Elsevier

Leukemia Research

Volume 23, Issue 11, November 1999, Pages 1055-1060
Leukemia Research

ABL-BCR expression in BCR-ABL-positive human leukemia cell lines

https://doi.org/10.1016/S0145-2126(99)00131-9Get rights and content

Abstract

Expression of normal ABL and BCR and of reciprocal fusion genes BCR-ABL and ABL-BCR was examined in a panel of 53 BCR-ABL-positive cell lines by RT-PCR to determine the influence of the various transcripts on leukemogenesis. Seventeen out of 18 lymphoid cell lines expressed ABL1a and/or ABL1b, whereas only 16 out of 35 myeloid cell lines expressed one or both normal ABL transcripts. Normal BCR was expressed in seven lymphoid cell lines; all cell lines from the m-bcr group (n=9) were BCR-negative. Among the myeloid cell lines, 77% expressed the BCR gene. The M-bcr and m-bcr translocations were equally distributed among cell lines with lymphoid phenotype. The m-bcr translocation was not found in myeloid cell lines. b3-a2 constitutes the predominant form of fusion gene in myeloid cell lines with an incidence of about 68%. One myeloid cell line exhibited the μ-bcr variant. An ABL-BCR transcript of the 1a splice variant was not detected in any of the cell lines. ABL1b-BCR was 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-a2 translocations. 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 be found with cell lineages and translocation types. Further work is required to determine the exact role of the reciprocal fusion gene transcripts on the pathophysiological mechanisms of leukemogenesis.

Introduction

Chronic myeloid leukemia (CML) is characterised by the occurrence of the Philadelphia (Ph) chromosome in about 95% of CML patients (for review see [1]). The Ph chromosome originates from the reciprocal translocation t(9;22)(q34;q11) by which the downstream portion of the ABL protooncogene from chromosome 9 is juxtaposed with the upstream portion of the BCR gene on 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 pathogenesis of the leukemia, but other thus far unidentified genetic changes are probably additionally involved in the progression of the disease, in particular for the transition to blast crisis [2].

The positions of the breakpoints in the genes are variable. In the ABL gene, the breakpoints are scattered between the 5′-end of the ABL gene and exon a2, resulting in unique transcripts all displaying ABL exon a2 in the BCR-ABL fusion mRNA [3]. Concerning the BCR gene, the majority of the breakpoints (M-bcr) found in Ph chromosomes lies within a 5.8 kb region spanning BCR exons 12 to 16 (also designated as b1 to b5). The bulk of breakpoints in the M-bcr results in one of two possible transcripts which differ by 75 bp and which either includes or deletes BCR exon b3. The expression of the M-bcr fusion gene leads to 210 kD proteins with enhanced tyrosine kinase activity (p210BCR-ABL). A second breakpoint cluster region, rarely found in CML patients but in two thirds of Ph+ acute lymphoblastic leukemia (ALL) patients (designated m-bcr), lies within a long intron between BCR exons 1 and 2 (termed e1 and e2). This m-bcr chimeric gene produces a 190 kD protein (p190BCR-ABL) [3]. Recently, a third breakpoint cluster region (designated μ-bcr) downstream of BCR exon 19 was discovered leading to the large p230BCR-ABL fusion protein which is found very rarely in CML patients [4].

The BCR-ABL fusion protein is thought to be involved in a defective β1-integrin function which might lead to the abnormal circulation and proliferation of the Ph+ progenitor cells. BCR-ABL is also involved in a number of signal transduction pathways including the activation of the RAS oncogene. The ABL part of the fusion protein appears to be responsible for the latter two functions as the normal ABL protein was described to have similar activities [5]. The normal ABL gene product (p145ABL) is an ubiquitously expressed non-receptor protein tyrosine kinase which fluctuates between the nucleus and the cytoplasm. The function of the normal BCR gene product is not yet entirely understood. The role of the reciprocal fusion gene, namely ABL-BCR, in tumorigenesis of CML is currently unknown. Melo et al. [6] and MacKenzie et al. [7] have shown that the ABL-BCR gene is transcriptionally active in a portion of CML patients and may have functional consequences due to the dysregulated GIPase activating proteins (GAP) activity. It is not yet clear whether the expression or absence of normal BCR and ABL transcripts as well as the chimeric break-region transcripts exert any influence on the phenotype of the leukemic cells. It should be noted that in primary CML cells, normal ABL and BCR transcripts were always found [8].

The aim of the present study was to investigate the incidence and distribution of the expression of the ABL-BCR transcripts in a panel of human BCR-ABL positive cell lines in relation to the occurrence of M-, m-, or μ-bcr breakpoint cluster regions as well as the detection of the alternative ABL splicing variants from exons 1b or 1a in normal and aberrant ABL genes (ABL1b, ABL1a, ABL1b-BCR and ABL1a-BCR).

Section snippets

Culture of cell lines

The continuous cell lines were taken from the stock of the DSMZ cell bank or were provided for research purposes by the original investigators [9], [10]. Cell lines were grown at 37°C in a humidified atmosphere of air containing 5% CO2. The basic growth media (Life Technologies, Eggenstein, Germany) were supplemented with 10–20% foetal bovine serum (Sigma, Deisenhofen, Germany). For factor-dependent cell lines, specific growth factors or conditioned media containing growth factors were added.

Expression of fusion gene mRNAs

The expression of the various gene products of BCR and ABL in t(9;22) positive leukemia cell lines, which include the normal BCR and ABL1b and ABL1a mRNAs, the transcripts of the fusion gene of the Ph chromosome (BCR-ABL) and the mRNAs of the reciprocal fusion gene (ABL-BCR), was investigated. The cell lines used for this study contain all described forms of translocations and were established from patients with ALL, acute myeloid leukemia (AML) and CML in blast crisis (CML-bc). Except for

Acknowledgements

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

References (18)

There are more references available in the full text version of this article.

Cited by (0)

View full text