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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1688-1699
The AML1/ETO(MTG8) and AML1/Evi-1 Leukemia-Associated Chimeric
Oncoproteins Accumulate PEBP2 (CBF) in the Nucleus More
Efficiently Than Wild-Type AML1
By
Kozo Tanaka,
Tomoyuki Tanaka,
Mineo Kurokawa,
Yoichi Imai,
Seishi Ogawa,
Kinuko Mitani,
Yoshio Yazaki, and
Hisamaru Hirai
From the Third Department of Internal Medicine and the Department of
Transfusion Medicine and Immunohematology, Faculty of Medicine,
University of Tokyo, Japan.
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ABSTRACT |
AML1, a gene on chromosome 21 encoding a transcription
factor, is disrupted in the (8;21)(q22;q22) and (3;21)(q26;q22)
chromosomal translocations associated with myelogenous leukemias; as a
result, chimeric proteins AML1/ETO(MTG8) and AML1/Evi-1 are generated, respectively. To clarify the roles of AML1/ETO(MTG8) and AML1/Evi-1 in
leukemogenesis, we investigated subcellular localization of these
chimeric proteins by immunofluorescence labeling and subcellular fractionation of COS-7 cells that express these chimeric proteins. AML1/ETO(MTG8) and AML1/Evi-1 are nuclear proteins, as is wild-type AML1. Polyomavirus enhancer binding protein (PEBP)2 (core binding factor [CBF]), a heterodimerizing partner of AML1 that is located mainly in the cytoplasm, was translocated into the nucleus with dependence on the runt domain of AML1/ETO(MTG8) or AML1/Evi-1 when
coexpressed with these chimeric proteins. When a comparable amount of
wild-type AML1 or the chimeric proteins was coexpressed with
PEBP2(CBF), more of the cells expressing the chimeric proteins showed the nuclear accumulation of PEBP2(CBF), as compared with the cells expressing wild-type AML1. We also showed that the chimeric proteins associate with PEBP2(CBF) more effectively than
wild-type AML1. These data suggest that the chimeric proteins are able
to accumulate PEBP2(CBF) in the nucleus more efficiently than
wild-type AML1, probably because of the higher affinities of the
chimeric proteins for PEBP2(CBF) than that of wild-type AML1.
These effects of the chimeric proteins on the cellular distribution of
PEBP2(CBF) possibly cause the dominant negative properties of the
chimeric proteins over wild-type AML1 and account for one of the
mechanisms through which these chimeric proteins contribute to
leukemogenesis.
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INTRODUCTION |
THE AML1 GENE was first
identified as the gene on chromosome 21 that is disrupted in the
(8;21)(q22;q22) translocation associated with acute myelogenous
leukemia.1 In t(8;21)(q22;q22), the gene rearrangement
results in the production of an AML1/ETO(MTG8) fusion
protein.2,3 Previously we reported that the AML1
gene is also disrupted and fused with the Evi-1 gene in the
(3;21)(q26;q22) translocation associated with the blastic crisis of
chronic myelogenous leukemia.4 Another group has also
reported that the AML1 gene is rearranged in the
(3;21)(q26;q22) translocation.5-7 Recently, it was reported
that the AML1 gene is rearranged in acute lymphoblastic leukemia carrying t(12;21)(p12;q22).8,9
PEBP2 B/CBF2, which is a mouse homolog of AML1,
was first identified as the gene encoding a member of the polyomavirus
enhancer binding protein (PEBP) 2 family or a core binding factor
(CBF) of Molony leukemia virus enhancer.10,11
PEBP2/CBF and PEBP2/CBF are components of the PEBP2/CBF
heterodimer, which binds to the cores of polyomavirus and Molony
leukemia virus enhancers.12,13 The mammalian
PEBP2/CBF subunits are encoded by three distinct genes:
AML1 (PEBP2B/CBF2), AML2
(PEBP2C/CBF3), and AML3
(PEBP2A/CBF1).1,10,11,14-16 A human homolog
of PEBP2/CBF is disrupted in inv(16)(p13q22) associated with
acute myelogenous leukemia.17 These facts suggest critical
roles of PEBP2/CBF in leukemogenesis.
AML1 has been shown to regulate the expression of several hematopietic
lineage-specific genes, such as those for myeloperoxidase, leukocyte
elastase,18 macrophage colony-stimulating factor
(colony-stimulating factor 1) receptor,19,20
granulocyte-macrophage colony-stimulating factor,21 and
T-cell receptors.22-26 We have shown that AML1 regulates
myeloid cell differentiation and transcriptional activation antagonistically by two alternative spliced forms, suggesting that a
transactivation property of AML1 is necessary for myeloid cell
differentiation.27 We also reported that the expression of
AML1 increases before morphological and functional differentiation of
U937 cells treated with all-trans retinoic acid.28
It was recently shown that mice lacking AML1 die during
midembryonic development because of extensive hemorrhaging and show the
complete absence of definitive hematopoiesis.29,30 These
findings suggest that AML1 contributes, by regulating the expression of
target genes, to hematopoietic cell differentiation and proliferation.
Within the AML1 protein, there are two functional domains that have
been identified. The runt domain, a 128-amino acids region of homology
with the Drosophila runt protein,31 is known to be
essential for DNA binding and heterodimerization with
PEBP2/CBF.16,32,33 AML1 specifically recognizes a
consensus sequence, designated as a PEBP2 site
(R/TACCRCA),33,34 whereas PEBP2/CBF binds to AML1 and
increases its affinity for DNA without interacting with DNA by
itself.12 In our recent study, we showed that a conserved
cysteine residue in the runt domain of AML1 is important for the DNA
binding ability and the transforming ability.35 The
proline-, serine-, threonine-rich (PST) region is essential for
transcriptional activation,27,36 and this region is missing in the chimeric proteins AML1/ETO(MTG8) and AML1/Evi-1. Recently, we
showed that AML1 is phosphorylated in vivo on two serine residues within the PST region with dependence on extracellular signal-regulated kinase (ERK) activation.37
Chimeric proteins generated as a result of chromosomal translocations
should play causative roles in leukemogenesis. However, little is known
about the mechanism for leukemic transformation in t(8;21) and t(3;21)
leukemias. We and other groups have shown that AML1/ETO(MTG8) and
AML1/Evi-1 dominantly suppress the functions of intact AML1 and inhibit
myeloid cell differentiation.38-41 It is a useful approach
for elucidation of the function of the chimeric proteins to study
subcellular localization of leukemia-associated chimeric proteins and
compare it with those of original wild-type proteins. For example, in
t(15;17) acute promyelocytic leukemia, the alteration of subcellular
localization of the wild-type protein (PML) by the chimeric protein
(PML/retinoic acid receptor [RAR]) plays an important role in
leukemic transformation.42-44 In the present study, we
investigated subcellular localization of AML1/ETO(MTG8) and AML1/Evi-1.
Lu et al reported that PEBP2A and PEBP2B are nuclear proteins,
whereas PEBP2(CBF) is present mainly in the cytoplasm.45 Interestingly, they also reported that the N-
or C-terminally truncated PEBP2A colocalizes with PEBP2(CBF)
in the nucleus, in contrast to the full-size PEBP2A, which does not
colocalize with PEBP2(CBF). To clarify the mechanism of leukemogenesis, it should be important to investigate how
AML1/ETO(MTG8) and AML1/Evi-1 change the subcellular localization of
PEBP2(CBF). Using immunofluorescence and subcellular
fractionation, we showed that these chimeric proteins are located in
the nucleus. It was suggested that both the runt domain and the
AML1/ETO(MTG8) or Evi-1 portion of the chimeric proteins are
responsible for their nuclear localization. We also found that these
chimeric proteins accumulate PEBP2(CBF) in the nucleus with
dependence on the runt domain. Then we showed that the chimeric
proteins show the higher abilities to accumulate PEBP2(CBF) in
the nucleus than wild-type AML1. It was also shown that the chimeric
proteins associate with PEBP2(CBF) more effectively than
wild-type AML1, implying the relationship between the binding affinity
for PEBP2(CBF) and the ability to accumulate it in the nucleus.
These data suggest that AML1/ETO(MTG8) and AML1/Evi-1 exhibit dominant
effects over wild-type AML1 owing to their efficient ability of nuclear
accumulation of PEBP2(CBF) and give some important clues for
elucidation of transformation mechanism in leukemic cells expressing
such chimeric proteins.
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MATERIALS AND METHODS |
Cell culture and DNA transfection.
COS-7 cells were grown in a 5% CO2 environment in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
penicillin/streptomycin and 5% fetal calf serum. Plasmids were
transfected by the DEAE-dextran method as described
previously.41
Plasmid constructions.
The human AML1(AML1b)46 and AML1/Evi-1 cDNAs4
were inserted into the EcoRI site of pME18S, an SR
promoter-driven expression plasmid,47 as described
previously.27,41 The AML1/ETO(MTG8) cDNA was kindly
provided by S.W. Hiebert (St Jude Children's Research Hospital,
Memphis, TN).39 The mouse PEBP2 cDNA was kindly provided by Y. Ito (Kyoto University, Tokyo, Japan).12 The
EcoRI site was created by site-directed
mutagenesis48 at positions 65 or 6 bp upstream from the
AML1/ETO(MTG8) or PEBP2 translation initiation site, respectively,
and these cDNAs were inserted into the EcoRI site of pME18S.
The runt domain deletion mutants of AML1 (AML1 RD) and AML1/Evi-1
(AML1/Evi-1RD) were constructed as described
previously.41,49 For construction of the runt domain
deletion mutant of AML1/ETO(MTG8) (AML1/ETORD), the
EcoRI-Apa I [519] (numbers in parentheses indicate nucleotide numbers from the start site of translation to the cutting site of the enzyme) fragment of AML1/ETO(MTG8) was replaced by the
fragment from EcoRI to mutagenic Apa I [141] derived
from AML1/Evi-1RD. For tagging AML1, AML1/ETO(MTG8), or AML1/Evi-1 at the NH2-terminus, the influenza virus hemagglutinin (HA)
epitope (YPYDVPDYA) was inserted after the first methionine codon by
polymerase chain reaction (PCR).37,50 These HA-tagged cDNAs
were inserted into pME18S.
Antibodies.
A rabbit polyclonal antibody to AML1 (anti-AML1 serum) was raised
against maltose-binding protein fusion of AML1 as described previously.27 Polyclonal antibodies to PEBP2
(anti-PEBP2 serum) were obtained as follows. A glutathione
S-transferase (GST) fusion of PEBP2 was constructed by
ligating a mutagenic EcoRI fragment of PEBP2 into the
EcoRI site of the pGEX-2T vector (Pharmacia, Uppsala,
Sweden). This construct was expressed in Escherichia coli BL21 cells, purified, and used to immunize a rabbit and
hamsters as described previously.27
Immunofluorescent cell staining.
A total of 3 × 104 COS-7 cells per 24 mm × 24 mm
coverslip were plated 12 hours before plasmid DNA transfection. The
cells were incubated for 40 hours after transfection and then fixed and
blocked as described.45 Briefly, the cells were fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS) for 20 minutes, permeabilized with 0.1% Nonidet P-40 in PBS for 10 minutes, and blocked in PBS containing 5% normal goat serum (GIBCO-BRL,
Gaithersburg, MD) and 1 mg of bovine serum albumin fraction V (Sigma
Chemical Co, St Louis, MO) per mL for 1 hour. Then the cells were
incubated with anti-AML1 serum (1:1,000 in dilution) or hamster
anti-PEBP2 serum (1:1,000 in dilution) for 1 hour, followed by
incubation with fluorescein isothiocyanate (FITC)-conjugated goat
anti-rabbit IgG (Zymed Laboratories, San Francisco, CA; 1:50 in
dilution) or Texas Red-conjugated goat anti-hamster IgG (Cedarlane
Laboratories, Ontario, Canada; 1:50 in dilution) for 1 hour. For double labeling, the mixture of anti-AML1 serum and hamster
anti-PEBP2 serum was used as the primary antiserum and the mixture
of FITC-conjugated goat anti-rabbit IgG, and Texas Red-conjugated goat
anti-hamster IgG was used as the secondary antiserum. The cells were
visualized under a Bio-Rad MRC 1024 confocal microscope (Bio-Rad
Laboratories, Richmond, CA).
Subcellular fractionation and Western blotting.
Cells were homogenized in 500 µL of hypotonic suspension buffer (10 mmol/L sodium phosphate buffer [pH 7.0], 5 mmol/L EDTA, 1 mmol/L
dithiothreitol [DTT], and 1 mmol/L phenylmethylsulfonyl fluoride
[PMSF]) using Dounce homogenizer and separated into the nuclear
(pellet) and cytoplasmic (supernatant) fractions by the centrifugation
at 1,000g. One tenth of each fraction was subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
electrotransferred onto polyvinylidene difluoride filters (Millipore
Corp, Bedford, MA), then reacted with anti-AML1 serum (1:500 in
dilution), HA. 11 anti-HA serum (BAbCO, Richmond, CA; 1:1,000 in dilution), or rabbit anti-PEBP2 serum (1:500 in
dilution). To verify the purity of subcellular fractionation, the
membranes were also reacted with the anti-actin monoclonal antibody
(Boehringer Mannheim, Mannheim, Germany; 1:100 in dilution) or the
anti-Rb monoclonal antibody (Pharmingen, San Diego, CA;
1:500 in dilution). The blots were visualized by Protoblot system
(Promega, Madison, WI) or ECL blotting system (Amersham, Arlington
Heights, IL).
In vitro binding assays.
The GST or GST-PEBP2 protein was expressed in E coli BL21
cells and purified as described previously.27 Approximately
5 µg of the GST or GST-PEBP2 protein immobilized on Glutathione Sepharose 4B (Pharmacia) was incubated with the lysates from COS-7 cells overexpressing HA-AML1, HA-AML1/ETO(MTG8), or HA-AML1/Evi-1 in
lysis buffer (50 mmol/L Tris-HCl [pH 8.0], 150 mmol/L NaCl, 0.02%
sodium azide, 1% Triton X-100, 100 µg of PMSF per mL, and 1 µg of
aprotinin per mL) for 1 hour at 4°C with gentle rotation. The
protein-GST beads were washed four times with the lysis buffer and
subjected to SDS-PAGE.
Immunoprecipitation and metabolic labeling.
COS-7 cells cultured for 40 hours after transfection were obtained in
250 µL of the lysis buffer (the same buffer as we used for in vitro
binding assay) per 100-mm-diameter culture dish. For
immunoprecipitations, 1 mg of cell lysates per lane were mixed with 20 µL of anti-AML1 serum and rotated for 1 hour at 4°C. Then samples
were incubated with protein-A-Sepharose (Sigma) for 1 hour at 4°C,
followed by washing three times with lysis buffer. Immunoprecipitates
were subjected to SDS-PAGE and Western-blotting with anti-AML1 serum or
rabbit anti-PEBP2 serum. For metabolic labeling, COS-7 cells were
cultured for 35 hours after transfection, transferred to and cultured
for 4 hours in methionine-free DMEM plus 50 µCi of
[35S]methionine (Tran-35S label; ICN
Pharmaceuticals Inc, Costa Mesa, CA) per mL, lysed, and
immunoprecipated with 5 µL (0.4 mg/mL) of 12CA5 anti-HA monoclonal antibody (Boehringer Mannheim) or 5 µL of rabbit anti-PEBP2 serum. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. All
transfection experiments were performed at least twice and similar
results were obtained.
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RESULTS |
Subcellular localization of AML1/ETO(MTG8) and AML1/Evi-1.
Generation of the chimeric proteins AML1/ETO(MTG8) and AML1/Evi-1 is
supposed to be essential for leukemic transformation in t(8;21) and
t(3;21) leukemias, respectively, and the investigation of their
cellular distributions should give an important clue to elucidate the
mechanism of leukemogenesis. We thus studied the subcellular
localization of these chimeric proteins by indirect immunofluorescence
using a rabbit polyclonal antibody to AML1 (anti-AML1) serum and
FITC-conjugated anti-rabbit IgG. By Western blotting with anti-AML1
serum, the 55-kD protein was specifically detected in COS-7 cells
transfected with the expression plasmid for AML1 (Fig 1A, lanes 1 to
4), as described in our other
reports.25,35,37,49 The endogenous AML1 was not detected.
There were two bands corresponding to AML1, and the upper band was
considered to represent the phosphorylated form, as we described
previously.37 In Kasumi-1 and SKH1 cell lines endogenously
expressing AML1/ETO(MTG8) and AML1/Evi-1, respectively, we could not
detect these chimeric proteins by immunofluorescence probably because
their expression levels are too low to be detected using this antiserum
(data not shown). Therefore, we overexpressed these chimeric proteins
in COS-7 cells to study their subcellular localization. The endogenous
AML1 proteins in COS-7 cells were below the detectable level, because
COS-7 cells transfected with the empty pME18S vector were not stained
with anti-AML1 serum (Fig 1B[c], see page 1690). COS-7 cells were
transfected with the expression vectors for the proteins listed in Fig
2 and investigated by immunofluorescence.
As reported previously, wild-type AML1 was present exclusively in the
nucleus (Fig 1B [d]).45 The specificity of the staining
was confirmed by using preimmune serum (Fig 1B[a and b]). Two
chimeric proteins, AML1/ETO(MTG8) and AML1/Evi-1, were also located in
the nucleus (Fig 3A [a and b]). When
observed in lower magnification, wild-type AML1 and the chimeric
proteins were located in the nucleus in more than 95% of the cells
successfully transfected with the plasmids (Fig 3B). The rest of the
cells were slightly stained diffusely throughout the cell. In
accordance with these results, Sacchi et al reported that
AML1/ETO(MTG8) is detectable in the nucleus of Kasumi-1 cells by
immunofluorescence labeling using antiserum that recognizes
ETO(MTG8).51 Because the runt domain is known to be
responsible for the nuclear localization of PEBP2A,45 we
examined whether the nuclear localization of these chimeric proteins is
dependent on the runt domain. The deletion mutant of AML1 lacking the
runt domain showed a perinuclear accumulation with a weak nuclear
fluorescence, which is compatible with the previous report (data not
shown).45 On the other hand, the mutant chimeric proteins
lacking the runt domain still remained mainly in the nucleus (Fig 3A
[c and d]). These data suggest that the ETO(MTG8) and Evi-1 portions
of the chimeric proteins play some roles in the nuclear localization of
the chimeric proteins.
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| Fig 1.
(A) Specificities of antibodies as revealed by Western
blotting. A total of 3 × 104 COS-7 cells were transfected
with 4 µg of pME18S (lanes 1, 3, 5, 7, 9, and 11), expression plasmid
for AML1 (lanes 2 and 4), or that for PEBP2 (lanes 6, 8, 10, and
12), lysed, and subjected to Western blotting. The blots were probed
with anti-AML1 serum (lanes 3 and 4), rabbit anti-PEBP2 serum (lanes
7 and 8), hamster anti-PEBP2 serum (lanes 11 and 12), as well as
with the respective preimmune sera (lanes 1 and 2, 5 and 6, and 9 and
10, respectively). The AML1 or PEBP2 protein is indicated by the
arrowhead. Molecular weight standards (in kilodaltons) are indicated.
(B) (see page 1690) Specificities of antibodies as revealed by
immunofluorescence. A total of 3 × 104 COS-7 cells were
transfected with 4 µg of pME18S (a, c, e, and g),
expression plasmid for AML1 (b and d), or that for PEBP2 (f and h).
The cells were analyzed by immunofluorescence labeling with anti-AML1
serum (c and d) or hamster anti-PEBP2 serum (g and h) as well as
with the respective preimmune sera (a and b, and e and f,
respectively). Original magnification × 600.
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| Fig 2.
A schematic representation of full-length or mutant AML1,
AML1/ETO(MTG8), and AML1/Evi-1. The runt domain and the PST region (see
text) of AML1 (also called AML1b) are shown by striped and dotted
boxes, respectively. Other regions of AML1/ETO(MTG8) (proline-rich regions and the zinc finger domain) and AML1/Evi-1 (the noncoding exon
of Evi-1, zinc finger domains and the acidic domain) are indicated.
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| Fig 3.
(A) (see page 1690) Subcellular localization of
full-length or mutant AML1/ETO(MTG8) and AML1/Evi-1. A total of 3 × 104 COS-7 cells were transfected with 4 µg of each
construct as indicated and analyzed by immunofluorescence labeling with
anti-AML1 serum. Original magnification × 600. (B) The staining
pattern of the cells overexpressing AML1 (upper panel), AML1/ETO(MTG8)
(middle panel), or AML1/Evi-1 (lower panel) in lower magnification (× 100). (C) Identification of full-length or mutant AML1/ETO(MTG8) and
AML1/Evi-1 in cytoplasmic (lanes C) and nuclear (lanes N) fractions of
COS-7 cells. COS-7 cells were transfected with the same amount of each
construct as indicated in (A), lysed, fractionated, and subjected to
SDS-PAGE and immunoblotting using anti-AML1 serum. Each protein was
expressed at the anticipated size, as marked by the arrowhead. The cell
lysate of untransfected COS-7 cells was also analyzed as a control
(mock). Western blotting of the actin and Rb proteins are shown as
known cytoplasmic and nuclear proteins, respectively. Molecular weight
standards (in kilodaltons) are indicated.
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To confirm these results obtained by immunofluorescence, we also
investigated subcellular localization of AML1/ETO(MTG8) and AML1/Evi-1
by a combination of subcellular fractionation and Western blotting.
Cell lysates were fractionated into cytoplasmic and nuclear fractions,
and the chimeric proteins were detected by anti-AML1 serum. The
majority of the control proteins, actin and Rb, were detected in the
cytoplasmic and nuclear fractions, respectively (Fig 3C).
AML1/ETO(MTG8) was found predominantly in the nucleus as previously
shown (Fig 3C, lanes 3 and 4).39 AML1/Evi-1 was also
present predominantly in the nucleus (Fig 3C, lanes 7 and 8). On the
other hand, a certain fraction of each deletion mutant lacking the runt
domain was present in the cytoplasm (Fig 3C, lanes 5, 6, 9, and 10),
indicating that the runt domain is partially required for the nuclear
localization of the chimeric proteins. The difference of subcellular
localization between AML1/ETO(MTG8) or AML1/Evi-1 and each deletion
mutant was not clear in immunofluorescence, probably because of the
sensitivity of the immunostaining.
AML1/ETO(MTG8) and AML1/Evi-1 accumulate PEBP2 in the
nucleus with dependence on the runt domain.
PEBP2, a heterodimerizing partner of PEBP2, intensifies the DNA
binding ability of PEBP2.12 However, PEBP2 is present mainly in the cytoplasm when it is overexpressed solely, and does not
colocalize with PEBP2A.45 Interestingly, the truncated PEBP2A protein, devoid of the region either upstream or downstream from the runt domain, colocalizes with PEBP2 in the
nucleus.45 The study on the cellular distribution of
PEBP2 in association with that of AML1/ETO(MTG8) or AML1/Evi-1 will
give a good clue to reveal the role of the chimeric proteins in
leukemogenesis. Therefore, we examined subcellular localization of
PEBP2 and the effects of AML1/ETO(MTG8) and AML1/Evi-1 on it. The
antibodies against PEBP2 (anti-PEBP2 serum) were raised in a
rabbit and hamsters. The reactivities of the antibodies were examined
by Western blotting, and they specifically detected the 23-kD protein in COS-7 cells transfected with the expression plasmid for PEBP2 (Fig 1A, lanes 5 to 12). The endogenous PEBP2 in COS-7 was below the
level of detection. AML1/ETO(MTG8) or AML1/Evi-1 was coexpressed with
PEBP2 in COS-7 cells, and subcellular localization of each protein
was determined by double staining. The chimeric proteins were detected
by anti-AML1 serum and FITC-conjugated anti-rabbit IgG. PEBP2 was
detected by hamster anti-PEBP2 serum and Texas Red-conjugated
anti-hamster IgG. We could not evidently detect an endogenous PEBP2
protein in COS-7 cells transfected with the empty pME18S vector by
immunofluorescence probably because of its low expression level (Fig 1B
[g]). In 100% of the cells overexpressing PEBP2 alone, it was
mainly located in the cytoplasm (Fig 1B [h]), in accordance with the
previous reports.45,52 The specificity of the staining was
confirmed by using preimmune serum (Fig 1B [e and f]). When
AML1/ETO(MTG8) or AML1/Evi-1 was coexpressed with PEBP2, PEBP2
colocalized with the chimeric proteins in the nucleus (Fig 4 [e and
g], see page 1690). We also examined the
effects of the deletion mutants of the chimeric proteins lacking the
runt domain on the localization of PEBP2. Although such deletion mutants of the chimeric proteins are located mainly in the nucleus, the
deletion of the runt domain almost completely abolished their abilities
to translocate PEBP2 into the nucleus (Fig 4 [f and h]). As
mentioned above, the deletion mutant of AML1 devoid of the runt domain
is distributed throughout the cell. When coexpressed with this mutant,
PEBP2 remained in the cytoplasm (data not shown). These data
indicate that AML1/ETO(MTG8) and AML1/Evi-1 have the abilities to
accumulate PEBP2 in the nucleus, and these abilities are dependent
on the runt domain.
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| Fig 4.
Double fluorescence labeling of COS-7 cells transfected
with the constructs of full-length or mutant chimeric proteins and PEBP2. A total of 3 × 104 COS-7 cells were transfected
with 4 µg of each construct together with 0.4 µg of the expression
plasmid for PEBP2. (a, b, c, and d) The chimeric proteins were
detected with anti-AML1 serum. (e, f, g, and h) PEBP2 was detected
with hamster anti-PEBP2 serum. Original
magnification × 600.
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AML1/ETO(MTG8) and AML1/Evi-1 associate with PEBP2
with dependence on the runt domain.
The runt domain of AML1 or PEBP2 was reported to be responsible for
the heterodimerization with PEBP2.16,32,33 As both AML1/ETO(MTG8) and AML1/Evi-1 also contain the runt domain, it is
likely that they are able to heterodimerize with PEBP2. To confirm
this, we examined the association of the chimeric proteins with
PEBP2 in COS-7 cells expressing AML1/ETO(MTG8) or AML1/Evi-1 together with PEBP2. COS-7 cells were transfected with expression plasmids for each chimeric protein and PEBP2 and lysed. After immunoprecipitation with anti-AML1 serum, these immunoprecipitates were
analyzed by immunoblotting using anti-AML1 serum or anti-PEBP2 serum. When the cells were transfected only with the expression plasmid
for PEBP2 and immunoprecipitated with anti-AML1 serum, no PEBP2
was detected (data not shown). PEBP2 was coimmunoprecipitated with
both AML1/ETO(MTG8) and AML1/Evi-1, showing the association between
each chimeric protein and PEBP2 (Fig 5,lanes 1 and 3). We also examined the association of the mutant chimeric
proteins devoid of the runt domain with PEBP2. As expected, PEBP2
was not coimmunoprecipitated with these deletion mutants (Fig 5, lanes 2 and 4), indicating that the chimeric proteins associate with PEBP2
through the runt domain. Together with the finding that the runt domain
is required for the accumulation of PEBP2 in the nucleus by
AML1/ETO(MTG8) and AML1/Evi-1, these data suggest that the chimeric
proteins accumulate PEBP2 in the nucleus by heterodimerization with
PEBP2.
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| Fig 5.
Association of AML1/ETO(MTG8) and AML1/Evi-1 with
PEBP2. COS-7 cells (1 × 106) were transfected with
10 µg of the expression plasmids for full-length or mutant chimeric
protein together with 1 µg of that for PEBP2. Cells were lysed,
immunoprecipitated with anti-AML1 serum, and subjected to Western
blotting with anti-AML1 serum or rabbit anti-PEBP2 serum. Arrowheads
show the positions of full-size and mutant chimeric proteins. The
position of PEBP2 is marked by the arrow. Molecular weight standards
(in kilodaltons) are indicated.
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AML1/ETO(MTG8) and AML1/Evi-1 accumulate PEBP2 in the
nucleus more efficiently than wild-type AML1.
The accumulation of PEBP2 in the nucleus by AML1/ETO(MTG8) and
AML1/Evi-1 may play a crucial role in leukemogenesis. To elucidate such
a role, it should be important to compare the abilities to accumulate
PEBP2 in the nucleus between wild-type AML1 and the chimeric
proteins. For this purpose, we tried to make the expression levels of
wild-type AML1 and the chimeric proteins approximately equal. We
constructed expression vectors in which the expressed wild-type AML1
and chimeric proteins contain HA epitope in their NH2-terminus and detected their expression by anti-HA serum
in Western blotting. The HA-tagged AML1, AML1/ETO(MTG8), or AML1/Evi-1 showed a subcellular localization similar to that of the original proteins (Fig 6B [b to d], see page
1690). In addition, HA-tagged AML1 showed a
similar transactivation ability compared with the original AML1 (M. Kurokawa and H. Hirai, unpublished observation, February
1995). COS-7 cells were transfected with various amounts of the expression plasmids for the HA-tagged proteins, and the expression levels of the proteins were compared. In conditions shown in
Fig 6A, the AML1, AML1/ETO(MTG8), and AML1/Evi-1 proteins were
expressed to the approximately same levels. Using these conditions, each of HA-tagged proteins was coexpressed with PEBP2 in COS-7 cells, and subcellular localization of both proteins was determined by
double staining. The expression levels of coexpressed PEBP2 were
also comparable when assessed by Western blotting (data not shown). We
observed nonspecific background signal in the nucleus when HA. 11 anti-HA serum was used for immunofluorescent labeling. Therefore,
HA-AML1, HA-AML1/ETO(MTG8), and HA-AML1/Evi-1 were detected by
anti-AML1 serum and FITC-conjugated anti-rabbit IgG. PEBP2 was
detected by hamster anti-PEBP2 serum and Texas Red-conjugated anti-hamster IgG. In these sets of experiments, COS-7 cells were transiently transfected and the expression levels of wild-type AML1 or
the chimeric proteins, and PEBP2 in each cell may be different.
Therefore, a statistical analysis is needed to estimate the ability to
accumulate PEBP2 in the nucleus. It is probable that the higher
amounts of wild-type AML1 or the chimeric proteins relative to that of
PEBP2 accumulate PEBP2 in the nucleus more efficiently than the
lower amounts, although we could not quantitatively compare the
expression levels of these proteins in individual cells. As we set up
conditions so that the average expression levels of wild-type AML1 and
the chimeric proteins are comparable, the proportion of the cells
showing the nuclear accumulation of PEBP2 is supposed to reflect the
ability of these proteins to translocate PEBP2 into the nucleus. So
we examined the percentage of the cells showing the nuclear
accumulation of PEBP2 among the cells expressing both each HA-tagged
protein and PEBP2 as a scale to estimate this ability. Some of the
cells with a strong fluorescence in the nucleus also showed a weak
cytoplasmic fluorescence, and they were counted as the cells showing
the nuclear accumulation of PEBP2. Less than 5% of the cells showed
faint and diffuse fluorescence throughout the cell, and they were
excluded from the counts. When coexpressed with HA-AML1, PEBP2
showed a nuclear accumulation in only 19% of the cells and remained in
the cytoplasm in the rest of the cells (Fig 6B [f] and C). On the
other hand, in 80% of the cells expressing both HA-AML1/ETO(MTG8) and
PEBP2, PEBP2 was located mainly into the nucleus (Fig 6B [g]
and C). HA-AML1/Evi-1 colocalized with PEBP2 in the nucleus in 66%
of the cells (Fig 6B [h] and C). The staining patterns of the cells with anti-PEBP2 serum in lower magnification shown in Fig 6D clearly
show the difference in the effect on subcellular localization of
PEBP2. Most of the cells overexpressing PEBP2 also expressed the
HA-tagged AML1 or chimeric proteins (data not shown). The transfection
efficiencies were comparable (15% to 20% of all the cells) among the
cells transfected with the HA-tagged AML1 and chimeric proteins. In the
majority of the cells expressing wild-type AML1, PEBP2 was located
mainly in the cytoplasm, especially in the perinuclear region (Fig 6D,
upper panel). In contrast, when coexpressed with HA-AML1/ETO(MTG8) or
HA-AML1/Evi-1, PEBP2 showed a nuclear pattern in most of the cells
(Fig 6D, middle and lower panels). These findings indicate that
AML1/ETO(MTG8) and AML1/Evi-1 have higher abilities to translocate
PEBP2 into the nucleus than wild-type AML1.
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| Fig 6.
(A) Expressions of the HA-tagged wild-type AML1 and
chimeric proteins in COS-7 cells. A total of 3 × 104
COS-7 cells were transfected with the expression plasmid for PEBP2
(0.4 µg) together with that for each HA-tagged protein. To obtain the
same expression level of HA-AML1, HA-AML1/ETO(MTG8), and HA-AML1/Evi-1,
based on several preparative experiments, we determined the amounts of
the transfected plasmids as follows; HA-AML1 (0.5 µg),
HA-AML1/ETO(MTG8) (1.2 µg), and HA-AML1/Evi-1 (4.0 µg). Cell
lysates were fractionated into cytoplasmic (lanes C) and nuclear (lanes
N) fractions and analyzed by immunoblotting using anti-HA serum.
Wild-type AML1 and the chimeric proteins were expressed at the
anticipated sizes, as marked by the arrowheads. The complete transfer
of these three proteins was verified by confirming that all molecular
weight standards were equally stained on the membrane and barely
detectable on the gel by Coomassie staining after blotting (data not
shown). Cell lysates of COS-7 cells transfected with only PEBP2
construct were also analyzed (mock). Molecular weight standards (in
kilodaltons) are indicated. (B) (see page 1690) Double fluorescence
labeling of COS-7 cells transfected with the constructs of each
HA-tagged protein and PEBP2. The amounts of the transfected
expression plasmids were the same as indicated in (A). (a, b, c, and d)
The HA-tagged proteins were detected with anti-AML1 serum. (e, f,
g, and h) PEBP2 was detected with hamster anti-PEBP2 serum.
Original magnification × 600. (C) The ability of each HA-tagged
protein to accumulate PEBP2 in the nucleus. COS-7 cells were
transfected with the expression plasmids as shown in (A). Two
hundred cells expressing both each HA-tagged protein and PEBP2 were
counted (see text). Bars show the percentages of the cells
showing stronger fluorescence detected by anti-PEBP2 serum in the
nucleus as compared with the fluorescence in the cytoplasm (obtained in
three independent experiments). Error bars indicate one standard
deviation. (D) The staining pattern of the cells with anti-PEBP2
serum overexpressing HA-AML1 (upper panel), HA-AML1/ETO(MTG8) (middle
panel), or HA-AML1/Evi-1 (lower panel) in lower magnification
(× 100).
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AML1/ETO(MTG8) and AML1/Evi-1 show the higher affinities for
PEBP2 than wild-type AML1.
If the accumulation of PEBP2 in the nucleus is dependent on its
binding to the chimeric proteins, it is anticipated that the chimeric
proteins associate with PEBP2 more effectively than wild-type AML1.
We performed in vitro binding experiments in which E
coli-expressed GST-PEBP2 immobilized on glutathione sepharose beads were incubated with the lysates from COS-7 cells overexpressing wild-type AML1 or the chimeric proteins. As shown in Fig
7A, the chimeric proteins bound to
GST-PEBP2 more strongly than wild-type AML1. To confirm this result
in vivo, we investigated whether the chimeric proteins show the higher
affinities for PEBP2 than wild-type AML1 when both proteins are
coexpressed. COS-7 cells were transfected with expression plasmids for
HA-AML1, one of the HA-tagged chimeric proteins, and PEBP2. The
cells were lysed and immunoprecipitated with anti-PEBP2 serum, and
we tried to compare the amounts of HA-AML1 and the HA-tagged chimeric
proteins coimmunoprecipitated with PEBP2. As the molecular weight of
HA-AML1 is close to that of the immunoglobulin heavy chains, it was
difficult to estimate the amount of immunoprecipitated HA-AML1 by
Western blotting in which anti-immunoglobulin antibody is used as a
secondary antibody for the protein detection. Therefore COS-7 cells
were metabolically labeled with [35S]methionine, and
immunoprecipitates were detected by autoradiography. In the first
place, the expression levels of wild-type AML1 and the chimeric
proteins were examined by Western blotting of total cell lysates with
anti-HA serum (Fig 7B [a]). The amounts of the HA-tagged chimeric
proteins were less than those of HA-AML1. To estimate quantitatively
the affinities for PEBP2, [35S]methionine-labeled
cell lysates were divided into two and subjected to the
immunoprecipitation using 12CA5 anti-HA monoclonal antibody and to the
coimmunoprecipitation with PEBP2 using anti-PEBP2 serum. Then the
ratios of the amount of each HA-tagged chimeric protein to that of
HA-AML1 were compared between the immunoprecipitates with anti-HA
monoclonal antibody and those with anti-PEBP2 serum. In the
immunoprecipitates with anti-HA monoclonal antibody, the ratios of the
amounts of HA-AML1/ETO(MTG8) and HA-AML1/Evi-1 to those of HA-AML1 were
0.53 and 0.72, respectively (Fig 7B [b]). These ratios are properly
assumed to reflect the ratios of the expression levels of these
proteins and [35S]methionine incorporation of the
proteins, because they are in accordance with the results of the
Western blotting of the total cell lysates with anti-HA serum (Fig 7B
[a]). On the other hand, in the immunoprecipitates with anti-PEBP2
serum, the ratios of the amounts of HA-AML1/ETO(MTG8) and HA-AML1/Evi-1
to those of HA-AML1 went up to 1.18 and 1.61, respectively (Fig 7B
[c]). These data suggest that these chimeric proteins associate with
PEBP2 more efficiently than wild-type AML1, and this is probably
related to the higher abilities of the chimeric proteins to accumulate PEBP2 in the nucleus as compared with wild-type AML1.
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| Fig 7.
Comparison of the affinities for PEBP2
between the chimeric proteins and wild-type AML1. (A) COS-7 cells
(1 × 106) were transfected with the expression
plasmid for HA-AML1, HA-AML1/ETO(MTG8), or HA-AML1/Evi-1. The amounts
of the transfected expression plasmids were the same as described in
the legend to Fig 6. The cells were lysed and incubated with GST (lanes
4 to 6) or GST-PEBP2 (lanes 7 to 9) linked to glutathione sepharose
beads and subjected to Western blotting with anti-HA serum. Ten percent
of the input was also run on the same gel (lanes 1 to 3). The positions
of wild-type AML1 and the chimeric proteins are indicated by the arrowheads. Molecular weight standards (in kilodaltons) are shown. (B)
COS-7 cells (1 × 106) were transfected with the
expression plasmid for PEBP2 together with the construct for HA-AML1
and that for HA-AML1/ETO(MTG8) or HA-AML1/Evi-1. The amounts of the
transfected expression plasmids were the same as described in the
legend to Fig 6. COS-7 cells transfected with only PEBP2 construct
were also analyzed (mock). Cells were subjected to
[35S]methionine labeling and lysed. (a) Expressions of
the HA-tagged chimeric proteins and wild-type AML1. Total cell lysates,
including 50 µg of protein, were subjected to SDS-PAGE and Western
blotting with anti-HA serum. Closed arrowheads show the position of
HA-AML1. Open arrowheads show the positions of HA-AML1/ETO(MTG8) (lane 2) and HA-AML1/Evi-1 (lane 3). Molecular weight standards (in kilodaltons) are indicated. (b) Comparison of the amounts of the HA-tagged chimeric proteins to that of HA-AML1 immunoprecipitated with
anti-HA monoclonal antibody. One milligram of each cell lysate was
immunoprecipitated with anti-HA monoclonal antibody and subjected to
SDS-PAGE and autoradiography. Closed arrowheads show the position of
HA-AML1. Open arrowheads show the positions of HA-AML1/ETO(MTG8) (lane
2) and HA-AML1/Evi-1 (lane 3). The radioactivities of the bands of
HA-AML1, HA-AML1/ETO(MTG8), and HA-AML1/Evi-1 were quantified by Fujix
BAS 2000 (Fuji Film Corp, Kanagawa, Japan), and the
ratios of the radioactivities of the HA-tagged chimeric proteins to
those of HA-AML1 are indicated. Molecular weight standards (in
kilodaltons) are shown. (c) Comparison of the amounts of the HA-tagged
chimeric proteins to that of HA-AML1 immunoprecipitated with
anti-PEBP2 serum. One milligram of each cell lysate was
immunoprecipitated with rabbit anti-PEBP2 serum and subjected to
SDS-PAGE and autoradiography. Closed arrowheads show the position of
HA-AML1. Open arrowheads show the positions of HA-AML1/ETO(MTG8) (lane
2) and HA-AML1/Evi-1 (lane 3). The position of PEBP2 is marked by
the arrow. The radioactivities of the bands of HA-AML1,
HA-AML1/ETO(MTG8), and HA-AML1/Evi-1 were quantified by Fujix BAS 2000 (Fuji Film), and the ratios of the radioactivities of the HA-tagged
chimeric proteins to those of HA-AML1 are indicated. Molecular weight
standards (in kilodaltons) are shown.
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DISCUSSION |
In this study, we showed that the two leukemia-associated chimeric
proteins, AML1/ETO(MTG8) and AML1/Evi-1, are proteins that are located
in the nuc |
Abstract
Introduction
Abstract