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The ABL-BCR Fusion Gene Is Expressed in Chronic Myeloid Leukemia
By Junia V. Melo, D.E. Gordon, N.C.P. Cross, and J.M. Goldman
Although the BCR-ABL hybrid gene on chromosome 22qplays a pivotal role in the pathogenesis of chronic myeloid
leukemia (CML), little is known of the reciprocal chimeric
gene, ABL-BCR, formed on chromosome 9q+. By reverse
transcription/polymerasechain reaction amplification (RT/
PCR) we have detected ABL-BCR mRNA in cells from 31
of 44 BCR-ABL positive CML patients and 3 of 5 CML cell
lines. Of the 34 positive samples, 3 1 had classical t(9;22)
(q34;qll) translocations; in 3 samples there was no Philadelphia (Ph) and/or 9q+ chromosomes. ABL-BCR expression consisted of ABL(lb)-BCR mRNA in 26 patients and of
both ABL(lb)-BCR and ABL(la)-BCR mRNA species in 6 patients. The ABL-BCR transcripts encoded one or, more
rarely, both of the two potentialjunctions, designated ABLb3 and ABL-b4. which differed in size by 75 bp. In 2 patients, the BCR exon b3 was not present in either the BCRABL or the correspondingABL-BCR transcript, whereas in
5 patients exon b3 was present in both transcripts. Direct
sequencing of PCR fragments representing the full-length
coding sequence of ABL-BCR cDNAs type lb-b3,la-b3, Ibb4, and la-b4 showed an open reading frame predicted to
encode fusion proteins of 3 7 0 to 4 1 4 amino-acids. If an
ABL-BCR gene product is produced in CML cells, it may be
relevant as a mechanism for deregulating the GTPase activating protein (GAP) function of BCR.
0 1993 by The American Society of Hematology.
C
the reciprocal t(9;22), is not known. Whereas most of the
presently available data suggest that the P2 10 fusion protein
encoded by the BCR-ABL hybrid gene is involved in the
pathogenesis of CML;-l2 it is likely that other genetic changes
are also necessary for defining the full leukemic phenotype.
The expression ofthe 5‘ABL-3‘BCRhybrid gene in this translocation has not so far been studied, but it may have functional consequences if it leads to abnormal activation of BCRGAP.
We show here that the reciprocal ABL-BCR gene is transcriptionally active in over two thirds of Ph-positive CML
patients, and that translation of its cognate chimeric mRNA
into an ABL-BCR fusion protein is compatible with its sequence.
HRONIC MYELOID leukemia (CML) is characterized
cytogenetically by the presence of the Philadelphia (Ph)
chromosome, which originates from the reciprocal translocation t(9;22) (q34;qll). In the formation of the Ph chromosome, the bulk of the ABL protooncogene is translocated
from chromosome 9 onto the BCR gene in chromosome 22.
This gives rise to a novel chimeric BCR-ABL gene, which
encodes a 210-Kd (P210) fusion protein with prominent tyrosine kinase activity and transforming ability.
The product of the normal BCR gene is a 160-Kd(P160BCR)
cytosolic phosphoprotein, whose physiological role is not
clearly defined. It was shown to form cytoplasmic complexes
with P2
in Ph-positive CML cells, as well as with a
53-Kd protein of unknown function in both Ph-positive and
Ph-negative cell lines. Sequences encoded by the first exon
of BCR are responsible for the P160BCRserine/threonine kinase a ~ t i v i t yThese
.~
sequences overlap the src-homology 2
(SH2)-binding regions of the BCR gene that are essential for
the activation of the ABL tyrosine kinase and the transforming potential of the chimeric BCR-ABL
In its
central segment, BCR has some homology to the dbl oncogene
and the yeast CDC24 gene.6 The product of the latter is involved in the control of cell division after DNA replication.’
The C-terminus of BCR has recently been shown to have a
GTPase-activating protein (GAP) activity for p2 1“‘, a member of the RAS family of small GTP-binding proteins.*
In the t(9;22) translocation, the p2 1“‘ GAP domain of
BCR is absent from the BCR-ABL chimeric protein. The 3’
end of the BCR gene containing the coding sequence for this
domain is, in turn, fused to the 5’ end of ABL on chromosome
9. The “fate” of the 9q+ chromosome, the other partner in
From the MRC/LRF Leukaemia Unit, Department of Haematology, Royal Postgraduate Medical School, London, UK.
Submitted July I , 1992; accepted September 3, 1992.
Address reprint requests to Junia V. Melo, MD, PhD, Department
of Haematology, Royal Postgraduate Medical School, Ducane Rd,
London W I 2 ONN, UK.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I993 by The American Society of Hematology.
0006-4971/93/81 01-0015$3.00/0
158
MATERIALS AND METHODS
Patients and cell lines. Cells from a total of 44 CML patients
were 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-eightpatients showed
characteristic Ph and 9q+ chromosomes, 1 had an atypical 22qwithout a 9q+, and 5 were Ph-negative. All patients had clonal BCR
gene rearrangement by Southern blot analysis.
Five BCR-ABL-positive cell lines were also investigated. These
were well characterized lines from patients with CML in blast crisis:
K562, KCL-22, KYO-1, BV173,I3and LAMA-84.I4The HL60 promyelocytic cell line was used as a negative control for BCR-ABL
expression in all tests.
Polymerase chain reaction (PCR) amplijication. Amplifications
of specific sequences on the ABL, BCR, BCR-ABL, and ABL-BCR
genes were performed by reverse transcription (RT) of cDNA, followed by PCR (RT/PCR) by standard methods. Briefly, PB white
blood cells (WBC) were obtained by dextran sedimentation or by red
cell lysis of centrifuged buffy coat ~reparati0ns.l~
The patients’ WBC
and freshly explanted cells from lines in culture were washed twice
in phosphate buffered saline and processed for RNA extraction by
the guanidinium thiocyanate/CsCl gradient method.16The RNA was
reverse transcribed into cDNA with Mo-MuLV reverse transcriptase
(GIBCO-BRL, Gaithersburg, MD) using random hexamers and, in
some samples, oligo-dT primers. PCR amplification of cDNA was
performed as described elsewhere.” Precautions towards eliminating
the possibility of false PCR results were based on the recommendations by Kwok and Higuchi.’’ In brief: (1) cells, RNA, and cDNA
preparations were always handled in a room separate from that specifically dedicated to the analysis of PCR products; (2) different sets
of pipettes were dedicated to sample preparation and PCR product
Blood, Vol 81, No 1 (January 1). 1993: pp 158-165
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159
ABL-BCR FUSION GENE IN CML
handling, and plugged tips (aerosol resistant) were used in all steps;
(3) all reagents were prepared under sterile conditions in a laminarflow cabinet, and stored as single-use aliquots; and (4) each PCR
experiment included 6 to 8 test (CML) cDNA samplesplus 2 known
negative controls: BCR-ABL- and ABL-BCR-negative cDNA from
HL60 cells, and a H20-blank (ie, no cDNA). In no instance were
ABL-BCR products detected in either negative control. Furthermore,
test samples showing no ABL-BCR amplification alongside samples
that did yield an ABL-BCR PCR product were always found in any
given experiment, reinforcing the validity of the positive results. Five
microliters from each PCR was electrophoresed through ethidium
bromide stained 1% to 2% agarose mini-gels, visualized, and photographed under UV light. Samples that showed no ABLBCR product were submitted to a second round of amplification with nested
primers and 1 pL of the original PCR products as template.
Primers for PCR, Southern hybridization, and sequencing. The
sequences of the synthetic oligonucleotideprimers used in this investigation are shown on Table 1. Some of the primers were designed
to contain natural or forced restriction enzyme sites at their 5’ ends
to facilitate future cloning of the PCR fragments into phage or plasmid
vectors. The size of PCR fragments amplified with each primer pair
is shown in Table 2.
Southern hybridization. Electrophoresed PCR products from
some samples were transferred from agarose gels to nylon membranes
by Southern blotting, and tested for hybridization to a synthetic oligonucleotideprobe (primer BS+)5’-labeledwith Y-~~P-ATP.
Hybridization and high-stringency washings were camed out at the appropriate discriminating temperaturesfor the oligonucleotideas described
elsewhere.”
Sequencing. Direct sequencingof PCR products was performed
by the linear amplification sequencing method” and/or by the Taq
cycle sequencing method [USB, Cleveland, OH]. PCR products were
used directly as templates on an estimated basis of 100 fmol ss-DNA
per base per primer. Denaturing sequencing gels were prepared, electrophoresed, and autoradiographed by conventional techniques.
RESULTS
BCR-ABL gene. BCR-ABL amplification of cDNA with
primers B2+ and CA3- yielded fragments 385 and 465 bp
Table 1. Synthetic Oligonucleotide Primers Used in This Study
Primer
Aa+
Ab+
PAa+
PAb+
JcCA381
B2+
B3+
B4+
+
B7+
C4’
84-
ecc5-
GPB-
+.
Sequence’ ( 5
3’)
TTGGAGATCTCCCTGAAG
CTTCTGGAAAGGGGTACCTATTA
tacggaattcATGTTGGAGATCTCCCTGAA
CGCTGAgaaTTCTGGAAGATCTTGAA
GGAGTGTTTCTCCAGACTGTTG
TGTTGACTGGCGTGATGTAGTTGCTTGG
GAGCGTGCAGAGTGGGGAGGGAGAACATCCGG
TTCAGAAGCTTCTCCCTGACAT
TCTGAATGTCATCGTCCACTCAGCCA
ttcaaATCTGTACTGCACCCTGGAGCT
GGTCgAaTTCAACAGCAGGGAGTTCA
attagatCTGAAGCTGTACTTCCGTGA
acgtgaaTTCCAGTTTGGCTCAGCTGT
tcaaggaTCCACGCACTGGCGCACGAT
ataggaTCCTTTGCAACCGGGTCTGAA
gatgggaTCCAGCTGCAGGAGTA
TCAGGAAGGaTCCCGCTCTACGGAT
Gene
LocatioPZ6
ABL (la)
ABL (Ib)
ABL (la)
ABL (Ib)
ABL (Ill)
ABL (Ill)
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 the
cDNA sequence, but incorporate restriction enzyme recognition sites.
Table 2. PCR Amplifications of ABL, BCR, BCR-ABL.
and ABL-BCR cDNA
--
Primers
Aa+
Ab+
Bl+
B2+
PAa+
CA3-
ABL (from la)
ABL (from Ib)
BCR
BCR-ABL
G-
ABL (la)-BCR
JcJc-
++
Gene
G-
-
Ab+ ++ G-
ABL (Ib)-BCR
PAa+
PB-
ABL (la)-BCR
PB-
ABL (Ib)-BCR
84- *
ABL (la)-BCR
84-
ABL (b-BCR
PAb+
PAa+
PAb+
84’
B7+
c4+
-
++
++
-
++
++
BcC5PB- *
BCR (in ABL-BCR)
BCR (in ABL-BCR)
BCR (in ABL-BCR)
PCR Product
(length in bp)
374
462
1,208
385 (b2a2)
4 5 6 (b3a2)
1,072 (la-b4)
1,147 (la-b3)
1,148 (lb-b4)
1,223 (lb-b3)
1,208 (la-b4)
1,283 (la-b3)
1.31 6 (lb-b4)
1,391 (lb-b3)
189 (la-b4)
2 6 4 (la-b3)
2 9 7 (lb-b4)
3 7 2 (lb-b3)
456
398
527
-
These were primers used for direct sequencing of overlapping segments of the template ABL-BCR amplified cDNAs as PCRs PAa+ PBand PAb+ PB-.
-
long, representing the b2a2 and b3a2 type transcripts respectively (Fig 1A).
The types of BCR-ABL transcript expressed in the 44 patients and the five CML cell lines were as follows. In the CP
group, 45% of patients expressed only b2a2 and 50% only
b3a2 BCR-ABL transcripts. A relatively similar distribution
for b2a2 and b3a2 patients was observed in the BC group,
which includes the five cell lines (59% and 38%, respectively).
One C P and one BC patient expressed both b2a2 and b3a2
transcripts.
Normal ABL and normal BCR genes. Expression of the
2 major alternative transcripts from the normal ABL allele
was observed in all the 44 patients. This was shown by RT/
PCR amplification of the sequence spanning exon Ia to exon
111(PCR PAa+ * Jc-) and, likewise, the sequence from exon
Ib to exon I11 (PCR Ab’ c-$ Jc-). Of the five cell lines studied,
only LAMA-84 and BV113 did not express either ABL transcript, which agrees with the absence of a normal chromosome
9 in these
The normal BCR allele was also found to be expressed in
all patients and cell lines, as assessed by PCR amplification
of a 1,208-bp long fragment spanning the major breakpoint
cluster region (M-bcr), and extending downstream of exon
~ 7 . This
~ ~fragment
3 ~ ~includes the BCR-GAP coding region
in the normal
ABL-BCR gene. The formation of a n ABL-BCR fusion
gene, the reciprocal product of the BCR-ABL translocation,
should theoretically yield different transcripts, depending o n
the positions of the breakpoints in both ABL and BCR (Fig
2). If the breakpoint in ABL occurs upstream of exon Ib, n o
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MELO ET AL
160
M
1
2
3
4
5
6
7 8 9 1 0 1 1 M
A
+b3a2
+b2a2
B
Fig 1. (A) BCR-ABL and (B)
ABL-BCR PCR products in 1 1
representative samples, as detected on ethidium bromidestained agarose gels. BCR-ABL
bands represent fragments of
3 8 5 bp (b2a2) andlor 465 bp
(b3a2). correspondingto amplifications with primers 82' and
CA3 ABL-BCR products span
sequences between primersAb'
(in ABLexon Ib) and G (in BCR),
representing fragments 1,223
bp (lb-b3) and/or 1,148 bp (Ibb4) long. Lanes 1 to 1 0 are CML
patient samples; lane 11 is the
HL60 cell line (negative control).
M, DNA molecular weight
marker (pEMBL digested with
Tw I ) .
eb-b3
lb-b4
ABL-BCR transcript is formed. If the breakpoint is between
exons Ib and la, only transcripts originating from the exon
Ib promoter are possible (Ib-BCR);and if between exons la
and 11, two RNA species can be transcribed from the independent promoters in exons Ib and la (Ib-BCR and la-BCR).
The 49 samples were tested for expression of ABL-BCR
transcripts by RT/PCR amplification using sense primers on
ABL exons Ib (Ah') and la (PAa'). and anti-sense primers
on BCR 3' end (G-or P B - ) . A total of 34 out of 49 patients
(69%).including 2 Ph-negative patients and the patient without a 9q+ chromosome, showed ABL-BCR amplification of
the Ib-BCR type and 6 of these 34 also expressed la-BCR.
No case expressed la-BCR alone (Table 3). Likewise, abnormally large ABL-BCR fragments containing both exons Ib
and la were never found. In 7 of the 34 samples the ABLBCR products were only detected by nested PCR. In all the
others, the level of ABL-BCR transcriptsseemed comparable
to that of ABL, BCR, and BCR-ABL amplified products, as
estimated within the limitations of a standard, nonquantitative PCR assay. Among the I5 ABL-BCR negative samples,
4 (3 patients and the cell line K562) were Ph-negative, although BCR-rearranged and BCR-ABL positive.
.
Like RCR-ABL. the ABL-BCR transcripts are predicted
to vary in length, depending on whether BCR exons b3 or
b4 are joined to the S'ABL. This was confirmed by Ib-BCR
products 1,148 bp and/or 1,223 bp long when primers
Ah'
G- were used for the PCR amplification (Fig IB).
The same 75 bp difference in the length of the PCR products was observed in la-BCR amplifications with primers
-
PAa'
-
G-.
Therefore, the ABL-BCR expressing cases fell into 3 categories (Table 4): 12 showing b b 4 junction, 19 with b b 3
junction, and 3 cases with double transcripts (Ibb4 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 presence or absence of BCR exon b3 in each transcript was confirmed by Southem hybridization of the ABL-BCR fragments
with a 26-mer oligonucleotide (primer B3') spanning a sequence specific for exon b3 (Fig 3) and, in some cases, by
direct sequencing of the junction region in each PCR fragment.
Overall, the proportion of ABLBCR-expressing patients
who showed only b2a2 (56%), only b3a2 (38%), and both
b2a2 and b3a2 (6%)BCR-ABL transcripts matched the pro-
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ABL-BCR FUSION GENE IN CML
161
BCR-ABL
b2a2
lb-b3
L
0
Ib
E2 1
W
I m"I--" . ---=TI-
~
1
ABL-BCR
Fig 2. Schematic representation of the ABL, BCR, BCRABL, and ABL-BCR genes. Arrows indicate the most frequent
regions for breakpoints in the
ABL and the BCR genes. The
possible ABL-BCR transcripts
arising from the different breakpoints in BCR are shown underneath the corresponding BCRABL transcripts type b2a2 and
b3a2, respectively.
U
El
BCR-ABL
Z
la
la-b3
E2 1
W
W N
XI
I 1 IJLU-
1
b3a2
lb-b4
E2 1
g
Ib
ABL-BCR
la-b4
la
X
E2 1
-
~~~
~
portion of patients in each BCR-ABL category in the whole
series. The frequency of ABL-BCR expression within each
BCR-ABL major group was 73% and 62%)in the b2a2 and
the b3a2 groups. respectively.
When the expression of the BCR-ABL and the ABL-BCR
fusion genes was compared in each individual sample (Table
5). it was found that, in 27 patients (79%). the junction type
in ABL-BCR was exactly reciprocal to the junction in BCRABL. Thus, from the 19 b2a2-positive samples, 15 expressed
an ABL-b3 type of junction, and 2 expressed both ABL-b3
and ABL-b4 transcripts. Similarly, 8 out of I3 patients with
single b3a2 and 2 patients with both b2a2 and b3a2 transcripts
from BCR-ABL showed ABL-BCR expression of the ABLb4 type, as expected. However. in 7 samples the junction
type in the ABL-BCR transcripts did not correlate with the
reciprocal BCR-ABL products: these were 2 b2a2 patients
who expressed only ABL-W transcripts, and 5 b3a2 patients
in whom ABL-b3 type of transcripts were found either alone
(4 samples) or together with ABL-W transcripts ( 1 sample).
The junction region of BCR-ABL and ABL-BCR products
from this group of patients was sequenced (primers PAa'
R 4 - ) and results confirmed that, in 2 cases, BCR exon b3
was not expressed in either BCR-ABL or ABL-BCR, whereas,
in 5 cases. this exon was present in both fusion-gene products.
The same result was obtained by Southern hybridization of
BCR-ABL and ABL-BCR products with the oligonucleotide
probe R3' (Fig 3). In 5 ofthe 7 cases it was possible to repeat
the BCR-ABL and ABL-BCR amplifications in duplicate
blood samples obtained at different times, and/or in duplicate
RNA/cDNA preparations from the original blood samples.
The initial results were always reproducible. In 2 patients
duplicate samples were not available for analysis.
Coding seqircvce qj'clie ABL-BCR gene. PCR amplified
ABL-BCR fragments from cDNA of 2 samples were sequenced in overlapping segments. using 4 pairs of sense and
antisense primers (Table 2). The full-length PCR products
represented ABL-BCR type Ibb3 and Ia-b3 from I patient.
and I b b 4 and la-b4 from another. Each fragment started 60
bp (Ib) or IO bp (la) upstream of ABL's initiation ATG. and
ended 86 bp downstream the stop codon for PI 60RCR.
Table 3. ABL-BCR Transcripts Found in CML Patients
Table 4. Junction Type of the ABL(Ib)-BCR Transcripts
in the Positive Cases
Stage of Disease
(no. of cases)
None
Ib-BCR
Ib-BCR and la-BCR
Chronic phase (20)
Blast crisis (29)'
TOTAL (49)
5 (25%)
10 (34%)
15 (31%)
13 (65%)
15 (52%)t
28 (57%)
2 (10%)
4 (14%)
6 (12%)
~
Including the 5 CML cell lines.
t Including the cell lines KCL-22. KYO-1, and BV173.
Stage of Disease
(no. of cases)
lb-b3
lb-b4
lbb3 and lbb4
Chronic phase (15)
Blast crisis (19)'
TOTAL (34)
7 (47%)
12 (63%)'
19 (56%)
6 (40%)
6 (32%)
12 (35%)
2 (13%)
1(5%)
3 (9%)
Including the cell lines KCL-22, KYO-1. and BV173.
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MELO ET AL
162
In the 4 products. the sequences matched exactly ABL
exon
or la.” which joined in phase BCR exons b3 or
b4, maintaining the open reading frame (ORF) up to the
normal BCR stopcodon.” The ORFs ofthe ARL-BCR hybrid
cDNAs code for predicted fusion proteins of 4 14 amino-acids
(AA) (Ihb3). 395 A A (la-b3). 389 AA (lkb4).and 370 AA
(Ia-b4).
Table 5. BCR-ABL Transcriot Versus ABLfIbl-BCR TranscriDt
BCR-ABL
(no. of cases)
b2a2 (19)
b3a2 (13)
b2a2 and b3a2 (2)
ABL(lb)-BCR
lb-b3
lb-b4
lbb3 and lbb4
15
2‘
8
2
2
1’
0
4’
0
DISCUSSION
Cases in which the ABL-BCR transcripts are not reciprocal to the
BCR-ABL transcripts.
In Ph-positive CML. the coding sequences of two genes,
ABL and BCR. are disrupted as a result of the reciprocal
exchange between chromosomes 9 and 22. and two hybrid
genes are formed. BCR-ABL (on 22q-. Ph) and ABL-BCR
(on 9q+). Expression of the BCR-ABL gene was shown in
all cases of Ph-positive CML when the sensitive method
of RT/PCR amplification was used.’“’’ However, it was
not known whether the reciprocal ABL-BCR hybrid gene
is likewise functionally active in this disease. This possibility was raised previously in a single report” when a 3.5
M
1
2
3
4
5 6
7 8 91011M
b3a2
.
)
2
3
4
5 6
7 8
9 10 11 M
+lb-b3
Fig 3. Southern-blot hybridization with oligonucleotide
(primer) B3’ of (A) BCR-ABL and
(B) ABL-BCR PCR products Of
the same representative samples shown in Fig 1. The oligonucleotide probe sequence is
specific for BCR exon b3. Note
that in samples 2 and 8 both
BCR-ABL and ABL-BCR transcripts hybridize to this probe,
indicating the presence of BCR
exon b3 in the two hybridgenes.
Conversely, neither BCR-ABL
nor ABL-BCR transcripts from
sample 9 hybridize to the probe,
which confirms the absence of
exon b3 in both genes.
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ABL-BCR FUSION GENE IN CML
163
kb transcript, presumed to be a S’ABL-3‘BCR fusion meshybrid genes are not part of the same clone, which implies
sage, was detected in Northern blots of the BV173 cell
that BCR exon b3 alone was duplicated in the t(9;22).
line.
Whether this b3 duplication took place before or during the
In the present study, we show that ABL-BCR expression
translocation is not known. The breakpoint regions of both
can be detected by RT/PCR in WBC from approximately
BCR-ABL and ABL-BCR in these 5 cases are being cloned
70% of CML patients. It is noteworthy that, among the ABLfor further studies.
BCR positive patients, 2 were Ph-negative and 1 had no 9q+
The nucleotide sequence of the 4 different ABL-BCR
chromosome, showing that, like BCR-ABL, the ABL-BCR
hybrid cDNAs found in our series shows that in each case
gene rearrangement can also take place in the absence of
the S‘ABL-3’BCR junction is in phase as expected. Therecytogenetic evidence of a t(9;22).
fore, ABL-BCR fusion proteins of about 390 AA could be
In one third of our cases, no ABL-BCR expression was
translated, the exact size of each varying according to the
detected, in spite of repeated PCR tests with several combiexon contribution of ABL and BCR to the transcript. The
nations of primers. This group contains 4 of the Ph-negafact that ABL-BCR amplifications were obtained from
tive, BCR-ABL positive samples that may represent comoligo-dT primed cDNAs indicates the presence of polyplex and not reciprocal chromosome trans location^.^^-^'
(A) on these transcripts and suggests that translatable RNA
The remaining 14 samples (26% of the Ph-positive CML)
is produced. Investigations on the presence of such ABLpresumably result from translocation breakpoints in chroBCR proteins in cells from patients with CML are in promosome 9 upstream of ABL exon Ib,” or from deletions
gress.
in BCR sequences 3’ to the chromosome 22 b r e a k p ~ i n t . ~ ~ The significance of ABL-BCR expression in CML is
In neither case would an ABL-BCR hybrid message be
still unclear. It seems unlikely that the hybrid ABL-BCR
present.
gene has any primary oncogenic role, because it is not
The higher proportion (82%)of patients expressing only
present in at least one third of the CML patients. FurABL-BCR transcripts of the Ib-BCR type, compared with
thermore, the fact that ABL-BCR is expressed in CP as
those ( 18%) with transcripts initiated from both exons Ib
well as in BC of CML argues against a role in causing
and Ia (Ib-BCR and Ia-BCR) is probably a reflection of
disease progression. On the other hand, ABL-BCR
the frequency of breakpoints between exons Ib and Ia, and
expression could well be associated with specific clinical
Ia and 11. Because the distance between exons Ib and Ia is
and/or hematological features in subsets of CML patients,
greater than 200 kb,24approximately I O times that between
which may reflect prognosis and response to treatment.
exons Ia and I1 ( 1 9 kb),34 the probability of a break ocData from our present series are still insufficient to address
curring within the first large ABL intron might be 10-fold
this question.
greater than within the second i n t r ~ n . ~The
~ - ~fact
’ that a
If a functional ABL-BCR fusion protein is indeed prosingle transcript carrying both exons Ib and Ia was never
duced,
as predicted from the cDNA coding sequences, it
found shows that the normal mechanism of alternative
would
contain
a GAP-BCR domain in its C-terminus linked
splicing of these two exons3’ is maintained in the ABLto an N-terminal ABL sequence. Such an arrangement could
BCR hybrid gene.
alter the rucGAP function of BCR’ leading to either conThe overall similar distribution of ABL-BCR-positive pastitutive activation or inactivation of the rucGAP activity.
tients among the b2a2 and b3a2 BCR-ABL categories suggests
Moreover,
the Ib-BCR-encoded fusion protein, like the ABL
that there is no simple correlation between the breakpoint
type
I1
P145,3’,41,42
would have a myristoylation site at its
sites in ABL and in BCR. On the other hand, the structure
N-terminus and, therefore, unlike p 160BCR,it could become
of the BCR moiety of the BCR-ABL and ABL-BCR tranassociated with the cell membrane. Although mRNA
scripts from 7 patients showed unexpected patterns. In 2 of
expression from the normal BCR allele can be detected in
these, neither BCR-ABL nor ABL-BCR transcripts include
CML
cells,43it is not clear whether the level of P160BCR
BCR exon b3. This means that the breakpoint may be in
production is the same as in normal leukocytes. Coexpresexon b3 itself, or that this exon was included in deletions at
sion of an abnormal ABL-BCR gene product could result
the breakpoint site.39Another possibility is that b3 was spliced
in competition for the same target protein, ruc, and imbalout in the mature mRNA from whichever of the two chimeric
ance in the rate of ruc activation. The biologic effects of a
primary transcripts that retained it. Precedents for alternative
deregulated BCR are unknown. However, because the ruc
splicing of exon b3 in BCR-ABL are well established in those
proteins display relative myeloid s p e c i f i ~ i t yand
~ ~ are inpatients who express both b2a2 and b3a2 transcript^^^,^^,^^
volved in activation of the NADPH oxidase system of neuand, in the present study, by the 3 patients with both Ib-b3
t r o p h i l ~it, ~is~tempting to speculate that an ABL-BCR proand Ib-b4 ABL-BCR. More surprising are the 5 patients in
tein with altered GAP activity might have a role in
this study in whom both BCR-ABL and ABL-BCR transcripts
granulocyte functional defects.
contain BCR exon b3. In 3 of these (3 BC), the karyotype
showed single Ph and 9q+ chromosomes, in 1 (CP), the 22qACKNOWLEDGMENT
was longer than a typical Ph and there was no 9q+, and in
the fifth (CP),. additional i(Ph) chromosomes probably arose
We thank Julie Bungey for reviewing the karyotypes of patients
from duplication of the original Ph. Therefore in these cases,
and cell lines, and Dr Alan Hall (Chester Beatty Institute for Cancer
there is no evidence that the BCR-ABL and the ABL-BCR
Research, London) for valuable discussion.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
MELO ET AL
164
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1993 81: 158-165
The ABL-BCR fusion gene is expressed in chronic myeloid leukemia
JV Melo, DE Gordon, NC Cross and JM Goldman
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