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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3841-3847
AF4 Encodes a Ubiquitous Protein That in Both Native and MLL-AF4
Fusion Types Localizes to Subnuclear Compartments
By
Quanzhi Li,
Joy L. Frestedt, and
John H. Kersey
From the University of Minnesota Cancer Center and Departments of
Pediatrics and Laboratory Medicine/Pathology, University of Minnesota,
Minneapolis, MN.
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ABSTRACT |
Acute leukemia with t(4;11)(q21,q23) translocation results from the
in-frame fusion of the MLL to the AF4/FEL gene. In previous studies,
we and others demonstrated that AF4 transcripts are present in a
variety of hematopoietic and nonhematopoietic human cells. To
further study the wild-type and leukemia fusion AF4, we used glutathione S-transferase (GST)-fusion proteins as immunogens to produce rabbit polyclonal antibodies that were specific for normal
and chimeric AF4 proteins. Using Western blotting analysis, we
demonstrated that the AF4 gene encodes proteins with apparent molecular
weight of 125 and 145 kD. A 45-kD protein coprecipitated with AF4
protein in immunoprecipitation. Also, the anticipated MLL-AF4-encoded
240-kD protein was detected in all cell lines with t(4;11)
translocations; fusion proteins were present in lesser quantity than
the wild-type AF4. The proteins recognized by the antibodies are of the
predicted sizes of the AF4 and MLL-AF4-encoded proteins based on
previous DNA sequencing analysis. The MLL-AF4 fusion protein had a
similar subcellular distribution as AF4. Both t(4;11) and non-t(4;11)
leukemic cells showed a similar pattern of punctate nuclear staining in
all cell lines tested using confocal immunofluorescence microscopy. AF4
antibodies should be useful for further elucidation of the function of
AF4 in normal cellular physiology, as well as the function of MLL-AF4
in leukemogenesis. The antibodies should also be helpful for the
diagnosis of the MLL-AF4 fusion proteins in t(4;11) leukemias.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
AF4, ALSO KNOWN AS FEL, is a gene which
was first described as a fusion partner with MLL in the t(4;11) acute
leukemia.1-3 Based on analysis by Northern blotting, AF-4
mRNAs were found to be widely expressed in hematopoietic cells and
normal human tissues.4,5 cDNA sequence analysis of the AF-4
gene showed that it encodes a serine/proline-rich protein with a
predicted size of 130 to 140 kD containing guanosine triphosphate
(GTP)-binding and putative nuclear-localization sequences
(NLS).2 Studies have demonstrated that a related gene,
LAF4, isolated from Burkitt's lymphoma, shares high degree of sequence
homology with AF4. LAF4 was shown to possess DNA binding ability and
transcriptional activation potential.6 Both AF4 and LAF4
were shown to be evolutionary conserved in vertebrates suggesting an
important functional role of the genes. Recently, a third gene (FMR2)
has been recognized as a member of AF4/LAF4 gene family. FMR2 maps to X
chromosome at position Xq28. Mutations of FMR2 are associated with mild
hereditary mental retardation.7,8 Members of the homologous
AF4/LAF4/FMR2 gene family are expected to have transcriptional
activation functions. We previously found that another DNA sequence
located on chromosome 5q31, probably is a member of this gene
family.9
Clinical studies have demonstrated that MLL (also known as HRX and
ALL-1) rearrangements in infant ALL are associated with very poor
prognosis.4 At the molecular level, important sequence characteristics and putative functional motifs of MLL and MLL-AF4 fusion genes have been defined. Molecular analysis of the MLL gene
indicates that it contains two central zinc-fingers, a C-terminal region with homology to Drosophila trithorax gene, three
N-terminal A/T hook regions, a domain with homology to
methyltransferase, and a proline-rich region.3,10-12 The
t(4;11) translocation separates the second and third DNA binding domain
of MLL from the zinc finger region.10 The AT-hook and
proline-rich regions of MLL are fused in-frame to the region in AF-4,
which contains the NLS and GTP-binding activity, resulting in a
chimeric mRNA of 12.5 kb that encodes a predicted fusion protein of 240 kD.2,4 Rabbit polyclonal antibodies specific to N-terminal
fragments of MLL-encoded proteins expressed in Escherichia coli
have been recently reported.13 The antibodies recognized
the MLL and MLL/AF-4-encoded proteins in a leukemic cell line and
cells transfected with portions of MLL. A monoclonal antibody specific
for N-terminal epitopes of MLL protein has also been reported. This
antibody detected MLL-AF4 fusion protein in Western
blotting.14 Recently, Nilson et al15 reported
rabbit antibodies against human AF4 protein; an unexpected 116-kD
protein was detected in cell lysates by western blottings with no
detection of the MLL-AF4 fusion proteins. The major
purpose of our study was to identify and characterize the proteins
encoded by AF4 and MLL-AF4 genes by developing antibodies specific for AF4 proteins using molecular and immunological approaches.
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MATERIALS AND METHODS |
Cell lines and construction of expression vectors.
The RS4;11 cell line was established in our laboratory16
and is available from ATCC (American Type Culture Collection,
Rockville, MD). Two t(4;11) cell lines, B117 and
AN4;114 have been described. Another t(4;11) line, Sem-k2,
was a gift from Dr F.E. Cotter (Department of Haematology and Oncology,
Institute of Child Health, London, UK). Other cell lines have been
described and used in our laboratory for a number of years including
Nalm-6, KM3, BLIN-1 (B-cell lines), Raji (B-cell lymphoma), Molt-4, CEM (T-cell lines), M418 (human neuroblastoma), MG-63 (human osteosarcoma), and K562 (myeloid cell line).4 The cells were grown and
maintained in RPMI 1640 tissue culture medium supplemented with 10%
fetal bovine serum (FBS) and penicillin/streptomycin. A 3.3-kb cDNA designated PL12 that contained most of AF4 gene open reading frame was
isolated from a human placenta cDNA library.9 Two regions of the PL12 clone, one from the 5 end (base pair 612-1110, designated C9) and another from the 3 end (base pair 1942-2242, designated C15) of the RS4;11 breakpoint and other breakpoints
(Fig 1) were amplified by polymerase chain
reaction (PCR) using standard protocols. The C9 and C15 clones encode
polypeptides of 18 kD and 10 kD, respectively. The selection of these
regions from AF4 gene for construction of expression vectors was based
on the "plotstructure analysis" using the GCG computer program
(Genetics Computer Group, Madison, WI). Both regions showed high
surface probability, low hydrophobicity, and high antigenic index,
suggesting a strong potential for stimulating immune responses. PCR
primers were selected using the oligos 4s computer software (National
Bioscience, Plymouth, MN) and engineered to include EcoR1 and BamH1
restriction sites for direct in-frame insertion into the GST expression
vector (Pharmacia, Piscataway, NJ). The PCR products were gel purified
and ligated into TA cloning vectors (Invitrogen, San Diego, CA). The
inserts were sequenced from both ends into the vector sequences to
assure proper orientation. The GST vectors with C9 and C15 inserts were expected to encode a 44- and a 36-kD protein, respectively (GST, 26 kD;
inserts, 18 kD and 10 kD).
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| Fig 1.
Schematic representation of predicted AF4 gene structure.
The relative location and size of C9, C15, and PL12 as compared with
AF4 gene are shown as dark lines; alternate splicing sites, breakpoints
of t(4;11) translocations, nuclear localization sequences (NSL), and
GTP binding motifs of AF4 are indicated. The figure was constructed
based on the data from Morrissey et al,2 Frestedt et
al,9 Tkachuk et al,11 and Hilden et
al.22
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Expression and purification of GST-fusion proteins.
GST expression vectors with or without the inserts were inserted into
DH5 alpha bacteria (GIBCO/BRL, Grand Island, NY) using standard
bacterial transformation procedures. The transformed colonies were
selected and grown in ampicillin-containing (100 µg/mL)
LB medium at 37°C overnight with vigorous shaking. GST-fusion proteins were induced with 0.1 mmol/L IPTG
(Isopropyl-B-D-thiogalactopyranoside, Sigma Chemical Co, St Louis, MO)
for 3 to 4 hours. The bacteria were harvested by centrifugation, washed
and resuspended in PBST-100, pH 7.4 (phosphate-buffered saline, 1%
Triton-X100, 1 mmol/L EDTA) and sonicated (Branson Ultrasonic Corp,
Brandury, CT) to release the proteins. The presence of the fusion
proteins in the cell lysates were determined by analyzing the samples
in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). GST and GST-fusion proteins were affinity purified by
passing the bacterial lysates through a glutathione sepharose 4B
(Pharmacia) column and washed thoroughly with PBST-100. The bound
proteins were eluted with 100 mmol/L NaCl solution containing 25 mmol/L reduced glutathione (Sigma). The proteins were concentrated to 2 to 3 mg/mL using a Centricon-10 protein concentrator (Amicon, Bedford,
MA) and buffer exchanged to 100 mmol/L HEPES buffer using a PD-10 desalting column (Pharmacia). The protein concentration was
determined by a protein assay reagent (Pierce, Rockford, IL). After
affinity purification, GST-fusion proteins were further purified by
preparative electrophoresis. The protein samples were separated in 10%
SDS-PAGE gels and surface-stained with Commassie blue staining solution
for 2 to 3 minutes. The bands of interest were cut from the gels and
eluted into SDS-PAGE electrode buffer using an electro-elution device.
SDS contained in the protein solution was removed by acetone
precipitation method of Hager and Burgess.18
Production and purification of antibodies.
Two hundred microliters (1 mg/mL) of the 44-kD or 36-kD GST-fusion
protein obtained from preparative electrophoresis were emusified with
an equal volume of complete Freund's adjuvant and injected into two
rabbits at multiple sites. Booster injections with the decreasing
amounts of antigen emusified with incomplete Freund's adjuvant were
performed at a 2-week interval over a period of 84 days. The immune
responses of the animals were monitored by indirect enzyme-linked
immunosorbent assay (ELISA)19 and Western blottings. The
rabbits were killed and the sera collected when the ELISA titers of the
immune sera against the immunizing antigen reached 1:5,000 or greater.
Our initial attempts to immunize the rabbits with glutathione sepharose
4B purified GST-fusion proteins in denatured or native forms failed to
generate useful antibodies. This initial problem led us to further
purify the proteins by preparative electrophoresis to obtain a higher
proportion of full-length fusion protein for immunizations. Rabbits
immunized with these highly purified and denatured antigens stimulated
strong antibody responses as determined by immunoblotting and ELISA
using native and denatured proteins as coating antigens. Specific
antibodies in the sera were purified by affinity chromatography.
Briefly, 2 mg of purified GST, GST-C9, or GST-C15 fusion proteins were conjugated to 1 mL Affigel-10 (Bio-Rad, Hercules, CA)
following manufacturer's recommendations. Protein-conjugated
Affigel-10 was packed into a minicolumn and equilibrated with PBS. The
antiserum diluted fourfold with PBST (PBS containing 0.1% Tween-20)
was passed through the column containing Affigel-10 conjugated GST to
remove antibodies against GST. The eluent was then passed several times
through the column containing conjugated GST-C9 or GST-C15 fusion
protein, washed extensively with PBST, and eluted with 50 mmol/L
glycine buffer (pH 2.8). The eluted antibody was neutralized by 1.89 mol/L Tris buffer (pH 8.9) in the collection tubes. Purified antibodies
were designated anti-C-9 and anti-C15 and stored at 4°C in a
solution containing 0.5% BSA, 0.4 mol/L L-arginine, and 0.02% sodium
azide. IgG fractions of preimmune sera were purified by a protein A
column (Pierce).
Western blotting.
Cultured cell lines in log phase were harvested by low speed
centrifugation (1,500g), washed twice with PBS, and subjected to sonication at maximum intensity for 10 seconds. The cell lysates were solubilized in SDS-PAGE sample buffer, heated in boiling water for
2 minutes, and separated on SDS-PAGE gels under reducing-conditions. After electrophoresis, the proteins in the gels were
electrophoretically transferred onto hydrated nitrocellulose membranes
for 2 hours. The membranes were blocked with 5% nonfat milk in PBS for
2 hours at 37°C or at 4°C overnight. The membranes were probed
with diluted primary antibodies in blocking buffer at room temperature
for 1 hour. For detection of AF4 and MLL-AF4-encoded proteins,
affinity purified anti-C15 and anti-C9 antibodies (50 to 100 µg/mL)
were diluted 1:100 to 1:200 and used in Western blotting. Mouse
antihuman  tubulin monoclonal antibody (Sigma) used as an internal
control in some experiments was diluted according to supplier's
recommendations. After incubating with primary antibodies, the blots
were washed either under high or low stringent conditions. Unless
specified, all Western blottings were performed under high stringent
washing conditions. Under high stringent conditions, the blots were
washed three times (15 to 20 minutes/each) with PBS containing 0.05% Tween-20. Low stringent wash consisted of three washes (5 to 8 minutes/each) with PBS containing 0.01% Tween-20. After washing, the
blots were incubated with goat antirabbit or goat antimouse IgG
conjugated to horseradish peroxidase (HRP, Amersham, Alington, IL) for
45 to 60 minutes at room temperature. The blots were washed as before
and incubated with enhanced chemiluminescence (ECL) reagents (Amersham)
for 1 minute. The blots were blotted dry with filter papers, enclosed
in transparent plastic sheets, and exposed to autoradiographic film.
Other antibodies used in this study were rabbit anti-LAF4 (A gift from
Dr Louis M. Staudt, Metabolism Branch, National Cancer Institute,
National Institutes of Health, Bethesda, MD) and monoclonal anti-MLL
antibody (a gift from Dr Lisa H. Butler, John Radcliffe Hospital,
Oxford, UK).
Blocking experiment.
The specificity of purified anti-C15 and anti-C9 were accessed by
blocking with their corresponding immunizing antigens in Western
blotting format. Total cell lysate of Km3 was separated in 8% SDS-PAGE
(20 to 40 µg/lane) and electrophoretically transferred to
nitrocellulose membrane, cut into strips, and incubated with different
preparations of primary antibodies. Affinity-purified stock solutions
(50 to 100 µg/mL) of anti-C15 and anti-C9 were diluted 1:100 with
PBST containing 5% nonfat milk and 80 µg of corresponding immunizing
antigens in a final volume of 10 mL. Preimmune IgG and the same amount
of antibodies blocked with GST (80 µg/sample) were used as controls.
After 30 minutes incubation at room temperature, the nitrocellulose
strips were incubated with primary antibodies and the rest of the
Western blotting procedures were performed as above.
Immunoprecipitation and pulse chase analysis.
A total of 1 × 107 cells were harvested by low speed
centrifugation and washed twice with cold Hank's balanced salt
solution. The cells were resuspended in 5 mL RPMI medium containing 5%
dialized FBS (Gibco) and 0.2 mCi 35S (ICN, Costa Mesa, CA)
and incubated at 37°C with gentle shaking for 3 hours. The labeled
cells were washed twice with cold Hank's balanced salt solution and
lysed with 600 µL of lysis buffer (PBS containing 1% Triton X-100,
0.5% SDS, 0.5% deoxycholate, 1% BSA, leupeptin [10 µg/mL] and
0.2 mmol/L phenylmethyl sulfonyl fluoride [PMSF], Sigma). The cell
lysate was incubated in ice for 10 minutes and centrifuged in a
microcentrifuge at top speed for 10 minutes to remove insoluble
materials. Three hundred microliter cell lysate was precleared by
incubating with 1 µg rabbit IgG and 30 µL protein G plus/protein
A-agarose (CALBIOCHEM, Cambridge, MA) for 1 hour at room temperature.
The supernatant was incubated with 0.5 to 1 µg anti-C15 and 30 µL
protein A/G agarose at 4°C for 3 hours. After incubation, the
agarose beads were washed three times with lysis buffer. The
immunoprecipitates were solubilized in 40 µL SDS-PAGE sample buffer,
separated by 10% SDS-PAGE, and visualized by autoradiography. Pulse
chase labeling was performed based on the methods of
Bonifacino.20 Briefly, 5 × 107 cells were
incubated with 5 mL methionine-free RPMI medium with 5% dialized FBS
(pulse medium) for 30 minutes at 37°C to deplete intracellular
methionine. The cells were pelleted and incubated in 5 mL fresh warmed
pulse medium containing 1 mCi/mL 35S for 0.5 to 1 hour. An
aliquot of 1 × 107 cells for time 0 were collected,
and the remaining cells were washed once with Hank's balanced salt
solution, resuspended in 20 mL complete RPMI medium supplemented with
excessive L-methionine (15 mg/mL) and incubated at 37°C for the
chase time 1, 2, 3, and 6 hours. At each time point, equal amount of
cells were washed, lysed, precleared, and immunoprecipitated with
anti-C15 as described above.
Confocal immunofluorescence microscopy.
Cultured cells were harvested by low speed centrifugation and
resuspended in complete RPMI 1640 medium. The cells (1 to 2 × 105/mL) were fixed onto microscopic slides by cytospin. The
slides were air dried and fixed in absolute methanol solution at
 20°C for 10 minutes. The fixed cells were blocked in
blocking solution (2% BSA, 10 mg/mL goat IgG in PBS) for 30 minutes
followed by incubation with affinity-purified anti-C15 (1:40) at room
temperature for 1 hour. The slides were washed extensively with PBS and
incubated with goat antirabbit IgG Fab2 conjugated to
fluorescein isothiocyanate (FITC) (Sigma) for 1 hour and washed
extensively as described. Stained slides were mounted in glycerol
mounting buffer (PBS, 10%; p-phenylenediamine, 10 mg/mL; and Glycerol,
90%)21 and examined by a Bio-Rad MR C600 confocal
microscope. The images were processed using Adobe photoshop software
(Adobe Systems Inc, San Jose, CA).
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RESULTS |
Specificity of anti-C15 and anti-C9 antibodies.
The specificity of affinity-purified anti-AF4 antibodies was first
evaluated by Western blotting. As shown in a high stringency experiment
(see Materials and Methods), affinity-purified anti-C15 recognized a
single protein band of about 145 kD (Fig
2A, lane 1) in Western blotting against total Km3 cell lysate. The
signal was absent using preimmune serum (lane 2) and was completely
blocked by incubating the antibody with immunizing antigen before
Western blotting (lane 3). Lane 4 shows lack of blocking with GST.
Under low stringent washing conditions, anti-C15 also binds to a 80-kD protein in Western blotting; however, the cross-reactivity could be
removed by extensive washing. In contrast to anti-C15,
affinity-purified anti-C9 detected four major proteins in Western
blotting (Fig 2B, lane 1) with an estimated molecular size of 184, 145, 75, and 50 kD. All four signals were completely blocked by the
immunizing antigen (Fig 2B, lane 3). Preimmune IgG did not show any
reactivities against the cell lysate (lane 2) and GST failed to block
immunoreactivity of the antibody (lane 4).
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| Fig 2.
Blocking of anti-C15 (A) and anti-C9 (B) by
immnunizing antigens. Km3 cell lysate was separated in 8% SDS-PAGE and
electrophoretically transferred onto a nitrocellulose membrane. The
blots were cut into strips and Western blotting was performed using
various preparations of primary antibodies. (A) Lane 1, anti-C15 versus
cell lysate; lane 2, preimmune IgG; lane 3, anti-C15 blocked by
immunizing antigen; lane 4, anti-C15 blocked with GST. (B) Lane 1, anti-C9; lane 2, preimmune IgG; lane 3, anti-C9 blocked by immunizing
antigen; lane 4, anti-C9 blocked by GST.
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Immunoprecipitation was performed using anti-C15 antibody. After
immunoprecipitation of Km3 lysate with anti-C15, a protein of about 145 kD was detected in the immunoprecipitates by Western blotting using the
same antibody (Fig 3A, lane 3).
The protein band was not seen using preimmune serum (lane 2) or without
lysate (lane 4). The protein signal seen in Fig 3A, lane 3, was
identical to that of nonprecipitated lysate (lane 1). We also conducted experiments to evaluate proteins that were physically associated with
AF4. Km3 cells were radiolabeled with 35S and the cell
lysate was immunoprecipitated by anti-C15. In results shown in Fig 3B,
the expected protein of about 145 kD and a second 45-kD protein were
detected (Fig 3B). The 45-kD protein was not detected in Western
blottings suggesting that the 45-kD protein was coprecipitated with the
larger protein.
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| Fig 3.
Immunoprecipitation using anti-C15. (A)
Immunoprecipitation followed by Western blotting. Lane 1, Km3 cell
lysate without immunoprecipitation; lane 2, unlabeled Km3 lysate
immunoprecipitated with preimmune IgG; lane 3, Km3 lysate
immunoprecipitated with anti-C15; lane 4, anti-C15 with no lysate. The
protein samples were separated in 8% SDS-PAGE, transferred to a
nitrocellulose membrane, and probed with anti-C15. (B)
Immunoprecipitation of radiolabeled proteins. 35S-labeled
Km3 lysate (300 µL) was precleared with preimmune IgG and incubated
with 1 µg anti-C15 for 3 hours at 4°C. The immune complex was
pelleted by protein A/G beads, washed, separated by 10% SDS-PAGE, and
autoradiographed.
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More than one AF4 protein is detected in Western blotting.
Because the immunizing antigens of anti-C9 and anti-C15 were from the
5 and 3 end of AF4 gene, respectively, both antibodies should recognize the full-length AF4 protein in total cell lysate. As
shown in Fig 4A, a side-by-side comparison
of anti-C15 and anti-C9 showed that anti-C15 recognized a single
protein band in the cell lysates and anti-C9 bound to several proteins.
One of the proteins detected by anti-C9 (lane A3) showed the same migration as that detected by anti-C15 (lane A1). When anti-C9 and
anti-C15 were mixed (lane A2) and used for Western blotting, the AF4
protein signal was stronger than the signals detected by either
antibody alone, while other protein bands detected by anti-C9 remained
comparable. These results strongly suggest that both antibodies
recognized the same AF4 protein. When the Western blottings were
performed using lower percentage gels (Fig 4B), the AF4 protein
migrated as doublets (145 kD and 125 kD), which were detected by both
anti-C15 (lane B1) and anti-C9 (lane B2). Repeated experiments showed
that anti-C15 generally reacted stronger with the 125-kD band, which is
most likely due to the results of alternate splicing of AF4 mRNA. In
previous studies, a 10.5-kb and a 12-kb mRNA transcript of AF4 gene was
observed.2,4 An alternate explanation for the doublets is
the possibility of precursor-product relationship of the doublets. To
evaluate this possibility, pulse chase analysis was conducted. Despite
repeated attempts, we were able to show the doublets in
autoradiographes using low percentage gels only after long periods of
incubation (data not shown). A time course study of cell lysates
labeled with 35S and precipitated by anti-C15 indicated
that the doublets were observed in autoradiographes only in cell
lysates that have been labeled for a longer period of time (>3
hours). The extented labeling time required to show the doublets
prevented us from obtaining interpretable results of pulse chase
analysis, which requires a short labeling window of 30 minutes to 1 hour.20 Low immunoprecipitation efficiency of anti-C15 may
also be in part responsible for the observations.
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| Fig 4.
Comparison of Western blotting patterns of anti-C15 and
anti-C9. (A) Km3 lysate (20 µg/lane) was separated in 8% SDS-PAGE,
transferred to nitrocellulose paper, and probed with anti-C15 (lane 1),
anti-C15 plus anti-C9 (lane 2), and anti-C9 (lane 3). (B) K562 lysate
(20 µg/lane) was separated in 4% SDS-PAGE, transferred to
nitrocellulose paper, and probed with anti-C15 (lane 1) and anti-C9
(lane 2).
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Anti-C15 and anti-C9 recognize MLL-AF4 and AF4-MLL reciprocal fusion
proteins, respectively.
When the cell lysates were run on low percentage SDS-PAGE gels (4% to
5%), a protein band with a calculated molecular weight of 240 kD was
detected by anti-C15 in cell lines containing MLL-AF4 fusion gene
(Fig 5). This protein was consistently
detected in all cell lines with known t(4;11) translocations (Fig 5A,
lanes 2, 4, and 6), but not in those without the translocations (lanes 1, 3, and 5). These data indicate that the 240-kD protein is the MLL-AF4-encoded fusion protein and is consistent with the predicted protein sizes.2 A side-by-side comparison of anti-C15 and
the anti-MLL/HRX antibody (HRX107)14 in Western blotting
showed that a 240-kD protein band were detected by both antibodies
(data not shown). In these low percentage gels, anti-C15 also generally detected the AF4 doublet in cells used (Fig 5A). Again, as shown in Fig
4B, the smaller protein species showed stronger signals than the larger
species.
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| Fig 5.
Western blotting patterns of anti-C15 and anti-C9 in 4%
SDS-PAGE. Cell lysates were separated by SDS-PAGE, electrophoretically
transferred to nitrocellulose membranes, and probed with
affinity-purified anti-C15 (A) or anti-C9 (B). Lane 1, Nalm-6; lane 2, RS4;11; lane 3, Km3; lane 4, Sem-k2; lane 5, K562; lane 6, B1.
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We also evaluated anti-C9 antibody for study of the fusion proteins.
Figure 5B shows the immunoblotting patterns of anti-C9 against total
cell lysates in 4% gel. In addition to 180 kD and 145 kD proteins, the
antibody also recognized a high molecular weight protein (>240 kD) in
cells with t(4;11) translocations, RS4;11 and SEM-k2 (lanes 2 and 4, respectively). The B1 cells, which were previously shown to have low to
absent levels of 14 kb der4 RNA transcript,4 were found to
have a very weak band as shown in Fig 5B, lane 6. Although the exact
size of this high molecular weight protein cannot be determined due to
the lack of proper molecular weight markers, by comparing its migration rate with that of the 240 kD MLL-AF4 fusion protein, the size of the
protein was estimated in the range of 300 to 350 kD. Because the high
molecular weight protein signal is much weaker than that of AF4
protein, the blot in Fig 5B was intentionally overexposed to show the
high molecular weight band. Because anti-C9 was raised using N-terminal
polypeptide of AF4 as immunogen, it might expect to recognize AF4 and
reciprocal AF4-MLL proteins, but not the MLL-AF4 fusion proteins. In
view of the observation that the high molecular weight protein detected
by anti-C9 was seen only with t(4;11) lines and that the size of the
protein falls in the calculated size range of AF4-MLL fusion protein
{(430 (MLL) + 140 (AF4) 240 (MLL-AF4) = 335 kD
(AF4-MLL)}, we suspect that anti-C9 recognizes the AF4-MLL reciprocal
fusion protein in all t(4;11) lines tested.
AF4 and MLL-AF4 fusion proteins are localized in the nucleus, but not
in the cytoplasm.
Purified anti-C15 was used to evaluate localization of the AF4 and
MLL-AF4-encoded fusion proteins in immunofluorescence studies. All
cell lines stained with the antibody showed a strong punctate fluorescence staining in nucleus, but not in cytoplasm
(Fig 6). The immunostaining patterns were
generally similar in all cells tested and there was no significant
difference in the patterns and distribution of the labeling for cell
lines with or without t(4;11) translocations. Further study will be
required to establish whether the AF4 and MLL-AF4 proteins colocalize
in the same subcellular compartment. It is also important to determine
whether wild-type AF4 and the fusion proteins function in
distinct cellular compartments.
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| Fig 6.
Indirect immunofluorescence microscopy. Cultured cells in
log phase were harvested and adjusted to 1 to 2 × 105/mL.
The cells were attached to microscope slides by cytospin. The cells
were probed with anti-C15 followed by goat antirabbit IgG conjugated to
FITC. The image of the stained cells was obtained by a Bio-Rad MR C600
confocal microscope and processed using Adobe photoshop software. The
cell lines used are indicated.
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DISCUSSION |
In this study, we have produced and characterized specific antibodies
to AF4-encoded proteins. We have also provided direct evidence that
AF4-encoded proteins are present in a variety of human leukemic and
nonleukemic cells, as demonstrated by immunoblottings and
immunofluorescence microscopy. In Western blottings, anti-C15 and
anti-C9 antibodies consistently recognized a protein of about 145 KD in
all cell lines tested. Under high stringency conditions of washing, the
anti-C15 antibody recognized a single 145-kD AF4 protein in Western
blottings, which migrated as doublets when lower percentage gels (4%)
were used, the antibody also detected a 240-kD protein in all cell
lines with t(4;11) translocations. The molecular weight of the 240- and
145-kD proteins are consistent with predicted sizes of MLL-AF4 and
AF4-encoded proteins based on previous DNA sequencing and Northern
blotting analysis.2,4,22 Previous studies demonstrated the
presence of 12 kb and 10.5 kb AF4 mRNA transcripts in cell lines
tested. The 12-kb and 10.5-kb RNA transcripts identified in the
previous studies are most likely to be the results of alternate
splicing of AF4 mRNA, which subsequently encode the 145-kD and 125-kD
proteins. The 12.5-kb mRNA transcript previously detected in all cell
lines with t(4;11) translocations corresponds to the 240-kD protein
detected in this study. Coprecipitation of the 45-kD protein with AF4
protein suggests the possibility that it may exert its biological
function through interaction with a second protein.
The anti-C9 that was raised against N-terminal end of AF4 protein
detected the protein of about 145 kD. However, it also bound to a
184-kD, a 75-kD, and a 50-kD protein in Western blottings. It is not
clear whether these proteins are AF4-related. Thus far, mRNA species
corresponding to these proteins have not been identified. It was of
interest that anti-C9 reacted to a high molecular weight protein
(>240 kD) in RS4;11 and Sem-k2 cells with t(4;11) translocations, while the protein was absent from non-t(4;11) cells. These observations suggest that anti-C9 is able to detect AF4-MLL reciprocal fusion protein if it is present.
Molecular analysis of the MLL and AF4 genes by our group and
others3,10,11,12,22 indicates that the N-terminal portion of MLL fuses to C-terminal portion of AF4. The C15 clone used for
expression and immunization was chosen to be downstream of all known
breakpoints. Thus, anti-C15 recognizes the chimeric MLL-AF4 protein in
all cell lines tested (B1, RS4;11, and Sem-k2). The MLL-AF4 chimeric
proteins in RS4;11, AN4;11, and B1 cells are calculated to contain
2319, 2276, and 2234 amino acid residues, respectively. Fusion proteins
seen in the Western blottings migrated as a single band in 4% SDS-PAGE
gel (Fig 5) with apparent similar molecular weight, which is consistent
with the predicted sizes and showing that the size difference of the
proteins was too small to be resolved under the experimental conditions
used.
Recently, Joh et al13 have generated rabbit polyclonal
antibodies specific for N-terminal epitopes of MLL. These antibodies recognized a 240-kD protein in Western blotting. Butler et
al14 produced a monoclonal antibody against a 15 amino acid
peptide at N-terminal of MLL gene and the antibody (HRX 107) also
recognized the 240-kD MLL-AF4 fusion protein in the Sem-k2 cell. It is
of some interest that the quantity of MLL-AF4 fusion protein detected by our Western blotting is significantly less than that of the AF4
protein. The significance of these differences is not clear at present.
A gene previously cloned by Ma and Staudt6 from a cDNA
library of a Burkitt's lymphoma is closely related to the AF4 gene. Both genes share a high degree of homology (75% homology at C-terminal and 62% at N-terminal), contained a proline/serine-rich region and
NLSs at virtually the same position. The LAF4 gene encodes a major
protein of 135 kD. However, the expression of LAF4-encoded protein was
shown to be restricted to lymphoid cells. As predicted from DNA
sequence analysis, our Western blotting results indicate that LAF4
encoded a smaller protein than that detected by AF4 antibodies (data
not shown).
Results of our experiments indicate that anti-C15 detects the AF4
protein in subcellular compartments in immunofluoresence microscopy.
The subcellular localization of AF4 with punctate distribution in the
nucleus is similar to that previously described for
transcription-associated proteins. Other proteins with similar distribution are LAF4, MLL,13 and several other MLL
partners including ENL/LT6,13,23 AF9/LT69,13
and ELL.24 ELL is of interest because of the demonstration
that the ELL gene encodes a RNA polymerase II enlongation
factor.25 Consistent with a role for AF4 and LAF4 in
transcriptional regulation is the previous observation by Ma and Staudt
that AF4 has domains that activate transcription when fused to GAL4
DNA-binding domain. Of note is the observation that the transactivation
domain of AF4 is retained within the MLL-AF4 fusion
protein.6 Further studies will be necessary to determine
the significance of the punctate compartmentalized staining seen with
anti-AF4 antibody and other antibodies to transcription-related molecules. Previous studies with another transcription factor have
demonstrated that phosphorylation of hepatic nuclear factor 4 is
required for nuclear compartmentalization.26 It has been shown that the compartmentalized nuclear staining of the protein of
Drosophila polycomb (related to Drosophila trithorax)
developed homogeneous nuclear staining after the protein was
mutated.27
Finally, the observation that the anti-C15 antibody was able to detect
the MLL-AF4 fusion proteins in leukemia lines studied demonstrates the
potential use of this AF4-specifc antibody as a diagnostic reagent
using Western blotting.
| |
FOOTNOTES |
Submitted March 23, 1998;
accepted July 10, 1998.
Supported in part by an Outstanding Investigator Grant Award No. R35 CA
49721 from the National Cancer Institute (to J.H.K.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to John H. Kersey, MD, University
of Minnesota Cancer Center, Box 86 MAYO, 420 Delaware St, SE,
Minneapolis, MN 55455.
| |
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Abstract
Introduction