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J Biol Chem, Vol. 275, Issue 5, 3438-3445, February 4, 2000
§¶,§
,§,§,§**,,§§, and§¶¶
From the Departments of Biochemistry and Medicine and
the § Vanderbilt-Ingram Cancer Center, Vanderbilt University
School of Medicine, Nashville, Tennessee 37232 and
Department of Pathology and the
§§ Department of Tumor Cell Biology, St. Jude
Children's Research Hospital, Memphis, Tennessee 38105
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The AML-1-encoded transcription factor, AML-1B,
regulates numerous hematopoietic-specific genes. Inappropriate
expression of AML-1-family proteins is oncogenic in cell culture
systems and in mice. To understand the oncogenic functions of AML-1, we established cell lines expressing AML-1B to examine the role of AML-1
in the cell cycle. DNA content analysis and bromodeoxyuridine pulse-chase studies indicated that entry into the S phase of the cell
cycle was accelerated by up to 4 h in AML-1B-expressing 32D.3 myeloid progenitor cells as compared with control cells or cells expressing E2F-1. However, AML-1B was not able to induce continued cell
cycle progression in the absence of growth factors. The DNA binding and
transactivation domains of AML-1B were required for altering the cell
cycle. Thus, AML-1B is the first transcription factor that affects the
timing of the mammalian cell cycle.
The largest form of acute myeloid leukemia-1
(AML-1),1 termed AML-1B (1)
(also known as Runx1, CBFA2, or PEBP2 AML-1 is one of the most frequently mutated genes in human leukemia.
For example, it is disrupted by the t(8;21) in AML and by the t(12;21)
in childhood B-cell acute lymphoblastic leukemia (17-19). AML-1 is
also targeted indirectly by the Inv (16), which fuses CBF Although these translocations are closely associated with acute
leukemia, it is the wild-type form of AML-1 that transforms cells.
Wild-type AML-1 (AML-1B) is transforming when expressed in fibroblasts,
and this activity requires the C-terminal transcriptional regulatory
domain (25, 26). Likewise, the closely related protein PEBP2 Overexpression of the fusion protein encoded by the Inv (16) inhibited
cell cycle progression (28). This protein can act as an AML-1 dominant
repressor, suggesting a role for AML-1 in the G1 phase of
the cell cycle. However, the Inv (16) fusion protein could also
interact with and regulate other factors. In addition, many proteins
are required for cell cycle progression, but few factors actually
promote transit through the cell cycle. Therefore, we have explored the
functions of AML-1 in regulating the cell cycle. We have enforced the
expression of AML-1B and found that it accelerates transit through the
G1 phase, whereas transcriptionally inactive forms of AML-1
do not affect the cell cycle. This phenotype is similar to that
observed upon overexpression of the D- and E-type cyclins (29-31).
Thus, AML-1 is one of the first transcription factors whose enforced
expression shortens the G1 phase of the cell cycle.
Cell Lines--
Parental 32D.3 myeloid progenitor cells were
maintained as suspension cultures in RPMI 1640 containing 10% fetal
bovine serum (BioWhittaker), 2 mM L-glutamine
(BioWhittaker), 1% penicillin G-streptomycin (Life Technologies,
Inc.), and 15 units/mL IL-3. 32D.3 cells stably transfected with AML-1B
or derivatives thereof were constructed as described previously (32)
using the sheep metallothionine promoter plasmid pMT-CB6. COS cells
were cultured in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum. The designation pMT indicates a control
neomycin-resistant clonal cell line that was co-selected with the
AML-1B-expressing cell lines that are designated pMT-AML-1B. The
numbers after each cell line name are the clone (e.g.
pMT.11, control cell line number 11).
Cell Cycle Analysis--
Asynchronously growing cells were
washed 2× in PBS and resuspended in media lacking IL-3. At the
indicated times after IL-3 removal, 1 × 106 cells
were centrifuged and resuspended in 1 ml of propidium iodide staining
solution (0.05 mg/ml propidium iodide, 5 µg/ml DNase-free RNase A,
0.1% sodium citrate, 0.1% Triton X-100) and analyzed by flow
cytometry as described (33).
For bromodeoxyuridine (BrdU) analysis, asynchronously growing cells
were resuspended at 0.5 × 106/ml in media containing
IL-3. Cells were pulse-labeled with 10 µM BrdU (Sigma)
for 30 min, washed twice to remove BrdU, and resuspended in normal
growth media containing IL-3. Aliquots of 2 × 106
cells were obtained at the indicated time points following the removal
of BrdU. BrdU detection was performed by standard procedures (33).
Briefly, cells were washed in cold PBS and fixed in cold 70% ethanol
for 30 min. Cell pellets were then incubated for 30 min in 4 N HCl, washed in 0.1 M borax (pH 9.1) (Sigma),
followed by a PBS wash. Cells were then resuspended in 50 µl of
PBS-TB (PBS containing 0.5% Tween 20 (Sigma) and 0.5% bovine serum
albumin Fraction V (Sigma)), and a 1:150 dilution of an anti-BrdU
antibody (BU-33) (Sigma) for 30 min at room temperature. After two
washes in PBS-TB, cells were incubated with a 1:150 dilution of sheep fluorescein isothiocyanate-conjugated anti-mouse F(ab')2
(Sigma) in PBS-TB for 30 min at room temperature. Cells were again
washed twice in PBS-TB, resuspended in PBS containing 5 µg/ml
propidium iodide (Sigma), 1 µg/ml RNase A, and analyzed on a
FACScalibur flow cytometer (Becton Dickinson).
Protein Analysis--
For protein analysis, cells were lysed in
microextraction buffer, sonicated, and clarified as described
previously (34, 35). Total protein concentration of lysates was
measured using the Bio-Rad protein assay reagent. Cell lysate (100 µg) was electrophoresed on a 10% polyacrylamide gel, transferred
onto nitrocellulose, and probed with the indicated antibodies. Immune
complexes were detected using the Super Signal detection system
(Pierce) and a peroxidase-labeled goat anti-rabbit secondary antibody
(Sigma). All proteins were detected using a purified rabbit polyclonal antibody specific to the runt homology domain (RHD) of AML-1. Gel
mobility shift analysis and antibodies recognizing the AML family of
proteins have been described (36). The antisera are available from
Calbiochem. Gel mobility shift analysis for E2F was performed as
described (35). Immunoblot analysis for cell cycle regulatory proteins
was performed with antisera purchased from Calbiochem.
Transcription Assays--
A 1.1-kilobase pair fragment of the
murine cyclin D2 promoter was amplified from genomic DNA by
polymerase chain reaction based on the published sequence (37). This
fragment was cloned into pGL2 basic, which contains a
polylinker sequence upstream of the firefly luciferase gene. The
indicated promoter deletions were made using convenient endonuclease
restriction sites. pGL2-D2 was co-transfected using the
Superfect reagent (Qiagen) with pCMV5-AML-1B or
pCMV5-AML-1B(L175D) into COS-7 cells, and luciferase
activity was measured 40 h later. Secreted alkaline phosphatase
activity expressed from the cytomegalovirus immediate early promoter
was used as an internal control to correct for transfection efficiency. Regulation of the human thymidine kinase promoter was performed in the
same manner but using the renilla luciferase as the reporter gene
(Promega). The cyclin D1-luciferase promoter plasmid was a kind gift
from Dr. Ronald Wisdom (Vanderbilt University School of Medicine)
(38).
Enforced Expression of AML-1B Accelerates Entry into S
Phase--
The observation that the Inv (16) fusion protein inhibited
cell cycle progression (28) suggested that AML-1 normally functions in
the G1 phase of the cell cycle. To test this possibility,
we enforced the expression of AML-1B, the largest AML-1-encoded
product, to determine its effects on the cell cycle. The diploid,
IL-3-dependent myeloid progenitor cell line 32D.3 was used
for this cell cycle analysis because these cells express relatively low
levels of AML-1B. We established 32D.3 cell lines expressing AML-1B
from the zinc-inducible sheep metallothionine promoter. Single cell clones expressing AML-1B were identified from three separate parental populations by immunoblot analysis using antibodies specific for AML-1
(Fig. 1). As a control for later
experiments, we also selected clonal cell lines that did not express
AML-1B (labeled pMT in later figures). In addition, we isolated cell
lines that expressed mutant AML-1B proteins. AML-L175D contains a
single amino acid change within the DNA binding domain (also known as
the runt homology domain or RHD) that disrupts DNA binding (leucine 175 changed to aspartic acid, labeled L175D, Fig. 1) (39), whereas AML-1a lacks the C-terminal transactivation domain (AML-1a.3, Fig. 1). Although the metallothionine promoter was used, high levels of protein
expression were found in the absence of zinc. To avoid complications in
the cell cycle experiments, metal induction was not used. Cell lines
with higher basal expression were used for further analysis
(e.g. lines 7 and 28).
To compare the rates of cell cycle progression of these cell lines, DNA
content analysis was performed. Enforced expression of AML-1B appeared
to affect the cell cycle, as fewer cells were present in the
G1 phase and more cells accumulated in S phase in
AML-1B-expressing cells versus a control cell line that was established at the same time (designated pMT, Fig.
2A).
The accumulation of AML-1-expressing cells in S phase could be due to a
shortening of another cell cycle phase or to a lengthening of S phase.
To address this issue, we determined the timing of the cell cycle
phases by performing pulse-chase analysis using the nucleotide analogue
BrdU. This method avoids the use of cell cycle poisons that grossly
disrupt cellular morphology and physiology. A 30-min pulse of BrdU was
followed over a 24-h chase period by flow cytometry using an anti-BrdU
antibody to quantitate BrdU incorporation and propidium iodide staining
of DNA to measure DNA content (Fig. 2, B and C).
By plotting BrdU incorporation versus DNA content we were
able to observe both BrdU labeled (Fig. 2B, panel
pMT.3, 0 Hour area above the horizontal
line, and see arrows in Fig. 2C) and
unlabeled cells (Fig. 2B, panel pMT.3, 0 Hour area below the horizontal line and areas
highlighted by arrowheads in Fig. 2C) traversing
the cell cycle during the chase period. This method of analysis gives
rise to a characteristic horseshoe-shaped plot (Fig. 2B,
panel pMT.3, 0 Hour). During the 30-min labeling period, all cells that were synthesizing DNA
incorporated BrdU (Fig. 2B, panel pMT.3, O
Hour, +BrdU). This includes cells at the G1
to S phase transition that appear to have a 2 N DNA content, and cells at the S to G2 phase transition that
have a 4 N DNA content (Fig. 2B, panel
pMT.3, 0 Hour). To measure the time required for cells
to traverse the G2/M and G1 phases, we measured
cells re-entering the S phase from 8 h to 16 h after labeling. Cells that were labeled with BrdU and that were re-entering the S phase had divided and contained one-half the levels of BrdU of
undivided cells. This difference ensured that we were observing cycling
cells (note that the plots in Fig. 2, B and C use
a logarithmic scale, making this 2-fold shift small). To measure cells
that were clearly in S phase versus those cells that had
just begun to synthesize DNA, the gate was set to quantitate the
central portion of the S phase plot. This area is equivalent to the
area between the unlabeled G1 and G2/M phase
cells immediately after the labeling (below the horizontal
line and labeled -BrdU at 0 Hour, Fig.
2B).
At 8 to 9 h post-BrdU labeling, labeled cells had exited S phase
and divided as judged by a reduction in fluoresence intensity, and the
first cells were beginning to re-enter S phase (Fig. 2, B
and C). By 10 h after labeling, only 17% of control
32D.3 cells containing BrdU were present in early S phase (Fig.
2B, pMT.3, 10 Hour). By 12 h
after BrdU labeling, 30% of cells that were originally in S phase had
traversed G2/M and G1 and were re-entering S
phase (Fig. 2B, pMT.3, 12 Hour). In
addition, 10 h and 12 h after labeling with BrdU, most of the
unlabeled cells that were initially in the G1 and
G2/M phases had moved into the S phase and the
G1 phase, respectively (e.g. Fig. 2B,
compare pMT.3 0 Hour and 10 Hour, and note areas
marked by arrowheads in Fig. 2C). Cells
expressing E2F-1 and its co-factor DP-1 showed similar cell cycle
profiles (38% S phase 12 h post-BrdU labeling; Fig. 2B, pMAM-Neo-E2F-1.1/DP-1.2, and see Hiebert
et al. (32)).
When AML-1B-expressing cells were assayed, nearly twice as many
BrdU-labeled cells expressing AML-1B were re-entering S phase 10 h
after labeling as control cells (34% and 30% versus 17%
for control cells; Fig. 2B compare panels
pMT-AML-1B.7 and pMT-AML-1B.28 with pMT.3 at
10 h). At 12-h post-labeling, nearly 50% more AML-1B-expressing cells were in mid-S phase (45% and 54% S-phase,
pMT-AML-1B.7 and pMT-AML-1B.28, respectively;
Fig. 2B). In addition, at the 12-h time point, the unlabeled
AML-1B-expressing cells showed accelerated cell cycle kinetics (Fig.
2B; note also the unlabeled cells in the 9- and 13-h
panels marked with arrowheads in Fig.
2C). 12-13 h after labeling the AML-1B-expressing cells had
exited the G1 phase and were in late S phase (Fig. 2,
B and C) (note the reduction in the number of
cells in early S phase of the unlabeled cell portions of the plots of
cells expressing AML-1B as compared with control and E2F-1-expressing
cell plots (Fig. 2B, and marked with brackets
Fig. 2C)). Similar results were obtained from more than 10 independently isolated single cell clones that were selected from three
different parental populations of 32D.3 cells. The percentages of cells
entering S phase 10 or 11 h after BrdU labeling from three
different experiments are shown in Table
I. Each AML-1B-expressing cell line
entered S phase faster than the control cell lines (Fig. 2B
and Table I).
The results of DNA content analysis (Fig. 2A) and BrdU
analysis of S phase suggested that AML-1B caused accelerated entry into
S phase. In principle, this could be due to a shortening of the
G1 or the G2/M phases. However, DNA content
analysis indicated that the total number of AML 1B-expressing cells in
the G2/M phase were similar to control cells, and the
number of cells in the G1 phase was reduced, suggesting a
shortening of the G1 but not G2/M phases (Fig.
2A). To extend this result and to directly determine whether
the G2/M phase was shortened by AML-1B expression, we used
BrdU pulse-chase analysis to determine the time required to traverse
G2/M (Fig. 3 and Table
II). This was accomplished by measuring
the time required for the first BrdU-labeled cells (those cells in late
S phase at the time of labeling) to traverse G2/M and enter
the G1 phase. Both control G418-resistant cells (pMT) and
AML-1B-expressing cells passed through the G2/M phase and
began entering the G1 phase after 3-4 h (Fig. 3 and Table II). Analysis of further time points confirmed this result. We conclude
that enforced expression of AML-1B does not affect G2/M but
shortens the G1 phase, consistent with the ability of an
AML-1 inhibitor to arrest cells in the G1 phase (28).
AML-1B Cannot Induce G1 to S Phase Progression in the
Absence of Growth Factors--
The observation that AML-1B-expression
accelerated S phase entry in myeloid progenitor cells was unexpected.
Cell cycle regulatory transcription factors and oncogenes that act in
the G1 phase of the cell cycle such as E2F-1 and c-Myc do
not alter the timing of the cell cycle (32, 33, 40). However, both
E2F-1 and c-Myc can induce S phase progression of 32D.3 cells in the
absence of IL-3 (32, 33, 40). Therefore, we tested whether AML-1B could
also induce continued cell cycle progression in the absence of
cytokine. Cell lines were cultured in media lacking IL-3 for 12 h,
and cell cycle progression was measured using propidium iodide staining
to determine their DNA content. E2F-1-expressing cells displayed no
apparent cell cycle defects in the presence of IL-3 (Fig.
4). In the absence of IL-3, E2F-1 induced
continued S phase entry in the absence of the cytokine (Fig. 4). By
contrast, there were fewer cells expressing AML-1B in the
G1 phase and more cells in S-phase in the presence of IL-3,
but in the absence of IL-3 these cells accumulated in the
G1 phase (Fig. 4). Thus, AML-1B requires cooperating
proliferative signals from IL-3 to accelerate S phase entry and affect
the cell cycle.
Expression of Cell Cycle-related Proteins Is Higher in
AML-1B-expressing Cells--
One of the major targets for regulation
of cell cycle progression is the Rb pathway (41). To determine whether
this pathway was affected either directly or indirectly by AML-1B
expression, we measured the levels of cyclin-dependent
kinase-2 (cdk2), cdk4, cyclin D2, and E2F. Cell lysates were prepared
from control and AML-1B-expressing cells, and antibodies to each of
these proteins were used to probe immunoblots to determine their
levels. Each cell line expressing AML-1B contained more cdk2, slightly
elevated cdk4, and substantially more cyclin D2 (Fig.
5A). Consistent with this
observation, the levels of histone H1 kinase activity present in cdk2
immunoprecipitates was 2-3-fold higher in the AML-1B-expressing cells
(data not shown). Electrophoretic mobility shift analysis indicated
that AML-1B-expressing cells also contained more overall E2F DNA
binding activity relative to control cells, with most of this increase
corresponding to the "free" or active form of E2F (Fig.
5B). This was in contrast to Sp1 DNA binding activity that
did not change upon AML-1B expression (Sp1, Fig. 5B).
AML-1 Activates Cell Cycle-regulated Promoters--
The shortening
of the G1 phase and the higher levels of cyclin D2
suggested that AML-1 might activate key cell cycle regulatory genes
that affect cell cycle progression. Therefore, we scanned the promoters
for the G1 cyclins and found several AML-1 binding sites in
the cyclin D2 promoter. To determine whether AML-1 could activate the
cyclin D2 promoter, we used this promoter linked to a luciferase
reporter gene in transient assays. In COS-7 cells, the cyclin D2
promoter was activated by wild-type AML-1B but not by an AML-1B
containing a point mutation that ablates DNA binding (L175D, changing
residue 175 from leucine to aspartic acid, Fig. 6A). Expression from the
internal control promoter was unaffected by AML-1B (data not shown, but
used to correct for transfection efficiency). Promoter deletion
analysis indicated that 5' promoter sequences containing several
potential and known AML-1 binding sites were required for full
AML-1B-dependent transactivation, whereas a perfect AML-1
binding site 3' to the first transcriptional start site was not
sufficient for full activation (Fig. 6B). The caveat in this
analysis is that cyclin D2 is regulated during the cell cycle. Thus,
the activation observed with AML-1B could be indirect if AML-1B was
stimulating cell cycle progression. Therefore, we compared the ability
of AML-1B to activate the cyclin D2 promoter with other cell cycle
regulated promoters. Although the cyclin D2 promoter was activated to a
greater extent than the cyclin D1 or human thymidine kinase promoter
(which are not known to contain AML-1 binding sites), these promoters
were also activated by AML-1B (Fig. 6C). Once again, the
internal control was unaffected by AML 1B (data not shown).
AML-1B Effects on the Cell Cycle Require DNA Binding and
Transcriptional Regulatory Domains--
Because we could not determine
whether AML-1B directly activated cell cycle regulatory promoters, we
probed the mechanism of AML-1B cell cycle control by asking whether
expression of AML-1a, which lacks the C-terminal transactivation domain
(3, 42), or AML-1B(L175D) could affect cell cycle progression. The
clonal 32D.3 cell lines expressing AML-1a or AML-1B(L175D) (Fig. 1 and 7A) were compared with control
and AML-1B-expressing cells using BrdU pulse-chase analysis (Fig.
7B). Once again, the AML-1B-expressing cells entered S phase
with accelerated kinetics as compared with G418-resistant control
cells. Cells expressing either AML-1a or AML-1B(L175D) did not show
gross alterations in the timing of S-phase entry, indicating that both
the C-terminal domain and DNA binding activity are required for AML-1B
cell cycle functions. We conclude that AML-1B shortens the
G1 phase by regulating the expression of downstream target
genes.
AML-1 is unusual in that it is an oncogene, yet it is disrupted by
chromosomal translocations in acute leukemia. We observed that enforced
expression of AML-1B induced accelerated S phase entry (Figs. 2 and 7
and Table I). Cultures of cells expressing AML-1B had fewer cells in
the G1 phase and more cells in S phase (Fig. 2). The time
needed to traverse G2/M remained constant in these cells
(Fig. 3, Table II), which is consistent with expression of AML-1B
accelerating transit through the G1 phase of the cell cycle. Thus, AML-1B is one of the first transcription factors described
that shortens the length of the G1 phase of the mammalian cell cycle. Furthermore, this work points toward a cell cycle mechanism
for the ability of AML-1 to transform cells.
The ability of AML-1B to alter the cell cycle led us to examine the
promoters of known components of the G1 phase cell cycle control machinery to determine whether any of these genes could be
direct targets of AML-1B. We found perfect AML-1B binding sites in the
p21Waf1/Cip1 cyclin-dependent kinase inhibitor
promoter (16, 43) and the cyclin D2 promoter but not in the E2F1, cdk4,
or cdk2 promoters. In AML-1B-expressing cells, endogenous
p21Waf1/Cip1 protein levels were slightly elevated
(approximately 2-3-fold, data not shown). Recent work indicates that
p21Waf1/Cip1 can both inhibit cdk activity and promote the
assembly of cyclin:cyclin-dependent kinase complexes (44).
Both the p21Waf1/Cip1 and cyclin D2 promoters can be
regulated by AML-1B (Fig. 6, data not shown, and Refs. 16 and 43),
consistent with the requirement of AML-1B transcriptional activation
for the cell cycle phenotype (Fig. 7). However, because AML-1B
accelerates the cell cycle, we could not definitively determine whether
up-regulation of these genes is directly responsible for the shortening
of the G1 phase or whether the levels of these proteins (or
the activation of their promoters) are elevated because the cells are
cycling faster.
Initially, we observed faster growth of the AML-1B-expressing cells,
suggesting that these cells had not compensated for the acceleration of
the G1 phase and were cycling faster. However, as these
lines were maintained in culture, this phenotype was lost. It is likely
that these cells eventually compensated for the shortening of
G1 by lengthening the S phase, as has been observed for
cells overexpressing the G1 cyclins (29, 30). We also found
that the cell culture conditions were critical for revealing the
AML-1B-cell cycle phenotype. For instance, at high cell densities, the
shortening of G1 was lost (data not shown),
suggesting density-dependent effects. The mechanisms behind
these phenotypes will require further investigation.
Transcription factors such as E2F-1 and c-Myc are capable of affecting
cellular proliferation, but enforced expression of these factors did
not affect the timing of the 32D.3 cell cycle (32, 40). Therefore, the
phenotype of enforced AML-1B expression is more similar to that of
overexpression of the G1 cyclins, accelerating the
G1 phase without affecting the entry to or exit from
quiescence (29-31). Although stimulation of cyclin D2 or
p21Waf1/Cip1 expression may contribute to AML-1B-mediated
G1 phase control, these genes are unlikely to be the only
mediators of this phenotype. We are actively searching for other cell
cycle control genes that may contribute to AML-1-cell cycle control.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B1(2-4)), activates the
transcription of numerous tissue specific genes, including genes
encoding cytokines and cytokine receptors, T cell receptors, and
myeloid-specific genes (e.g. neutrophil peptide-3 and
myeloperoxidase) (5-7). When transfected alone, AML-1B activates the
transcription of these genes to low levels, but it cooperatively activates transcription to high levels in concert with tissue-specific factors (e.g. C/EBP, AP-1, ets-1, PU.1, and c-Myb) that
regulate cellular proliferation and differentiation (7-12).
Conversely, AML-1 can repress transcription by associating with the
Groucho and mSin3 co-repressors (13-16).
, an
AML-1-interacting protein, to a smooth muscle myosin heavy chain (20).
However, the Inv (16) fusion protein retains the ability to interact
with AML-1 and inhibits expression of AML-1 target genes (21-23).
Together these chromosomal translocations account for nearly one-third
of all AML and one-fourth of all childhood B-cell acute lymphoblastic
leukemia cases containing discernable chromosomal abnormalities
(24).
A1
(AML-3) is up-regulated by retroviral insertions that cooperate with
c-Myc to induce T-cell lymphomas (27). Therefore, in fibroblasts and in
mice, AML-1-family proteins are oncogenes. Thus, AML-1 has the unusual
property that both the wild-type and the translocated alleles can
affect cellular proliferation and differentiation pathways.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Establishment of AML-1B-expressing 32D.3 cell
lines. Immunoblot analyses of extracts from cell lines expressing
AML-1B, the DNA binding defective mutant AML-1B L175D, and AML-1a are
shown. Extracts from the indicated cell lines were analyzed by
immunoblot using an affinity-purified rabbit polyclonal antibody
specific to the runt homology domain (RHD) of AML-1 (45). Note that for
simplicity the pMT designation shown in later figures is not shown
here. The number following each name indicates the single
cell clone number.
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Fig. 2.
Cell cycle analysis of AML-1B-overexpressing
32D.3 cells. A, cell cycle analysis of
AML-1B-expressing and control cell lines using propidium iodide
staining and flow cytometry to measure DNA content. The pMT
designates a control cell line that expresses only the neomycin
resistance gene. B, BrdU pulse-chase analysis. Bivariate
distributions of BrdU incorporation (y axis)
versus DNA content (x axis) from the indicated
cell lines following pulse-labeling (30 min) with BrdU (0 hr) and following a 10- or 12-h chase period in the absence of
BrdU. Cells were washed, resuspended, and fixed as described under
"Experimental Procedures" before analysis on a FACScalibur flow
cytometer (Becton Dickinson) for DNA content (propidium iodide) and
BrdU incorporation (fluorescein isothiocyanate). Quantitation of the
numbers of BrdU-positive cells in S phase of the cell cycle was
performed by applying the ModFit analysis algorithm to the indicated
population of cells. The gates used for this quantitation are shown in
the first panel. C, BrdU pulse-chase analysis was
performed as in B but analyzed at 9 and 13 h after
labeling. The arrows indicate the leading edge of the
BrdU-labeled cells progressing through S phase. The
arrowheads indicate the leading edge of unlabeled cells
progressing though the cell cycle. The brackets in the 13-h
samples indicate the trailing portion of the unlabeled cells passing
though the S phase. The pMT designation indicates that the cells
express AML-1B from the sheep metallothionine promoter, and the single
cell clone number is indicated after the period. E2F-1 and DP-1 were
expressed from the pMAM-Neo expression vector in the absence of
dexamethasone (32).
Cell cycle analysis of AML-1B overexpressing 32D.3 cells
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Fig. 3.
AML-1B expression does not affect the
G2/M phase. BrdU pulse-chase analysis for the timing
of the G2/M phase is shown. Bivariate distributions of BrdU
incorporation (y axis) versus DNA content
(x axis) from the indicated cell lines following labeling
with BrdU for 30 min (0 h) and following a 3- or 4-h chase period in
the absence of BrdU is shown. Time to traverse G2/M was
measured by determining the time required for the first BrdU-labeled
cells to enter the G1 phase. The gates used for
quantitation of the number of cell in the G1 phase are
shown as open boxes.
Determination of G2/M transit time in 32D.3-overexpressing cell
lines
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Fig. 4.
AML-1B does not induce cell cycle progression
in the absence of IL-3. Cell cycle analysis is shown of
AML-1B-expressing and control cell lines using propidium iodide
staining and flow cytometry to measure DNA content. The indicated cell
lines were cultured in media containing IL-3 or lacking IL-3 for
16 h. Cell cycle profiles were determined using the ModFit
algorithm. Apoptotic cells (cells with less than a 2 N DNA
content) were gated out of the E2F-1/DP-1 sample.
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Fig. 5.
Components of the cell cycle machinery are
up-regulated in AML-1-expressing cells. A, 32D.3 cells
expressing AML-1B have more cdk2 and cyclin D2. Whole cell lysates of G
418-resistant control 32D cells (pMT) and the indicated
AML-1B-expressing cells were immunoblotted with antibodies directed to
cdk2, cyclin D2, or cdk4. B, AML-1B-expressing cells have
more E2F DNA binding activity. Cell extracts were prepared and analyzed
for DNA binding activities using a consensus E2F binding site as a
probe (upper panel) or an Sp1 binding site as probe
(bottom panel) in gel mobility shift assays. Whole cell
extracts were prepared from cells cultured in the absence of zinc
sulfate. The positions of E2F-p107-containing and E2F complexes lacking
a pRb family protein have been determined previously (33) and are
identified as E2F/p107 and Free E2F. The
numbers at the top of each lane indicate the
clonal cell line examined.
View larger version (19K):
[in a new window]
Fig. 6.
AML-1 activates cell cycle-regulated
promoters. A, AML-1B can activate the cyclin D2
promoter. Increasing amounts of AML-1B or the AML-1B L175D mutant
plasmid were co-transfected with 700 ng of pGL2-cyclin D2 plasmid. Fold
activation was calculated after correcting for transfection efficiency
using a plasmid expressing a secreted form of alkaline phosphatase
(SEAP) from the cytomegalovirus IE promoter. The numbers
below the bars indicate the amount of the plasmid in
nanograms. For the AML-1B 800-ng sample, the internal control was not
used, as this level of input plasmid suppressed the expression of SEAP,
probably due to promoter competition. The levels shown are the average
of duplicate experiments. In those samples lacking error
bars, the error was too small to graph. For subsequent
experiments, 200-300 ng of AML-1B plasmid was used. B,
schematic representation of the murine cyclin D2 promoter and fold
activation by AML-1B. A perfect AML-1 binding site is shown as a
hatched box, and 5 of 6 base pair matches of the consensus
binding site are shown as dark boxes. Numbers
above or below each box indicate the number of nucleotides
from the first major transcriptional start site. Fold activation is
shown on the right hand side. C, AML-1 activates the cyclin
D1 and thymidine kinase (TK) promoters. The ability of
AML-1B to activate the cyclin D2 promoter was compared with the TK and
D1 promoters that lack perfect AML-1 binding sites. Note that both TK
and D1 were activated but to a lesser degree than the D2
promoter.
View larger version (37K):
[in a new window]
Fig. 7.
Cell cycle analysis of 32D.3 cells expressing
mutant AML-1B proteins. BrdU pulse-chase analysis was performed on
control (pMT), AML-1B, AML-1B-L175D, and AML-1a-expressing cells.
A, schematic diagram of the expressed proteins.
B, bivariate distributions of BrdU incorporation
(y axis) versus DNA content (x axis)
from the indicated cell lines following pulse-labeling with BrdU for 30 min (0 h) and following a 10- or 12-h chase period in the absence of
BrdU.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dana King and Yue Hou for technical assistance and the Vanderbilt Cancer Center DNA sequencing facility and Randy Fenrick for insightful discussions and critical evaluation of data.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health (NCI) Grants RO1 AG13726, RO1-CA64140, RO1-CA77274 (to S. W. H.), and F32 CA77167 (to J. J. W.), by NCI Center Grant CA68485, and by the Vanderbilt-Ingram Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Dept. of Biology, University of South Carolina, Aiken, SC 29801.
A Special Fellow of the Leukemia Society of America
(3827-99).
** A Fellow of the Leukemia Society of America (5669-99).
¶¶ To whom correspondence should be addressed: Dept. Of Biochemistry, Vanderbilt Cancer Center, Vanderbilt University School of Medicine, Medical Research Building II, Rm. 512, 23rd and Pierce, Nashville, TN 37232. Tel.: 615-936-3582; Fax: 615-936-1790; E-mail: scott.hiebert@mcmail.vanderbilt.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: AML, acute myeloid leukemia; IL, interleukin; PBS, phosphate-buffered saline; PBS-TB, PBS containing 0.5% Tween 20 and 0.5% bovine serum albumin; BrdU, bromode- oxyuridine.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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