Influence of BCR/ABL fusion proteins on the course of Ph leukemias.
ABSTRACT The hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a result of the t(9;22) translocation. This gene rearrangement results in the production of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is compelling evidence that the malignant transformation by BCR/ABL is critically dependent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes part in realization of the malignant phenotype. We supposed that additional mutations accumulate in this region of the BCR/ABL oncogene during the development of the malignant blast crisis in CML patients. In ALL patients having p210 fusion protein the mutations were supposed to be preexisting. Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that along with single-nucleotide substitutions other mutations, mostly deletions, had occurred. In an ALL patient a deletion of the 5th exon was detected. The size of the deletions varied from 36 to 220 amino acids. For one case of blast crisis of CML changes in the character of actin organization were observed. Taking into account the functional role of these domains in the cell an etiological role of such mutations on the disease phenotype and leukemia progression is plausible.
Article: Abl tyrosine protein kinase.[show abstract] [hide abstract]
ABSTRACT: Mammalian c-Abl belongs to an evolutionary conserved family of non-receptor tyrosine kinases. It is distributed both in the cytoplasm in association with F-actin, and in the nucleus where it binds chromatin. The normal function of c-Abl is poorly understood. Nevertheless, there has been rapid progress in the characterization of the structural features, signal transduction pathways, substrates and ligands involved in the action of c-Abl and Abl-derived oncogenes. These developments suggest that several mechanisms co-operate to allow regulation of normal cell growth by c-Abl and induction of leukemias by Bcr-abl.Seminars in Immunology 09/1995; 7(4):255-66. · 5.93 Impact Factor
Influence of BCR/ABL fusion proteins on the course of Ph
Gennady D. Telegeev, Anna N. Dubrovska, Mykhaylo V. Dybkov and
Stanislav S. Maliuta?
Department of Molecular Genetics, Institute of Molecular Biology and Genetics of the NAS of
Ukraine, Kyiv, Ukraine
Received: 25 October, 2002; revised: 30 July, 2003; accepted: 26 February, 2004
Key words: Philadelphia chromosome, leukemia, BCR/ABL gene, actin cytoskeleton
The hallmark of chronic myeloid leukemia (CML) and a subset of acute
lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a
result of the t(9;22) translocation. This gene rearrangement results in the production
of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is
compelling evidence that the malignant transformation by BCR/ABL is critically de-
pendent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes
part in realization of the malignant phenotype. We supposed that additional muta-
tions accumulate in this region of the BCR/ABL oncogene during the development of
the malignant blast crisis in CML patients. In ALL patients having p210 fusion pro-
tein the mutations were supposed to be preexisting.
Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that
along with single-nucleotide substitutions other mutations, mostly deletions, had oc-
curred. In an ALL patient a deletion of the 5th exon was detected. The size of the de-
letions varied from 36 to 220 amino acids. For one case of blast crisis of CML
changes in the character of actin organization were observed. Taking into account
the functional role of these domains in the cell an etiological role of such mutations
on the disease phenotype and leukemia progression is plausible.
Vol. 51 No. 3/2004
?Corresponding author: Stanislav Maliuta, Department of Molecular Genetics, Institute of Molecular Bi-
ology and Genetics of the NAS of Ukraine, 150 Zabolotnogo str., Kyiv, Ukraine, 03143; tel.: (38 044) 266
0729; fax: (38 044) 266 0759; e-mail: email@example.com
Abbreviations: ALL, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; CNL, chonic
neutrophilic leukemia; DH, Dbl homology; GEF, guanine nucleotide exchange factor; PTK, protein
More than 90% of cases of chronic myeloid
leukemia (CML)and 10–25% of cases of acute
lymphoblastic leukemia (ALL) are character-
ized by a reciprocal translocation between
chromosomes 9 and 22 (Clark et al., 1988;
Cortes et al., 1995). As a result, a BCR/ABL
hybrid gene is formed on the derivative Phila-
delphia chromosome (Ph). Depending on the
location of the breakpoint in BCR, three types
of fusion protein can be formed, all of which
exhibit deregulated protein tyrosine kinase
(PTK) activity compared to normal ABL
(Konopka et al., 1984; Quackenbush et al.,
1997). As a result, there is excessive tyrosine
phosphorylation of many intracellular pro-
teins including the BCR/ABL protein itself
(Laneuville, 1995; Liu et al., 1993).
Several forms of the BCR/ABL oncogene re-
sponsible for the pathogenesis of Philadel-
phia chromosomes positive (Ph+) human
leukemias are generated by this trans-
location. The breakpoint locations in the BCR
gene and the specific parts of BCR that are
left intact determine the forms of the
BCR/ABL oncogene. The main forms of
BCR/ABL are as follows: P210, which is
found in most cases of CML and in 50% of
cases of ALL, P185, which is found in ALL
and P230, found in chronic neutrophilic leu-
kemia (CNL) (Pane et al., 2000; Martinelli et
The differences between the p210 and
p185 proteins are associated with additional
Dbl homology (DH) and PH domains (exons
3–12 of the BCR/ABL gene) which are pres-
ent only in p210. The Dbl family proteins ca-
talyses guanine nucleotide exchange on the
Rho family of small GTPases. Members of
this family control cell progression, transcrip-
tion and actin cytoskeletal arrangement. DH
functions together with the PH domain
(membrane targeting module).
CML is a biphasic disease with an initial
chronic phase during which the disorder is
easilycontrolled. However, chronic-phase
CML is followed by a terminal blastic phase
that resembles acute leukemia and is usually
refractory to treatment. Transformation of
the chronic phase to blast crisis is accompa-
nied by secondary cytogenetic changes in ap-
proximately 85% of cases (Gribble et al.,
1999). However, the genetic events responsi-
ble for the transformation of CML are poorly
understood. We supposed that additional mu-
tations accumulate in the Dbl, PH region of
BCR/ABL oncogene during the development
of the malignant blast crisis in CML patients.
In ALL patients, having the p210 fusion pro-
tein, we suppose that these mutations were
Patient samples and cell culture. Periph-
eral blood cells from patients with a referring
diagnosis of CML were obtained for molecu-
lar studies as part of diagnosis workup. The
Ph-positive cell line K562 and the Ph-negative
cell line U937 were cultured in the presence
of 10% fetal calf serum in RPMI 1640 medium
in a humidified atmosphere with 5% CO2at
RT-PCR, DNA amplification, cloning
and sequencing. Total RNA was extracted
from white blood cells as well as from the con-
trol cell lines by the method of Chomczynski
and Sacchi (1987). The Ph-chromosome was
detected as described (Kawasaki et al., 1988).
For amplification of the Dbl homology region
bp 1955–2810 of BCR/ABL cDNA were: 30 s
of denaturation at 94?C, 30 s annealing at
56?C and 90 s of extension at 72?C (30 cycles).
The following primers were used: ext1 dbl
and extr1 dbl (5?-GATGTTGGGCACTGCC-
TCCAGTTC-3’) for the first round and ext dbl
and extr dbl (5?-GAATTCTGCCTCCAGTTCA-
TCCAC-3?), for the second round. PCR prod-
ucts were gel separated, excised and after pu-
rification were cloned in pUC19 and se-
quenced using the T7-sequencing TM Kit
846G.D. Telegeev and others2004
Fluorescence microscopy. Cells were
plated onto cover slips and grown overnight
before preparing them for immunofluo-
rescence. Cells were fixed in paraformal-
dehyde, washed with phosphate-buffered sa-
line (PBS) and permeabilized with 0.1% Tri-
ton X-100. FITC-labeled phalloidin (Sigma-
Aldrich, U.S.A.) was applied to the cells as
previously described (Wulf et al., 1997.)
The medical diagnoses “Ph-positive CML”
and “Ph-positive ALL” were confirmed by
nested RT-PCR. Polymorphonuclear cells of
peripheric blood from patients C. and K.
(CML, blast crisis), patients F. and Y. (ALL),
normal donors A. and B., cell lines K562
(CML, erythrocyte blast crisis), U937 (Ph-neg-
ative promonocytic leukemia) were used for
fluorescence microscopy. Comparative analy-
sis of actin distribution in polymorphonuclear
leukocytes of the different donors made it
possible to distinguish the following types of
cell staining: 1) diffuse distribution (normal
donors) (Fig. 1, D); 2) paramembrane actin
distribution in the cells of patients C., F., Y.,
cell line K562 and U937 (Fig. 1, AB); 3) forma-
tion of amorphous cytoplasm accumulation,
“dot-like structures” in one case of patient K.
(CML, blast crisis) (Fig. 1, C). The BCR/ABL
Vol. 51 BCR/ABL fusion proteins 847
Figure 1. Comparative analysis of actin distribution in cells of patients with Ph-positive leukemias, cells
lines K562 (Ph-positive) and U 937 (Ph-negative).
A, B, paracortical actin distribution which is peculiar to cells from lines K562, U937 and to cells of patients C.
(CML, blast crisis), Y. (ALL), F. (ALL); C, “dot-like” structures, which were detected in patient K. (CML, blast cri-
sis); D, diffuse actin distribution in cells of normal donors.
protein is a polyfunctional protein composed
of several domains with diverse properties
(Gishizky et al., 1996; Butturini et al., 1996).
This functional diversity of the BCR/ABL
protein enables its participation in different
signalling pathways. The DH domain of is pre-
sented in p210 BCR/ABL but not in p185
BCR/ABL. This domain encodes a guanine
nucleotide exchange factor activity specific
for Rho GTPases which modulate the cell
actin structure (Chuang et al., 1995). The
modulatory effect of the Dbl domain on actin
structure may underlay the different trans-
forming properties of the two types of
BCR/ABL fusion proteins. We supposed here
that the observed dot-like distribution of actin
correlated with some mutation, which could
arise in the Dbl-homology part of the
BCR/ABL gene. Indeed, sequencing of the
BCR/ABL amplification product from patient
K. confirmed the presence of mutational
changes in the Dbl homology region in posi-
tions 2127 (replacement T?C) and 2449 (re-
placement C?A) of BCR/ABL cDNA that cor-
responded to substitution in position 547
(Phe?Leu) and 654 (Thr?Lys) of the protein
molecule. Therefore the mutations were likely
to influence GEF function of the Dbl domain
and, as a consequence, change the BCR/ABL
transforming potential leading to progression
in CML. The data obtained may shed light on
the nature of CML blast crisis development
and can be used for early detection of CML tu-
mor progression as well as for elaboration of
more effective treatment protocols.
During electrophoretic analysis in some
samples together with the full-length amplifi-
cation products shorter fragments were de-
tected. Alteration of PCR conditions (i.e. in-
crease in temperature of primer annealing,
decrease of elongation time) did not signifi-
cantly affect the pattern of the PCR products.
For further analysis the PCR products were
cloned in pUC19 vector and sequenced. Anal-
ysis of those clones revealed that the changes
in length are caused by deletions in the Dbl
848G.D. Telegeev and others2004
The form of diseaseClone numberThe deleted domain, amino-acid residues
Case 1 (ALL)CL2514–733 (220)
CL6 585–620 (36)
115 556–683 (129)
Case 2 (CML)2K10 558–712 (155)
2K19-1517–573 (57) + 581–734 (154)
2K23 493–660 (168)
Case 3 (CML) 2L1582–729 (148)
Table 1. Analysis of Dbl domain of BCR/ABL protein from clones obtained from blood samples of
patients with CML and ALL.
Figure 2. Localization of deletions in Dbl domain
of BCR/ABL gene.
Numbers of clones are on the left. Numbering of nu-
cleotides is according to human bcr protein mRNA 5?
end (Genbank, HUMBCRD, Accession: M24603).
domain. These deletions did not affect the
reading frame. Localization of the deletions
in comparison with the structure of full-
length Dbl domain of the BCR/ABL gene is
presented in the Fig. 2. The respective dele-
tion-dependent alterations in the p210 BCR/
ABL protein are shown in the Table 1.
It is well known that in 50–60% of CML pa-
tients simultaneous expression of p210
BCR/ABL and p185 BCR/ABL genes is ob-
served (Saglio et al., 1996; Lichty et al., 1998).
Our study found novel mutations in the Dbl
region of p210 encoding transcripts of CML
patients. It may be suggested that the differ-
ent functional forms of the fusion proteins
studied affect the factors determining cell
morphology and this may influence the
course of the disease. The results obtained
show that a mutational change occurred in
the Dbl region of the BCR/ABL gene giving
rise to a p210 protein whose properties could
be similar to those of p185. Long term moni-
toring of patients with such mutations as well
as increasing the number of such cases may
contribute to our understanding of progres-
sion in this disease and aid in patients man-
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