial: 50% of the lymphoblastoid cell lines (9/18) displaythe m-bcr translocation (e1-a2), whereas this transloca-tion was not found in the myeloid lineage (0/35). Cell lineMR-87 was described to contain the m-bcr translocation,and cell line HNT-34 was reported to carry both translo-cations (M-bcr and m-bcr); we were not able to amplifythe specific m-bcr PCR products in these cell lines. m-bcrwas detected at low levels in cell line HNT-34 by the verysensitive nested RT-PCR method by Hamaguchi et al.[15]. But van Rhee et al. [16] were able to demonstrateby RT-PCR that p190 BCR-ABL mRNA is expressed atlow levels in p210-positive CML and ALL even whenthere is no cytogenetic evidence for both translocations.Additionally, the karyotype of the cell line HNT-34shows only one Ph chromosome, which makes it unlikely

that both translocations occurred in the cell line. Cell lineMR-87 was described to exhibit the Ph chromosome butno BCR rearrangement was detected [17]. This points toanother chromosomal aberration different from theknown BCR-ABL abnormality. Among the M-bcr rear-rangements about 75% belong to the b3-a2 type oftranslocation, found in lymphoid (7/9) as well as inmyeloid cell lines (24/33).

Cell line AR-230 displayed the rarely found m-bcrtranslocation with breakpoints downstream of exon 18as previously described by Wada et al. [18]. This cell lineappears to be presently the only available model for theserare breakpoints.

3.4. Expression of reciprocal ABL-BCR transcripts

Depending on the breakpoint occurring upstream ofexon 1b, 1a or a2 in the ABL gene, none, one or two ofthe alternatively spliced mRNAs can be formed by thereciprocal fusion product, respectively. Additionally, thebreakpoint in the BCR gene determines the structure ofthe transcript. The b2-a2 and b3-a2 translocations wereamplified by oligonucleotides for exon 17 of the BCRgene, whereas for the e1-a2 translocation a reverse primerin exon 2 was chosen (Table 1).

Fifty-six percent (10/18) of the cell lines assigned to thelymphoid lineage expressed the ABL1b-BCR splice vari-ant. These ABL1b-BCR positive cell lines were dis-tributed among the different translocation types asfollows: 67% (6/9) of the e1-a2 fusion genes, 43% (3/7)of the b3-a2 fusion genes and 50% (1/2) of the b2-a2fusion genes. This distribution does not correspond to theABL1b expression of the normal ABL allele (except forBV-173, all lymphoid cell lines expressed normalABL1b). No information has been reported on the exactbreakpoints in the ABL gene.

Within the myeloid lineage, 66% (23/35) of the cell linesexpress the ABL1b-BCR transcript. The expression isequally distributed among the b2 and b3 translocationsof the BCR gene. Here, also no correlation can be foundbetween the ABL1b-BCR expression of the translocatedgene and of the normal ABL1b gene. Since no cell linewas positive for the ABL1a-BCR transcript, the majorityof the breakpoints within the ABL gene might be locatedupstream of exon 1a; additionally, more than one thirdof the breakpoints may even lie upstream of exon 1b.Melo et al. [6] reported that 34/49 samples (69%; 44 fromCML patients and 5 cell lines) were positive for theABL1b-BCR transcript (including 3 cell lines); six sam-ples (12%; all patient material) were positive for theABL1a-BCR transcript. Our results confirm these dataon the leukemia cell lines. MacKenzie et al. [7] reportedthat 7/12 CML samples (58%) were positive for ABL-BCR. No information about the nature of the transloca-tion or the splice variant is given.

In summary, we show that no normal BCR transcriptcould be found in any cell line with m-bcr translocation

C.C. Uphoff et al. / Leukemia Research 23 (1999) 1055–1060 1059

Table 2Expression of ABL, BCR, and resulting fusion genes in human BCR-ABL positive cell linesa

BCR–ABL fusionCell type Expression ofCell line

ABL1b–BCR ABL1a–BCR ABL1b ABL1a BCR

Lymphoid cell linese1-a2 + −ALL/MIK +BCP + −b3-a2 − −BCP +ALL-1 + +b2-a2 + − − −BV-173 +BCPb2-a2 − −T-cell +CML-T1 + −b3-a2 − −NALM-1 +BCP + +b3-a2 + −BCP +NALM-24 + –b3-a2 − −NALM-27 +BCP + +e1-a2 − −BCP +NALM-29 + –b3-a2 − −NALM-30 +BCP + +e1-a2 + −BCP +OM9;22 + –

PhB1 b3-a2B-LCL + − + + +e1-a2 + −B-LCL +SD-1 – –e1-a2 + −SUP-B15 +BCP + –b3-a2 + −T-cell +TK-6 + +e1-a2 + −TOM-1 +BCP + –e1-a2 − −BCP +Z-119 + –e1-a2 − −Z-181 +BCP + –e1-a2 + −BCP +Z-33 + –

Myeloid cell linesb3-a2 + −Ery.-mega. –AP-217 – +m-bcr − −AR-230 –Myelocytic – –b3-a2 + −Myelocytic –EM-2 – +

MyelocyticEM-3 b3-a2 − − – – +b3-a2 − −Myelocytic –GM/SO – –

MyelocyticHNT-34 b3-a2 + − + + +JK-1 b2-a2Erythrocytic + − – + +

b3-a2 + −Ery.-mega. –JURL-MK1 – +b3-a2 + −JURL-MK2 –Ery.-mega. – +b3-a2 − −Erythrocytic +K-562 + +b3-a2 + −KBM-5 –Monocytic – (+)b2-a2 + −Myelocytic –KBM-7 – –b2-a2 + −KCL-22 +Myelocytic + +b3-a2 + −Erythrocytic +KH88-B4D6 + +

ErythrocyticKH88-C2F8 b3-a2 + − + + +b3-a2 + −Myelocytic –KOPM-28 – +b3-a2 + −KU-812 –Myelocytic – –b2-a2 + −Myelocytic –KYO-1 – +

Ery.-mega.LAMA-84 b3-a2 – − – – +b3-a2 + −Ery.-mega. –LAMA-87 – –

Ery.-mega.LAMA-88 b3-a2 + − – – –b3-a2 − −MC3 +Megakaryocytic + +b2-a2 + −Megakaryocytic –MEG-01 – –b3-a2 + −MEG-A2 +Megakaryocytic (+) –b2-a2 − −Megakaryocytic +MEG-J + +b2-a2 + −MOLM-1 +Megakaryocytic + +– − −Myelocytic +MR-87 + +b3-a2 − −NS-Meg –Megakaryocytic – +b2-a2 − −Monocytic –RWLeu-4 – +

Ery.-mega.SAM-1 b3-a2 − − + + +b3-a2 + −Megakaryocytic –SKH-1 + +b3-a2 + −T-33 +Megakaryocytic (+) +b3-a2 − −Megakaryocytic –TS9;22 – +

MyelocyticYOS-M b2-a2 + − + + +b3-a2 + − +Megakaryocytic +YS9;22 +

a BCP, B-cell precursor; B-LCL, B-lymphoblastoid cell line; ery.-mega., erythroid-megakaryocytic; −, no expression; (+), weak expression; +,expression.

C.C. Uphoff et al. / Leukemia Research 23 (1999) 1055–10601060

Fig. 1. Expression of the various ABL, BCR, BCR-ABL and ABL-BCR transcripts in cell lines exhibiting m-bcr (TOM-1) or M-bcr(KCL-22, KOPM-28) detected on ethidium bromide-stained agarosegels after RT-PCR amplification. The sizes of the amplicons can beestimated by the 100 bp marker lanes and the exact sizes aresummarised in Table 1. As found for all e1-a2 m-bcr cell lines,normal BCR (indicated as BCR) is not expressed in TOM-1. None ofthe cell lines show a band in the ABL1a-BCR lane, indicating that notranscript of this reciprocal fusion gene is expressed. In KOPM-28,none of the possible two normal ABL transcripts could be detected.The different sizes of the BCR-ABL and ABL1b-BCR fragmentsillustrate the use of different primer pairs for m-bcr and M-bcr casesand the different breakpoints (exons b2 and b3 of BCR) in M-bcr.

References

[1] Melo JV. The diversity of BCR-ABL fusion proteins and theirrelationship to leukemia phenotype. Blood 1996;88:2375–84.

[2] Vickers M. Estimation of the number of mutations necessary tocause chronic myeloid leukaemia from epidemiological data. BrJ Haematol 1996;94:1–4.

[3] Melo JV. The molecular biology of chronic myeloid leukaemia.Leukemia 1996;10:751–6.

[4] Pane F, Frigeri F, Sinodna M, Luciano L, Ferrara F, Cimino R,Meloni G, Saglio G, Salvatore F, Rotoli B. Neutrophilic chronicmyeloid leukemia: a distinct disease with a specific molecularmarker (BCR/ABL with C3/A2 junction). Blood 1996;88:2410–4.

[5] Gotoh A, Broxmeyer HE. The function of BCR/ABL and relatedproto-oncogenes. Curr Opinion Hematol 1997;4:3–11.

[6] Melo JV, Gordon DE, Cross NCP, Goldman JM. The ABL-BCR fusion gene is expressed in chronic myeloid leukemia.Blood 1993;81:158–65.

[7] MacKenzie E, Stewart E, Birnie GD. ABL-BCR mRNAs tran-scribed from chromosome 9q+ in Philadelphia-chromosome-positive chronic myeloid leukaemia. Leukemia 1993;7:702–6.

[8] Melo JV, Hochhaus A, Yan XH, Goldman JM. Lack of correla-tion between ABL-BCR expression and response to interferon-al-pha in chronic myeloid leukaemia. Br J Haemat 1996;92:684–6.

[9] Drexler HG, Dirks W, MacLeod RAF, Quentmeier H, SteubeK., Uphoff CC, DSMZ Catalogue of Human and Animal CellLines, 7th ed. Braunschweig, Germany:DSMZ, 1999.

[10] Drexler HG, MacLeod RAF, Uphoff CC. Leukemia cell lines: invitro models for the study of Philadelphia chromosome-positiveleukemia. Leuk Res 1999;23:207–15.

[11] Rowley JD. Rearrangements involving chromosome band 11q23in acute leukaemia. Sem Cancer Biol 1993;4:377–85.

[12] Drexler HG, MacLeod RAF, Borkhardt A, Janssen JWG. Re-current chromosomal translocations and fusion genes inleukemia-lymphoma cell lines. Leukemia 1995;9:480–500.

[13] Shurtleff SA, Buijs A, Behm FG, Rubnitz JE, Raimondi SC,Hancock ML, Chan GC-F, Pui C-H, Grosveld G, Downing JR.TEL/AML1 fusion resulting from a cryptic t(12;21) is the mostcommon genetic lesion in pediatric ALL and defines a subgroupof patients with an excellent prognosis. Leukemia 1995;9:1985–9.

[14] Andersson BS, Collins VP, Kurzrock R, Larkin DW, Childs C,Ost A, Cork A, Trujillo JM, Freireich EJ. KBM-7, a humanmyeloid leukemia cell line with double Philadelphia chromo-somes lacking normal c-ABL and BCR transcripts. Leukemia1995;9:2100–8.

[15] Hamaguchi H, Suzukawa K, Nagata K, Yamamoto K, YagasakiF, Morishita K. Establishment of a novel human myeloidleukaemia cell line (HNT-34) with t(3;3)(q21;q26),t(9;22)(q34;q11) and the expression of EVI1 gene P210 and P190BCR/ABL chimaeric transcripts from a patient with AML afterMDS with 3q21q26 syndrome. Br J Haematol 1997;98:399–407.

[16] Van Rhee F, Hochhaus A, Lin F, Melo JV, Goldman JM, CrossNCP. p190 BCR-ABL mRNA is expressed at low levels inp210-positive chronic myeloid and acute lymphoblasticleukemias. Blood 1996;87:5213–7.

[17] Okamura J, Yamada S, Ishii E, Hara T, Takahira H, NishimuraJ, Yumura K, Kawa-Ha K, Takase K, Enomoto Y, Tasaka H.A novel leukemia cell line, MR-87 with positive Philadelphiachromosome and negative breakpoint cluster region rearrange-ment coexpressing myeloid and early B-cell markers. Blood1988;72:1262–8.

[18] Wada H, Mizutani S, Nishimura J, Usuki Y, Kohsaki M, KomaiM, Kaneko H, Sakamoto S, Delia D, Kanamaru A, Kakishita E.Establishment and molecular characterization of a novelleukemic cell line with Philadelphia chromosome expressing p230BCR/ABL fusion protein. Cancer Res 1995;55:3192–6.

and that no ABL1a-BCR transcript was detectable in anyof the cell lines examined. The results indicate that noconsistent ABL-BCR or even normal ABL or BCRexpression can be found for any type of translocation orcell lineage. Further studies are required to investigatethe possible influence of the lack of ABL1a-BCR.

Acknowledgements

S. Fombonne was supported by the European Union-sponsored OFAG program. C.C. Uphoff assembled thedata, provided the analysis and data interpretation anddrafted the paper. S. Habig and S. Fombonne helped tocollect the data and provided technical support. Y.Matsuo provided study materials and H.G. Drexlerprovided the original concept, design, helped with datainterpretation and the drafting of the paper.