Immunocytochemical study of BCR and bcr-abl localization in K562 cells
Aim: To obtain polyclonal antibodies against recombinant proteins recognizing Bcr domain and fusion region of Bcr-Abl and analyze the patterns of intracellular distribution of Bcr and Bcr-Abl proteins in K562 cells of chronic myelogenous leukemia. Methods: The coding sequences of DН and РН domains o...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Cite this: | Immunocytochemical study of BCR and bcr-abl localization in K562 cells / G.D. Telegeev, A.N. Dubrovska, V.A. Nadgorna, M.V. Dybkov, M.P. Zavelevich, S.S. Maliuta, D.F. Gluzman // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 81-83. — Бібліогр.: 13 назв. — англ. |
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nasplib_isofts_kiev_ua-123456789-1386032025-02-10T01:20:46Z Immunocytochemical study of BCR and bcr-abl localization in K562 cells Telegeev, G.D. Dubrovska, A.N. Nadgorna, V.A. Dybkov, M.V. Zavelevich, M.P. Maliuta, S.S. Gluzman, D.F. Original contributions Aim: To obtain polyclonal antibodies against recombinant proteins recognizing Bcr domain and fusion region of Bcr-Abl and analyze the patterns of intracellular distribution of Bcr and Bcr-Abl proteins in K562 cells of chronic myelogenous leukemia. Methods: The coding sequences of DН and РН domains of Bcr-Abl were cloned, and the recombinant proteins were expressed in E. coli. The rabbit polyclonal antibodies were produced and used for immunocytochemical study of Bcr and Bcr-Abl localization in K562 cells. Results: The gene constructs containing sequences coding for DН and РН domains of Bcr-Abl have been obtained. The antibodies with relative specificity to corresponding recombinant proteins differ by the patterns of their intracellular reactivity with Bcr- and Bcr-Abl related structures. While Bcr protein is located predominantly perinuclearly, antibody against hybrid Bcr-Abl protein is reacted with the structures in cell periphery, namely on cell membranes. Conclusion: Antibodies against DН and РН domains of Bcr-Abl react with proteins located differently in chronic myelogenous leukemia cells. The difference in intracellular localization of Bcr and Bcr-Abl may be attributable to the different domains interacting with different multiprotein complexes. 2010 Article Immunocytochemical study of BCR and bcr-abl localization in K562 cells / G.D. Telegeev, A.N. Dubrovska, V.A. Nadgorna, M.V. Dybkov, M.P. Zavelevich, S.S. Maliuta, D.F. Gluzman // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 81-83. — Бібліогр.: 13 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/138603 en Experimental Oncology application/pdf Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions Telegeev, G.D. Dubrovska, A.N. Nadgorna, V.A. Dybkov, M.V. Zavelevich, M.P. Maliuta, S.S. Gluzman, D.F. Immunocytochemical study of BCR and bcr-abl localization in K562 cells Experimental Oncology |
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Aim: To obtain polyclonal antibodies against recombinant proteins recognizing Bcr domain and fusion region of Bcr-Abl and analyze the patterns of intracellular distribution of Bcr and Bcr-Abl proteins in K562 cells of chronic myelogenous leukemia. Methods: The coding sequences of DН and РН domains of Bcr-Abl were cloned, and the recombinant proteins were expressed in E. coli. The rabbit polyclonal antibodies were produced and used for immunocytochemical study of Bcr and Bcr-Abl localization in K562 cells. Results: The gene constructs containing sequences coding for DН and РН domains of Bcr-Abl have been obtained. The antibodies with relative specificity to corresponding recombinant proteins differ by the patterns of their intracellular reactivity with Bcr- and Bcr-Abl related structures. While Bcr protein is located predominantly perinuclearly, antibody against hybrid Bcr-Abl protein is reacted with the structures in cell periphery, namely on cell membranes. Conclusion: Antibodies against DН and РН domains of Bcr-Abl react with proteins located differently in chronic myelogenous leukemia cells. The difference in intracellular localization of Bcr and Bcr-Abl may be attributable to the different domains interacting with different multiprotein complexes. |
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Article |
| author |
Telegeev, G.D. Dubrovska, A.N. Nadgorna, V.A. Dybkov, M.V. Zavelevich, M.P. Maliuta, S.S. Gluzman, D.F. |
| author_facet |
Telegeev, G.D. Dubrovska, A.N. Nadgorna, V.A. Dybkov, M.V. Zavelevich, M.P. Maliuta, S.S. Gluzman, D.F. |
| author_sort |
Telegeev, G.D. |
| title |
Immunocytochemical study of BCR and bcr-abl localization in K562 cells |
| title_short |
Immunocytochemical study of BCR and bcr-abl localization in K562 cells |
| title_full |
Immunocytochemical study of BCR and bcr-abl localization in K562 cells |
| title_fullStr |
Immunocytochemical study of BCR and bcr-abl localization in K562 cells |
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Immunocytochemical study of BCR and bcr-abl localization in K562 cells |
| title_sort |
immunocytochemical study of bcr and bcr-abl localization in k562 cells |
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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2010 |
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Original contributions |
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https://nasplib.isofts.kiev.ua/handle/123456789/138603 |
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Immunocytochemical study of BCR and bcr-abl localization in K562 cells / G.D. Telegeev, A.N. Dubrovska, V.A. Nadgorna, M.V. Dybkov, M.P. Zavelevich, S.S. Maliuta, D.F. Gluzman // Experimental Oncology. — 2010. — Т. 32, № 2. — С. 81-83. — Бібліогр.: 13 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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2025-12-02T10:56:44Z |
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Experimental Oncology 32, 81–83, 2010 (June) 81
Bcr-Abl, a constitutively active cytoplasmic tyrosine
kinase essential for the initiation of chronic myeloge-
nous leukemia (CML) is coded by the gene derived from
the fusion of the breakpoint cluster region (BCR) gene
on chromosome 22 and the Abelson leukemia onco-
gene (ABL) on chromosome 9. At diagnosis, up to 95%
of CML cases have the characteristic t(9; 22)(q34; q112)
reciprocal translocation giving the origin to Philadelphia
chromosome [1]. Depending on the breakpoint region
of the BCR gene implicated in the translocation, various
Bcr-Abl chimeras have been observed: p190 Bcr-Abl,
p210 Bcr-Abl and p230 Bcr-Abl [2]. The most frequent
one is p210 Bcr-Abl, which is responsible for CML, while
p190 Bcr-Abl is responsible for acute lymphoblastic
leukemia (ALL). Rarely, the breakpoint in BCR gene
occurs in μ-BCR region spanning exons 17–20 and
a larger fusion protein p230 is encoded. Patients with
this fusion demonstrate prominent neutrophil matura-
tion and/or conspicuous thrombocytosis.
The only structural difference between p190 and
p210 Bcr-Abl is the presence of Dbl homology (DH) and
pleckstrin homology (PH) domains in p210 Bcr-Abl. Thus,
a complete understanding of the biological mechanisms
underlying the origin of ALL and CML requires the charac-
terization of the signaling activities that reside within Bcr.
The isolated recombinant DH domain of Bcr is suggested
to be an activator of Rho GTPases [3]. The function of PH
domain has not yet been clarified in details. Recently, PH
domain has been shown to bind various phospholipids
and to take part in protein-protein interactions [4]. The
resulting Bcr-Abl fusion protein acts as an oncoprotein
and the constitutive activation of tyrosine kinase activi-
ty contributes to Bcr-Abl mediated leukemogenesis.
p210 Bcr-Abl unlike normal p145 c-Abl has been shown
to localize predominantly in cytoplasm [5]. The ectopic ex-
pression of p210 Bcr-Abl seems to be a factor contributing
to leukemic transformation of hematopoietic cells. In the
cytoplasm Bcr-Abl interacts with multiple signal transduc-
tion pathways that transmit anti-apoptotic and mitogenic
signals. The key pathways involve Ras, MAP kinases, the
STAT family, PI3 kinase, and myc, among others [2, 6].
The aim of the study is to obtain the specific anti-
bodies recognizing Bcr domain and fusion region of
Bcr-Abl and to study immunocytochemically the pat-
terns of intracellular distribution of Bcr and Bcr-Abl
proteins in the cells of chronic myelogenous leukemia.
In this study, the gene constructs containing se-
quences coding for DН and РН domains of Bcr-Abl
have been obtained, the corresponding fusion proteins
were produced in bacteria, purified and used for the
immunization. The polyclonal antibodies to Bcr domain
and to region of Bcr-Abl fusion were obtained and the
subcellular localization of Bcr-Abl and Bcr in K562 cells
was examined. Our data should help us to understand
the molecular mechanisms underlying the phenotypes
of Bcr-Abl positive leukemias and to find new targets
for therapeutic intervention.
MATERIALS AND METHODS
Cell line. Bcr-Abl positive K562 cells of blast phase
of human chronic myelogenous leukemia were obtained
from cell line depository of R.E. Kavetsky Institute of Ex-
perimental Pathology, Oncology and Radiobiology NAS
of Ukraine. The cells were cultured in suspension in 5%
CO2 — 95% air mixture at the temperature of 37 °C in RPMI
1640 medium supplemented with 10% fetal bovine serum
(Hyclone Laboratories, Logan, UT), 0.1% gentamycin
(Hyclone), and 1% L-glutamine (Hyclone).
IMMUNOCYTOCHEMICAL STUDY OF BCR AND BCR-ABL
LOCALIZATION IN K562 CELLS
G.D. Telegeev1, A.N. Dubrovska1, V.A. Nadgorna2, M.V. Dybkov1,
M.P. Zavelevich2, S.S. Maliuta1, D.F. Gluzman2, *
1Institute of Molecular Biology and Genetics of NAS of Ukraine, Zabolotnogo 150, Kiev 03143, Ukraine
2RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of NAS of Ukraine,
Vasylkivska 45, Kiev 03022, Ukraine
Aim: To obtain polyclonal antibodies against recombinant proteins recognizing Bcr domain and fusion region of Bcr-Abl and analyze
the patterns of intracellular distribution of Bcr and Bcr-Abl proteins in K562 cells of chronic myelogenous leukemia. Methods: The
coding sequences of DН and РН domains of Bcr-Abl were cloned, and the recombinant proteins were expressed in E. coli. The
rabbit polyclonal antibodies were produced and used for immunocytochemical study of Bcr and Bcr-Abl localization in K562 cells.
Results: The gene constructs containing sequences coding for DН and РН domains of Bcr-Abl have been obtained. The antibodies
with relative specificity to corresponding recombinant proteins differ by the patterns of their intracellular reactivity with Bcr- and
Bcr-Abl related structures. While Bcr protein is located predominantly perinuclearly, antibody against hybrid Bcr-Abl protein
is reacted with the structures in cell periphery, namely on cell membranes. Conclusion: Antibodies against DН and РН domains
of Bcr-Abl react with proteins located differently in chronic myelogenous leukemia cells. The difference in intracellular localization
of Bcr and Bcr-Abl may be attributable to the different domains interacting with different multiprotein complexes.
Key Words: Philadelphia chromosome, Bcr-Abl, CML, ALL, polyclonal antibodies, K562 cells.
Received: May 25, 2010.
*Correspondence: E-mail: butenko@onconet.kiev.ua
Abbreviations used: ABL — Abelson leukemia oncogene; BCR —
breakpoint cluster region; CML — chronic myelogenous leukemia.
Exp Oncol 2010
32, 2, 81–83
82 Experimental Oncology 32, 81–83, 2010 (June)
Cloning of coding sequences of DН and РН do-
mains of BCR-ABL. Expression construct рDDВА3 based
on рЕТ32b vector coding for DH domain of Bcr was de-
signed in the following way: The amplification product
of cDNA corresponding to the region 1955–2810 b.p.
according to GenBank X02596.1 was cloned in pET32b
at EcoRI site. The recombinant protein was expressed in
ВL21/DЕЗ strain of E. coli. Another expression construct
pPDBA 138 designed on the basis of рЕТ32b vector
was used to obtain the recombinant protein comprising
PH domain of Bcr and Abl fragment corresponding to
2744–3333 b.p. region of Bcr-Abl according to GenBank
X02596.1. The details of cloning procedures are presented
in [3]. The alignment of the coding sequences in both
constructs is schematically given in Fig. 1.
60 b.p.
DH region
582 b.p.
PH region
197 b.p.
рРDBA138
рDDBA3
Overlapping
region 66 b.p.
2744 2810 3087 3099 3270 3333
Bcr sequence Abl
1955 2015 281026132597
PH region
343 b.p.
C2coding region
171 b.p.
Abl
63 b.p.
Fig. 1. Schematical alignment of the cloned fragments of Bcr-Abl
in expression constructs рDDBA3 and рРDBA138
Purification of recombinant proteins and pro-
duction of polyclonal antibodies. The recombinant
proteins DDВА3 and РDВА 138 were purified by affini-
ty chromatography on Ni-NTA agarose and dialyzed
against PBS pH 7.4. The purity of antibodies was
assessed by electrophoresis in agarose gel (Fig. 2).
a
b
1 2 3
1 2 3 4 5 6 7
Fig. 2. Electrophoregrams of purification stages of recombinant
protein РDBA138: a, 1 — LMW standards; 2 — purified inclusion
bodies; 3 — soluble fraction of recombinant protein РDBA138 af-
ter dialysis against PBS; b, isolation of recombinant proteins by
affinity chromatography under the native conditions on Ni-NTA
agarose: 1–7 stages of elution with step-wise imidasol gradient
40, 60, 80, 100, 150, 200, 250 mМ
The purified proteins were used for producing poly-
clonal antibodies. The recombinant proteins mixed with
complete Freund adjuvant were injected to the male
rabbits, and in 8 and 12 weeks booster immunizations
followed. The titer of specific antibodies was assessed
in serum by immunoenzyme technique. The antibodies
were depleted on BrCN-activated Sepharose (Аmersham
Рhаrmасіа Віоtесh Іnс., USA) with immobilized total
bacterial proteins of Е. соlі ВL21(DЕЗ) as well as the
peptide expressed by pET32 itself. The specificity of
the antibodies obtained was assessed by Western blot.
Immunochemical technique. K562 cells were
used as a model for immunocytochemical study of
Bcr and Bcr-Abl localization. The cells were fixed
with formalin-acetone pH 7.6 and permeabilized with
0.1% Triton X-100. The specimens were treated with
anti-DDBA3 or anti-PDBA138 antibodies. EnVisionAP
system (DAKO, Denmark) was used as the secondary
antibody. The immunochemical staining was visuali-
zed upon cytochemical reaction with naphtol-AS-BI-
phosphate as the substrate of alkaline phosphatase.
RESULTS AND DISCUSSION
The results of Western blot with purified antibo dies
against DDВАЗ and РDВА 138 proteins are given in
Fig. 3. The lysates from K562 cells, the peripheral
blood cells of CML patients and the healthy donors
were used. Both anti-DDBA3 and anti-PDBA 138 reveal
p210 and p160 proteins in K562 cells and in cells from
CML patients. The cross-reactivity may be explained
by the presence of the short overlapping Bcr se-
quence, 22 amino acid residues in length (see Fig. 1),
in both recombinant proteins. Nevertheless, the affinity
of binding seems to be more pronounced in the case
of anti-PDBA 138 antibody. Therefore, we attempted
to use both antibodies in the analysis of intracellular
distribution Bcr and Bcr-Abl in K562 cells.
1 2 3 4 5 6 7
— 210 kD
— 160 kD
— 108 kD
a b
Fig. 3. Western blot with polyclonal anti-DDBA3 and anti-PD-
BA138 antibodies against the corresponding recombinant proteins.
а, b — detection of chimerical protein Bcr-Abl (210 kD) and its
normal analogue (160 kD) in polymorphonuclear cells of healthy
donors and CML patients with polyclonal anti-DDBA3 (a) and anti-
PDBA138 (b) antibodies: 1, 4 — healthy donors; 2, 3, 6 — CML
patients; 5 — K562 cells; 7 — HMW protein standard (Sigma, USA)
The polyclonal antibodies against DDВАЗ and РDВА
138 proteins recognizing different regions of Bcr-Abl hy-
brid protein allows one to analyze the intracellular location
of Bcr-Abl as well as Bcr proteins in CML cells in vivo. The
results of immunocytochemical analysis of K562 cells
with antibodies against DDВАЗ and РDВА 138 proteins
are presented in Fig. 4. Bcr protein is located predomi-
nantly perinuclearly, while anti-PDBA138 antibody against
Experimental Oncology 32, 81–83, 2010 (June) 83
hybrid Bcr-Abl protein is revealed predominantly in cell
periphery, namely on cell membranes. Therefore, the
anti-DDBA3 and anti-PDBA138 antibodies with relative
specificity to corresponding recombinant proteins differ by
the patterns of intracellular reactivity with Bcr- and Bcr-Abl
related structures.
a
b
c
Fig. 4. Topography of Bcr and Bcr-Abl in K562 cells assessed with
polyclonal anti-DDBA3 and anti-PDBA138 antibodies against the
corresponding recombinant proteins: a — K562 cells, MGG stain-
ing, x 900; b — perinuclear pattern of Bcr distribution (anti-DDBA3);
c — membrane pattern of Bcr-Abl distribution (anti-PDBA138)
In K562 cells transformed with pEGFP-Bcr-Abl con-
struct, the protein has predominantly cytoplasmic localiza-
tion generating cortical-F-actin ring [7] due to actin-bind-
ing domain in Abl moiety of Bcr-Abl hybrid protein [3, 8].
Also, in 32D myeloid cells Bcr-Abl locates on vesicle-like
structures that lacked detectable F-actin [9]. Such bind-
ing is not affected by mutations in actin-binding domain.
Our previous data [10] seem to hint at the mechanisms
of such location. Namely, PH domain has been shown to
bind to the membrane of Golgi complex, in particular with
PIP(3) and PIP(4). The different patterns of the intracellular
localization of Bcr and Bcr-Abl may be attributable to the
different domains interacting with different multiprotein
complexes. PDZ-binding domain in C-terminal part of Bcr
provides for its interaction with AF-6 protein facilitating Ras
binding producing the multimeric complex. As a result, Ras
and ERK are down-regulated [11]. Besides, PDZ-binding
domain provides for the interaction between Bcr and the
apical PDZ-K-1and Mint 3, the latter being the component
of the vesicular trafficking in the secretory pathway [12].
For understanding the role of Bcr in cell biology, the fact
of its displacement to the membrane upon the effects of
growth factors is of high importance [13].
Thus, our data show that in K562 CML cells Bcr-Abl
is predominantly localized to the cell periphery. This
fact seems to be explained by the presence of actin-
binding domain in Bcr-Abl hybrid protein. On the other
hand, the localization of Bcr may be partly explained by
the presence of PH and PDZ-binding domains.
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