From the Fels Institute for Cancer Research and
Molecular Biology and the § M.D./Ph.D. Program, Temple
University School of Medicine, Philadelphia, Pennsylvania 19140 and the
¶ Department of Medicine, Montefiore Medical Center, Bronx, New
York 10467
Received for publication, January 5, 2003
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ABSTRACT |
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The c-myb proto-oncogene plays a
central role in hematopoiesis and encodes a major translational product
of 75 kDa. c-Myb is highly expressed in immature hematopoietic cells,
and its expression is down-regulated during terminal differentiation.
Deregulated expression of c-Myb has been shown to block terminal
differentiation of hematopoietic cells. Here we have studied the
mechanism of action and the nature of target genes through which c-Myb
mediates the block in differentiation of 32Dcl3 murine myeloid cells.
We show that the ectopic overexpression of c-Myb in 32Dcl3 cells results in the overexpression of c-Myc. However, enforced expression of
c-Myc in 32Dcl3 cells did not alter the normal pattern of
differentiation. In addition, expression of dominant-negative mutants
of c-Myc relieved c-Myb-mediated block in differentiation. These
results led us to conclude that c-myc is a target gene of
c-Myb and activation of the c-myc gene is a necessary event
in Myb-mediated transformation. However, c-Myc expression alone is
inadequate to elicit the phenotypic effects seen with
Myb-mediated block in differentiation of myeloid cells, suggesting that
activation of additional transcriptional targets by c-Myb plays a
critical role in this process.
The hematopoietic system is composed of a variety of cells that
range from fully differentiated nonproliferating cells to pluripotent
stem cells with extensive capacity to self-renew, proliferate, and
differentiate (1). In response to a demand by the body for mature blood
cells, the stem cells generate a group of progenitor cell population,
that express specific cytokine receptors on their surface and
proliferate or differentiate in response to the cognate cytokine
binding (1). It is becoming increasingly evident that the
proto-oncogene c-myb plays the role of a master regulator of
hematopoietic cell development. Thus, in mammalian cells,
c-myb is highly expressed in immature hematopoietic cells,
and its expression is down-regulated during terminal differentiation of
these cells (2-7). Constitutive expression of c-Myb has been shown to
block Me2SO-induced terminal differentiation of
erythroleukemic cells (8) as well as GCSF- or
IL1-6-induced terminal
differentiation of mouse myeloid cell lines (9-12). In addition,
inhibition of c-Myb expression using antisense oligonucleotides has
been shown to cause growth arrest of several hematopoietic cell lines
and interfere with normal human hematopoiesis in vitro
(13-15). Targeted disruption of c-myb locus results in neonatal death of mice because of a failure of fetal hepatic
hematopoiesis (16). While the role of c-Myb in hematopoiesis is well
established, its mechanism of action is yet to be defined.
The myb gene product codes for a major translational product
of 75 kDa, which is localized to the nucleus and acts as a mediator of
transcriptional regulation (7, 17-19). Like most transcription factors, the protein encoded by this gene exhibits a modular structure, with an N-terminal DNA binding domain, a central transactivation domain
and a C-terminal negative regulatory domain (reviewed in Ref. 20). Many
target genes have been identified for c-Myb, which include genes such
as mim-1, TCR The interrelationship between c-myb and c-myc was
best studied in the myeloid leukemic M1 cell line, which is a
transformed mouse myeloid cell line that proliferates autonomously in a
factor-independent manner and forms tumors in nude mice (12, 27).
However, this cell line was found to undergo terminal differentiation
and growth arrest, when cultured in the presence of IL-6 or leukemia
inhibitory factor (LIF) (28-30). Under conditions that allow
continuous proliferation, these cells were found to express high levels
of c-Myc and c-Myb, and the expression of these two proto-oncogenes is
down-regulated following treatment with IL-6 or LIF, which induce
terminal differentiation (31). In addition, it was found that
constitutive expression of c-Myb or c-Myc in these cells results in a
block to IL-6- or LIF-induced terminal differentiation (4, 12).
However, while constitutive expression of c-Myb in these cells lead to
a block in terminal differentiation at an early stage, similar
expression of c-Myc was found to result in a block leading to an
intermediate stage myeloid cells, suggesting that the two
proto-oncogenes mediate differentiation by different mechanisms (4,
12).
However, in a recent study, it was observed that conditional expression
of a Myb-estrogen receptor fusion protein (Myb-ER) in IL-6-treated M1
cells resulted in the restoration of expression of c-Myc expression
(that was down-regulated by IL-6) and restored the proliferative
activity of IL-6-treated M1 cells (27). Similarly, expression of a
dominant-negative mutant of c-Myb in a mouse myeloid leukemic cell
line, RI-4-11 resulted in the down-regulation of c-Myc expression
leading to the suggestion that c-Myc may be solely responsible for
Myb-mediated proliferation (27).
While these studies conducted in the M1 cell line are very informative
for our understanding of the role of myc and myb
in the differentiation of myeloid cells along the monocytic pathway, it
is at present unclear whether they play a similar role in the differentiation of myeloid cells along the granulocytic pathway. In
addition, these studies suffer from the problem that M1 cells are
tumorigenic and, unlike normal hematopoietic cells, proliferate in a
factor-independent manner. To gain an understanding of the relationships that exist between myb and myc in
myeloid cell differentiation along the granulocytic pathway, we
investigated the role of these two genes in GCSF-induced terminal
differentiation of 32Dcl3 cells. These cells, derived from normal mouse
bone marrow, have been found to be strictly dependent on IL-3 for
growth and undergo terminal differentiation when placed in an IL-3-free
medium containing GCSF (32, 33). However, when the cells were incubated
in the absence of IL-3 or GCSF, they were found to become arrested in the G1 phase of the cell cycle and rapidly enter apoptotic
pathways loosing viability in 48-96 h (34). We and others (9-11) had previously shown that overexpression of v-Myb or c-Myb in these cells
results in a block to their ability to terminally differentiate into
granulocytes in the presence of G-CSF. This cell line was particularly
useful for studies aimed at understanding the effects of oncogenes and
proto-oncogenes on myeloid differentiation as it is not predisposed
to develop factor-independent clones (34). Results presented in this
communication show that while ectopic overexpression of c-Myb in 32Dcl3
cells (32D/Myb) resulted in a block to the GCSF-induced terminal
differentiation of these cells, ectopic overexpression of c-Myc
(32D/Myc) failed to achieve the same effect. However, ectopic
overexpression of c-Myb was found to result in elevated expression of
c-Myc, and expression of dominant-negative mutants of c-Myc in 32D/Myb
cells was found to result in a block to Myb-mediated effects on the
terminal differentiation program of these cells. Similarly, in M1
cells, c-Myc dominant-negative mutants were found to relieve
c-Myb-mediated block in terminal differentiation. These results led us
to conclude that c-myc is a target of c-Myb, and activation
of the c-myc gene is a necessary event in Myb-mediated
transformation. However, ectopic overexpression of c-Myc expression
alone was inadequate to elicit the phenotypic effects seen with
Myb-mediated block in differentiation of myeloid cells along the
granulocytic pathway, suggesting that activation of additional
transcriptional targets by c-Myb is necessary for this process.
Construction of Plasmids--
The c-myc
dominant-negative constructs, mycRX and max2RX, were generously
provided by Dr. Bruno Amati and described previously (35). The
constructs LK444A (36) and LK444A/c-myc (28) were kindly provided by
Dr. Dan Liebermann. The constructs pMT-neo and pMT/c-myb have been
described previously (37). The pOPRSVI-Puro/MCS was constructed by
replacing the neomycin cassette in pOPRSVI/MCS (Stratagene) with a
puromycin cassette (38). The myc dominant-negative constructs, mycRX and max2RX, were subcloned into pOPRSVI-Puro/MCS to
express these constructs in 32Dcl3 cells.
Cell Lines and Culture Conditions--
The murine myeloid
progenitor cell line, 32Dcl3 (32, 33), was maintained in Isocove's
modified Dulbecco's medium (IMDM) supplemented with 10% fetal bovine
serum, 0.5% penicillin/streptomycin, and 10% WEHI3B cell-conditioned
medium as a source of IL-3 (39). Since all the cell lines established
had metallothionein promoter-based constructs, 100 µM
ZnCl2 was also used in all experiments. The M1 cell line
was maintained in RPMI 1640 with 10% horse serum and 0.5%
penicillin/streptomycin.
Growth and Differentiation Assays--
To induce granulocyte
differentiation, parental or transfected 32Dcl3 cells were washed twice
in IL-3-free medium and plated at a density of 1 × 105 cells/ml in IMDM containing 10% fetal bovine serum and
10% GCSF (32). The viability and proliferation of the cell cultures
were monitored for the indicated days. Morphological analysis of
GCSF-treated cells was performed using an aliquot of cells that was
cytospun and stained with May-Grunwald-Geimsa stain.
The antibiotic-resistant M1 cells were induced to differentiate with
conditioned medium containing IL-6. After 4 days, cytospin smears of
the IL-6-treated cells were prepared and stained with Wright-Giemsa stain.
Transient Transfection and Luciferase Assay--
For
transfection into QT6 fibroblasts, cells were seeded into 6-well plates
at a density of 0.5 × 105 cells/well. The following
day, DNA were transfected by calcium phosphate method (Invitrogen)
using the manufacturer's protocol. In each transfection, 5 µg of
reporter plasmid (LK444A/c-myc) and 5 µg of effector plasmid were
transfected along with 0.5 µg of pRL-CH110 as an internal standard.
The effector plasmid, pODC (228-281), was constructed by ligating
firefly luciferase gene with the ODC promoter region Establishment of Stable Cell Lines Expressing
Transgenes--
32Dcl3 cells expressing pMT-Neo (empty vector) and
pMT/c-myb have been described previously (37). For stable transfection of 32Dcl3 cells, exponentially growing 32Dcl3 cells were electroporated with various linearized plasmids DNAs using a Gene-Pulser (BioRad) at a
pulse of 230 V, 960 mF. The surviving cells were selected in 1 mg/ml of
G418 (pMT-neo based constructs) or in 1 mg/ml of G418 and 2 µg/ml of
puromycin (pMT-neo/pOPRSVI-Puro-based constructs) for 2-3 weeks.
For M1 cells, the cDNA encoding for c-Myb was subcloned into the
retroviral vector MSCVneo, while the cDNAs encoding for MycRX and
Max2RX were subcloned into MSCVpac (38). The vectors were transfected into packaging cell line Phenix together with the expression vector VSV-G (Stratagene). Vector, MycRX, and Max2RX retroviral supernatants were harvested to infect M1 cells. The infected
cells were selected in puromycin (2 µg/ml). The puromycin-resistant cells were further infected with c-Myb retroviral supernatant. The
infected cells were then selected in puromycin and G418 (0.4 mg/ml).
Northern Blot Analysis--
Total RNA from each cell line in the
presence of IL-3 was isolated and purified using the Trizol Reagent
(Invitrogen). 20 µg of total RNA was used per sample, and Northern
blot analysis was performed as described previously (37). Full-length
c-myb, c-myc, mycRX, and max2RX cDNAs were
used to detect c-myb, c-myc, mycRX, and max2RX
mRNA transcripts. As an internal control for RNA loading, gels were
stained with ethidium bromide to compare levels of 18 S/28 S ribosomal
RNAs across lanes.
Western Blot Analysis--
Anti-Myc and anti-Max primary
antibodies were purchased from Santa Cruz. To analyze protein products
of the transfected c-myb gene in 32Dcl3 cells, normalized
amounts of proteins from each cell lysate were separated by
SDS-polyacrylamide gel electrophoresis (37), and separated proteins
were transferred to a nitrocellulose membrane in transfer buffer (10 mM CAPS, 10% methanol, pH 10.5). The filter was blocked
with 5% nonfat milk in TBS-T buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween-20) for 1 h at room
temperature, incubated with primary anti-c-Myb polyclonal antibody (37)
overnight at 4 °C and washed three times in TBS-T solution. The
secondary antibody reaction was performed by incubating the filters
with horseradish peroxidase-conjugated anti-rabbit Ig (Amersham
Biosciences) and washed in a similar manner as was described for the
primary antibody reaction. The Amersham Biosciences ECL detection
system was used for visualization as specified by the manufacturer. To detect protein products of c-Myc, MycRX and Max2RX, anti-c-Myc N262,
anti-Max C124, and anti-c-Myc 9E10 (Santa Cruz Biotechnology) primary
antibodies and 3% nonfat milk were used in the protocol described above.
Analysis of DNA Fragmentation--
DNA fragments released from
10 × 106 cells were extracted and separated by
electrophoresis in agarose gels (40). After experiments, cells were
lysed in a buffer containing 0.5% Triton X-100, 50 mM
Tris, 10 mM EDTA, pH 7.4, and 200 mg/ml proteinase K. Cell lysates were then incubated at 42 °C for 90 min, treated with 0.2 mg/ml RNase A and incubated at 37 °C for an additional 30 min and
centrifuged at 14,000 × g for 20 min. The resulting
supernatant was then extracted twice with phenol/chloroform (1:1). DNA
fragments were precipitated with one-tenth volume of 3 M
NaOAc and 2.5 volumes of ethanol at Viability Assays--
Mock-transfected and
c-myc-transfected 32Dcl3 cells were analyzed for cell
viability in the absence of IL-3 at different time points. At the
indicated time points, viable and non-viable cells were identified by
trypan blue exclusion/inclusion, counted in triplicates on a
hemocytometer and percentage of viable cells calculated.
Constitutive Expression of c-Myb and c-Myc in 32Dcl3
Cells--
The role of c-Myb on myeloid cell growth and
differentiation has been studied previously (37). To study the effect
of c-Myc on myeloid differentiation along the granulocytic pathway and compare it with that of c-Myb, we used the 32Dcl3 cell line. This cell
line is derived from normal mouse bone marrow and was found to be
strictly dependent on IL-3 for proliferation (33, 41). In the absence
of IL-3, the cells undergo death via apoptosis over a period of
24-72 h. Upon addition of GCSF, these cells terminally differentiate
into granulocytes over a period of 10-12 days (10, 32, 33). The
linearized c-myb cDNA construct in pMT-Neo (under the
control of metallothionein promoter) and c-myc cDNA
construct in LK444A (under the control of actin promoter) were
transfected into 32Dcl3 cells by electroporation. As negative controls,
empty vector DNAs, pMT-Neo, and LK444A, was similarly introduced into 32Dcl3 cells. Following selection with G418, mass cultures were established from stable transfections. The derived cell lines were
tested for the expression of each transgene. In the case of pMT-Neo
based constructs, the expression levels were analyzed in the presence
of Zn2+, which acts as an inducer of transcription from the
metallothionein promoter.
The profiles of mRNA induction in these cell lines are shown in
Fig. 1, A and B.
The parental 32Dcl3 cells (32D/wild-type) and those transfected with
empty vector DNA (32D/pMTneo) showed the presence of a 3.4-kb
endogenous c-myb band (Fig. 1A). In cells transfected with c-myb (32D/pMT/c-myb in Fig.
1A), transgenic c-myb expression was seen as a
2.3-kb transcript. Similarly, the parental 32Dcl3 cells (32D/wild-type)
and the cells transfected with empty vector DNA (32D/LK444A) showed the
presence of a 2.4-kb endogenous c-myc band (Fig.
1B). In cells transfected with c-myc (32D/LK444A/c-myc/1 and 32D/LK444A/c-myc/2 in Fig. 1B),
transgenic c-myc expression was seen as a 1.4-kb transcript.
The smaller size of the transgenic transcripts are due to the absence
of the 3'-untranslated sequence, which was deleted during the
construction of these expression plasmids. To definitely demonstrate
expression of c-Myb and c-Myc proteins from the transgenes, we compared
the protein levels following incubation of 32D cells in the presence of
GCSF for 10 days. It was shown previously that, in 32Dcl3 cells, the
endogenous levels of c-Myb and c-Myc proteins remain relatively high
until day 6-8 of GCSF treatment followed by a downward regulation of
mRNA and protein, such that they become undetectable by day 10 of
GCSF treatment (9, 10). Taking advantage
of this observation, we analyzed the levels of c-Myb and c-Myc proteins
on the 10th day of GCSF treatment in the transfected cells to determine
the levels of the transgenic proteins expressed in the absence of the
endogenous protein product. As shown in Fig. 1C, on the 10th day of GCSF treatment, the parental and empty vector-transfected cells
were found to express little c-Myb protein. On the other hand, in the
c-myb-transfected cells, high levels of c-Myb protein were
detected. Similarly, as shown in Fig. 1D, after incubation in medium containing GCSF for 10 days, the empty vector-transfected cells were found to express little c-Myc protein. However, the c-myc-transfected cells showed the presence of high levels
of c-Myc protein product.
Effect of Ectopic Overexpression of c-Myb and c-Myc on GCSF-induced
Myeloid Cell Growth and Differentiation--
Following the
verification of the expression of transgene, we tested the effects of
overexpression of c-Myb and c-Myc on GCSF-induced terminal
differentiation of 32Dcl3 cells. 32Dcl3 cells transfected with empty
vectors or with c-myb and c-myc transgenes were
removed from IL-3-containing medium, washed twice, and cultured in the presence of GCSF. The results of these experiments are shown in Fig.
2. Parental 32Dcl3 cells or cells
transfected with the empty vectors (32D/pMT-Neo in Fig. 2A
and 32D/LK444A in Fig. 2B) undergo several rounds of cell
division followed by growth arrest around day 8 of GCSF treatment. On
the other hand, 32D/c-myb cells showed a significantly higher
proliferative potential (Fig. 2A) and continued to undergo
cell division exponentially. Interestingly, 32D/c-myc cells
(32D/c-myc/1 and 32D/c-myc/2 in Fig. 2B) behaved very
similar to mock-transfected cells, i.e.
c-myc-transfected cells underwent cell divisions until day 8 after which their proliferative potential declined. Thus c-Myb and
c-Myc conferred different properties to 32D cells in response to GCSF.
While constitutive expression of c-Myb markedly enhanced the growth
potential of 32D cells in response to GCSF, the expression of c-Myc
apparently did not alter the normal pattern of 32D cell growth in
GCSF.
Morphological analysis of the cells treated with GCSF is presented in
Fig. 3. Cells transfected with empty
vector plasmids (32D/pMT-Neo and 32D/LK444A), when treated with GCSF,
showed a progressive increase in the appearance of mature forms, which included the myelocytes, metamyelocytes, and granulocytes. By day 10, the entire population was completely replaced by terminally differentiated cells, consisting of metamyelocytes and granulocytes (Fig. 3). In contrast, 32D/c-myb cells showed little evidence of
progression to a differentiated phenotype and continued to proliferate
indefinitely in GCSF-containing medium as myelocytes/promyelocytes. Interestingly and in agreement with data presented in Fig. 2, both the
vector-transfected cells and 32D/c-myc cells progressed through the
normal pattern of differentiation when treated with GCSF and by day 10 were terminally differentiated into granulocytes. Again, c-Myb and
c-Myc had different effects on 32D cell differentiation program.
Whereas ectopic expression of c-Myb completely blocks the ability of
these cells to differentiate in response to GCSF, c-Myc by itself
apparently had no effect on 32D cell differentiation.
Effect of Ectopic Overexpression of c-Myc on the Apoptotic Program
of 32Dcl3 Cells--
Since c-Myc does not appear to have any
phenotypic changes on 32Dcl3 cell differentiation, we then investigated
if the overexpressed c-Myc protein is in fact functional in these
cells. It has been previously demonstrated that ectopic overexpression
of c-Myc in 32Dcl3 cells results in accelerated apoptotic death of
cells following IL-3 withdrawal (34). c-Myc has also been shown to
induce apoptosis in M1 myeloid leukemia cells following treatment with
TGF Induction of c-myc by c-myb in 32Dcl3 Cells in Response to
GCSF--
As noted earlier, when 32Dcl3 cells are treated with GCSF,
the expression of c-myc is detected for 6-8 days.
Thereafter the expression of c-myc declines and by day 10 is
detectable only at minimal levels. Using Atlas Gene Arrays
(Clontech), we had noticed high levels of
c-myc mRNA transcript in 32D/c-myb cells even at day 10 of GCSF treatment (data not shown). Sequence analysis of the
c-myc promoter/enhancer region had earlier revealed the presence of multiple Myb-binding sites in this region and in
vitro transcriptional transactivation assays had shown that c-Myb
can mediate transcriptional transactivation of this promoter leading to
the suggestion that c-myc could be a target gene of c-Myb
(43-45). Recently, the regulation of resident chromosomal copy of
c-myc by c-Myb was demonstrated in M1 myeloid leukemia cells
where c-Myb prevented IL-6 induced growth arrest and restored
expression of c-Myc in nearly terminally differentiated M1 cells
(27).
To determine the expression of c-myc in 32D/c-myb cells in
the presence of IL-3 and GCSF, we analyzed the protein lysates from
these cells by Western blot. As shown in Fig.
5A, in the presence of IL-3,
parental, mock-transfected and 32D/c-myb cells had approximately equal
levels of c-Myc protein expression. However, when the cells were
incubated in GCSF for 10 days, the expression of c-Myc was markedly
down-regulated in wild-type and empty vector-transfected cells while
its expression was elevated in cells that were transfected with
c-myb expression vectors (Fig. 5B). It should be
noted that after 10 days of incubation in GCSF, the parental and empty
vector-transfected cells were terminally differentiated into
granulocytes, while the cells transfected with c-Myb expression plasmid
were found to be promyelocytes/metamyelocytes. Thus, as seen in M1
leukemia cells, ectopic expression of c-Myb induces the expression of
c-Myc in 32D myeloid cells despite the presence of GCSF in the
medium.
Constitutive Expression of c-Myc Dominant Negatives in
32D/c-myb Cells--
The above observation that the
expression of c-Myc is induced by c-Myb in myeloid cells during their
differentiation led us to examine the precise role of c-Myc in
mediating the phenotypic effects of ectopic expression of c-Myb.
Specifically, we wanted to determine whether or not c-Myc is necessary
and sufficient for c-Myb-mediated block to the GCSF-mediated terminal
differentiation of 32Dcl3 cells. Our earlier observation that
deregulated expression of c-Myc does not change the normal pattern of
growth and differentiation of 32Dcl3 cells in GCSF allows us to
conclude that induction of c-Myc by itself is inadequate to bring about
the changes that result from the overexpression of c-Myb in this system.
To address the question as to whether or not c-Myc is necessary for
c-Myb-mediated block in 32D cell differentiation, we constitutively expressed c-Myc dominant-negatives mutants, MycRX and Max2RX, in
32D/c-myb cells. These dominant-negative mutants were previously constructed based on the observation that the biological and
biochemical activity of Myc is dependent on its ability to
heterodimerize with Max, which facilitates the binding of the Myc
protein to DNA via the bHLH-LZ motif (35). As depicted in Fig.
6A, the dominant-negative
mutants were generated by reciprocal exchange of HLH-LZ regions. Thus
MycRX contains the HLH-LZ region of Max and Max2RX contains the HLH-LZ
region of Myc. MycRX sequesters wild-type Myc into inactive complexes
whereas Max2RX forms stable binding complexes with wild-type Max (46).
These mutants were found to behave as strong dominant suppressors of
Myc activity by decreasing the levels of functional endogenous Myc-Max
complexes (35).
To further verify that the c-Myc dominant negatives suppress the
transcriptional activity of c-Myc, we performed transactivation assays
using QT6 fibroblasts. As a reporter, we used a fragment of ODC
promoter, pODC (228-281) shown to have maximal transactivation by
c-Myc (51). As depicted in Fig. 6B, exogenous
c-myc gives ~2-fold transactivation over that of empty
vector alone. A high level of activity was observed in empty
vector-transfected cells due to the presence of high level of
endogenous c-myc in these cells. When c-myc was
transfected along with the c-myc dominant-negative constructs, the transactivation activity of c-Myc was suppressed and
found to almost the same as that observed for empty vector alone. Thus
the dominant-negative mutants of c-myc inhibit its transcriptional activity.
The linearized mycRX and max2RX constructs in pOPRSVI-Puro/MCS (under
the control of RSV promoter) were transfected into the c-Myb-expressing
32Dcl3 cells via electroporation and their expression by Northern (Fig.
6, C and D) and Western blot analysis (Fig. 6,
E and F).
Effect of c-Myc Dominant Negatives on c-Myb-induced Proliferation
of 32D Cells--
Following the verification of the expression of
c-Myc dominant negatives in 32D/c-myb cells, we tested the effects of
constitutive expression of these Myc mutants on c-Myb-mediated block in
GCSF-induced terminal differentiation of 32Dcl3 cells. As described
before, these cells were removed from IL-3-containing medium, washed
twice, and cultured in the presence of GCSF. The growth curves are
shown in Fig. 7. 32D/c-myb cells
transfected with empty vector (32D/pOPRSVI-MCS/1 and 32D/pOPRSVI-MCS/2)
continued to proliferate beyond day 10 as seen in 32D/c-myb cells. In
contrast, 32D/c-myb cells transfected with c-Myc dominant-negative
mutants (32D/MycRX/1 and 32D/MycRX/2 in Fig. 7A;
32D/Max2RX/1 and 32D/Max2RX/2 in Fig. 7B) underwent several
rounds of cell division until about day 8-9 after which they ceased
proliferation. The growth pattern of these cells was very similar to
that observed in parental 32D cells. Thus, inhibition of the activity
of c-Myc in 32D/c-myb cells results in a reduction of the proliferative
potential conferred by c-Myb.
Effect of c-Myc Dominant Negatives on c-Myb-induced Block of
Differentiation of 32D Cells--
Fig. 8
shows the morphological analysis of cells
treated with GCSF. As expected, 32D/c-myb cells transfected with empty
vector (32D/pMT-c-myb/pOPRSVI/MCS/1 and
32D/pMT-c-myb/pOPRSVI/MCS/2) were blocked in their
progression to a differentiated phenotype and continued to proliferate
in GCSF containing medium at the myelocytic/promyelocytic stage.
Interestingly, 32D/c-myb cells transfected with c-Myc dominant-negative
mutants (32D/ pMT-c-myb/pOPRSVI/MycRX/1 and
32D/pMT-c-myb/pOPRSVI/ MycRX/2; 32D/pMT-c-myb/pOPRSVI/Max2RX/1 and 32D/pMT-c-myb/pOPRSVI/Max2RX/2) resumed normal pattern of differentiation, and by day 10, the cell population predominantly consisted of growth-arrested and terminally differentiated granulocytes (Fig. 8). These results suggest that c-Myc is an essential mediator of
the property of c-Myb to block terminal differentiation of 32D myeloid
cells.
Effect of c-Myc Dominant Negatives on c-Myb-induced Block of
Differentiation of M1 Cells--
It has been previously shown that
ectopic overexpression c-myb also blocks IL-6-induced
terminal differentiation of M1 cells into monocytes. To verify whether
this process is also dependent on the expression of c-Myc, we expressed
c-Myb together with either MycRX or Max2RX in M1 cells by retroviral
transduction. Following selection, parental M1 cells together with the
infected M1 cells V/c-Myb were treated with IL-6 for 4 days. The
parental M1 cells differentiated into mature cells with monocytic
morphology confirming the previous observations that IL-6 treatment was
sufficient to induce differentiation in this cell line (Fig.
9). When c-Myb was expressed in these
cells, differentiation was blocked and cells (V/c-Myb) remained as
blasts (Fig. 9). We next expressed the dominant-negative mutants of Myc
(MycRX and Max2RX) in these cells using a retroviral expression system.
Following the verification of expression of c-Myb as well as MycRX and
Max2RX, we exposed the cells to IL-6 for 4 days. These results
presented in Fig. 9 show that when the dominant-negative mutant MycRX
or Max2RX was expressed in the presence of c-Myb, the blockage of
differentiation by c-Myb was inhibited and the cells (MycRX/c-Myb and
Max2RX/c-Myb) differentiated into mature cells (Fig. 9). The results
were consistent with those observed in 32D cells and suggest that in
both systems c-myc gene expression is essential for
c-myb-induced transformation.
In this article, we have examined the molecular mechanisms
associated with Myb-mediated transformation of the myeloid precursor cell line, 32Dcl3. This cell line, which is derived from normal mouse
bone marrow is strictly dependent on IL-3 for growth (32, 33) and is
not predisposed to developing factor-independent clones (34). These
cells have been found to undergo terminal differentiation when placed
in an IL-3-free medium containing GCSF (32, 33). However, when the
cells were incubated in the absence of IL-3 or GCSF, they were found to
become arrested in the G1 phase of the cell cycle and
rapidly enter apoptotic pathways loosing viability in 48-96 h (34).
Results presented here as well those published earlier show that
ectopic overexpression of v-Myb or c-Myb in these cells results in a
block to their ability to terminally differentiate into granulocytes in
the presence of GCSF (9-11). In addition, the results presented in
this communication show that ectopic overexpression of c-Myb in these
cells results in an elevated expression of c-Myc further supporting the
notion that at least some of the effects mediated by c-Myb are achieved via the activation of c-Myc. A number of earlier studies have shown
that the promoter/enhancer region of c-myc contains several Myb-binding sites and in vitro transactivation studies
showed that the c-myc promoter is activated by c-Myb (43,
44, 45). However, the expression pattern of c-Myc and c-Myb in
mammalian tissues is very different suggesting that the regulation of
c-myc by c-Myb is not universal and may occur, if any, in a
limited number of tissues. However, at least in one cell line, the
myeloid leukemic cell line, M1, it was found that expression of c-Myc is dependent on the expression of c-Myb, suggesting that in myeloid cells, this relationship might exist (27). This notion is further confirmed by our observations presented here, which show that ectopic
overexpression of c-Myb results in a block to GCSF-induced down-regulation of c-myc.
To determine whether elevation of c-Myc expression alone is adequate
for Myb-mediated effects on GCSF-induced granulocytic differentiation
program, we ectopically overexpressed c-Myc in 32Dcl3 cells and studied
the effects of this gene on the differentiation program. Our results
show that unlike with c-Myb, ectopic overexpression of c-Myc had little
or no effect on GCSF-induced terminal differentiation of 32Dcl3 cells,
suggesting that Myb-mediated effects are not solely manifested through
c-Myc regulation. This is in sharp contrast to the results obtained
with the M1 leukemic cell line, whose terminal differentiation program
was found to be blocked by ectopic overexpression of c-Myb and c-Myc
(12, 31). Such differences are seen in other cell types as well and the
role of c-Myc in differentiation appears to be
tissue-dependent. For example, c-Myc-expressing primary
quail myoblasts are unable to form myotubes or express muscle-specific
genes, such as myosin, and show severely reduced expression of the
myogenic regulatory factors myoD, myogenin, and myf5 (47). However,
activation of c-Myc in primary human keratinocytes caused a progressive
reduction in growth rate and a marked stimulation of terminal
differentiation (48). In a recent study, it was shown that,
although c-Myc expression was downregulated in human leukemia K562
cells during differentiation and ectopic overexpression of c-Myc in
these cells did not affect the monocytic-macrophagic and magakaryocytic
differentiation induced by 12-O-tetradecanoyl phorbol-13
acetate (TPA) and staurosporine respectively (49). However, c-Myc is
able to inhibit the erythoid differentiation of K562 cells when induced
with 1- To gain a further understanding of the role of c-Myc in Myb-mediated
transformation of myeloid precursor cells, we expressed dominant-negative mutants of Myc in 32Dcl3 cells that have been earlier
transfected with a c-myb expression vector. These
dominant-negative mutants take advantage of the fact that the Myc and
Max proteins heterodimerize and bind to nuclear DNA via the b-HLH-LZ
motifs. As depicted in Fig. 5A, the dominant-negative
mutants were generated by reciprocal exchange of HLH-LZ regions (35).
As a result of this exchange, these mutant molecules sequester
endogenous wild-type Myc and Max into inactive complexes (35, 46).
These mutants were previously shown to act as strong dominant
suppressors of Myc activity by decreasing endogenous active Myc-Max
complexes (35). Interestingly, expression of these dominant-negative
mutants of Myc in Myb-transfected cells resulted in a block to the
effects of c-Myb and resulted in the re-initiation of the GCSF-induced terminal differentiation program. To investigate whether this relationship between c-Myc and c-Myb holds true in another cell line,
we used the M1 myeloid leukemic cells. As shown before, expression of
c-Myb in M1 cells blocked IL-6-mediated terminal differentiation of M1
cells. Moreover, when c-Myc dominant negatives were expressed in
Myb-transfected M1 cells, Myb-induced block was relieved, and the cells
were able to differentiate in a normal manner. These results taken
together suggest that while the activation of c-Myc by itself is
inadequate to mimic the phenotypic effects of c-Myb, its expression is
essential for the Myb-mediated block to GCSF-induced terminal
differentiation of 32Dcl3 cells. These observations suggest that c-Myb
activates additional signal transduction pathways that in concert with
c-Myc bring about the phenotypic effects exerted by c-Myb.
Identification of these transcriptional targets of c-Myb and a detailed
understanding of the signal transduction pathways that these genes are
associated with is likely to provide an understanding of the role
played by proto-oncogenes in the regulation of hematopoiesis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, the neutrophil elastase gene,
the homeobox gene GBX2 as well as several proto-oncogenes such as c-kit and c-myc (reviewed in Refs.
20-22). Of these the most interesting is the c-myc gene,
which also codes for a nuclear transcription factor and has been shown
to be intimately associate with cell growth, differentiation, and
apoptosis (reviewed in Refs. 23 and 24). The c-Myc protein belongs to
the family of transcription factors that contain the basic
helix-loop-helix leucine zipper (bHLH-LZ) domains at the C-terminal
end, which mediate the DNA binding and homo- and heterodimer formation
(reviewed in Ref. 25). Thus the c-Myc protein was found to require the formation of heterodimers with its partner, Max, for sequence-specific DNA binding as well as for biological activity (reviewed in Ref. 25).
While the c-myc and c-myb genes are co-expressed
in many hematopoietic cells, the c-myc gene expression is
seen more ubiquitously than that of c-myb, suggesting that
c-myb does not regulate c-myc expression in all
cell types (reviewed in Refs. 20 and 26).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
228 to
281
(51). The internal control plasmid, pRL-CH110, was constructed by
ligating the Renilla luciferase gene to the RSV promoter.
For transient transfections with the myc dominant-negatives,
15 µg of the corresponding dominant-negative construct was also added
to the DNA mixture above to get a final ratio of 1:3 between
myc transcriptional activator and dominant-negative transcriptional suppressors. Following incubation for 24 h, cells were washed and incubated in fresh medium for additional 24 h. After 48 h of post-transfection, cells were lysed in 500 µl/well of Passive Lysis buffer (Promega). Luciferase activity was assayed using Dual-Luciferase Reporter Plasmid System (Promega). Firefly luciferase activities were normalized against Renilla
luciferase activity to determine relative luciferase activity, and the
activation fold was obtained by setting the value of the empty vector
control as 1.0.
20 °C overnight and
resuspended in 10 mM Tris/HCl, 1 mM EDTA, pH
8.0. 2 mg of DNA were then analyzed on a 2% agarose gel.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Constitutive expression of c-Myb and c-Myc in
32Dcl3 cells. Mouse c-myb cDNA cloned into pMT-neo
vector (pMT/c-myb) and c-myc cDNA in LK444A vector
(LK444A/c-myc) were transfected into 32Dcl3 cells. Empty
vectors (pMT-Neo and LK444A) were also transfected as negative
controls. A and B, Northern blot analysis of
total RNA extracted from different cell lines using full-length
c-myb and c-myc cDNAs as probes. Endogenous
c-myb transcript (upper band) and exogenous
c-myb transcript (lower band) are shown in
A and endogenous c-myc transcript (upper
band) and exogenous c-myc transcript (lower
band) are depicted in B. As an internal control for RNA
loading, gels were stained with ethidium bromide to compare levels of
18 S/28 S ribosomal RNAs across lanes (shown below). C,
expression of c-Myb protein in the presence of GCSF for 10 days in
wild-type, mock-transfected, and c-myb-transfected 32Dcl3
cells. D, expression of c-Myc protein in the presence of
GCSF for 10 days in mock-transfected and c-myc transfected
32Dcl3 cells.
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Fig. 2.
Growth and differentiation of 32Dcl3 cells
overexpressing c-Myb and c-Myc in the presence of GCSF. 32Dcl3
cells transfected with empty vectors (32D/pMT-Neo and 32D/LK444A),
c-myb (32D/pMT c-myb), and c-myc (32D/LK444A
c-myc/1 and 32D/LK444A c-myc/2) expression vectors were analyzed for
their growth in the presence of GCSF. Cells were washed twice in
IL-3-free medium and cultured in the medium containing GCSF and were
split 1:4 with complete medium containing GCSF when the cells reached a
density of 5-7 × 105/ml. On each indicated day, the
number of cells were determined by trypan blue exclusion. Growth curves
of 32Dcl3 cells transfected with empty vector pMT-neo,
c-myb, and wild-type cells (A) and empty vector
LK444A and c-myc (B) are shown.
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Fig. 3.
Effect of ectopic expression of c-Myb and
c-Myc on GCSF-induced granulocyte differentiation of 32Dcl3 cells.
32Dcl3 cells overexpressing c-Myb (32D/pMT-c-myb) and c-Myc
(32D/LK444A-c-myc/1 and 32D/LK444A-c-myc/2) and mock-transfected cells
(32D/pMT-Neo and 32D/LK444A) were induced to differentiate by the
addition of GCSF. Following incubation for 0, 5, and 10 days in the
presence of GCSF (Day 0, Day 5, Day 10), aliquots of cells were
cytospun and stained with May-Grunwald-Geimsa. Representative samples
of cytospin slides of cells overexpressing c-myb and
c-myc constructs and mock-transfected cells at different
time points are shown in each block.
1 (42). As reported by other groups, the 32D/c-myc cells showed
accelerated kinetics of cell death (Fig.
4A). This observation could be
further verified by examining the degradation of DNA into
oligonucleosomal fragments, which is shown in Fig. 4B. In
empty vector-transfected cells, the onset of DNA ladder formation did
not occur for 24 h and the most intense ladder formation was seen
at the 48-h time point. In contrast, in 32Dcl3 cells transfected with
c-myc, the DNA ladders appeared much earlier with high
intensity ladders appearing around 18 h following IL-3
withdrawal.
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Fig. 4.
Effect of c-Myc on IL-3 withdrawal-mediated
apoptosis of 32Dcl3 cells. A, analysis of the viability
of c-myc-transfected cells in the absence of IL-3.
Mock-transfected and c-myc-transfected 32Dcl3 cells were
washed in IL-3-free medium and incubated up to 5 days. At each
indicated time point, the cells were analyzed for viability by trypan
blue exclusion/inclusion. The curves represent a mean of three
experiments. B, analysis of DNA fragmentation. At indicated
times following IL-3 withdrawal, DNA fragments released from 10 × 106 cells were extracted and separated by electrophoresis
and stained with ethidium bromide.
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Fig. 5.
Regulation of c-Myc by c-Myb in 32Dcl3
myeloid cells in the presence of IL-3 and GCSF. A,
analysis of expression of c-Myc protein by Western blot in presence of
IL-3 in wild-type (32D/wild-type), mock-transfected (32D/pMT-Neo) and
c-myb-transfected (32D/pMT/c-myb) 32D cells. B,
cells were washed twice in IL-3-free medium and induced to
differentiate in medium containing GCSF for 10 days. Western blot
analysis of c-Myc protein in wild-type (32D/wild-type),
mock-transfected (32D/pMT-Neo), and c-Myb-expressing (32D/pMT/c-myb)
32D cells at day 10 of incubation in medium containing GCSF.
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Fig. 6.
Constitutive expression of c-Myc
dominant-negative MycRX and Max2RX in 32Dcl3 cells with deregulated
expression of c-Myb. A, schematic representation of
Myc, Max, Max2, MycRX, and Max2RX. Max2 is a naturally occurring
variant of Max differing by a 9-amino acid insert upstream of the basic
domain. The dominant-negative MycRX and Max2RX were obtained from Dr.
B. Amati (35) and were constructed by swapping helix-loop-helix leucine
zipper domains in Myc and Max2, respectively. B, inhibition
of the transcriptional activity of c-myc by
dominant-negative mycRX and max2RX. Each dominant negative was
transfected into QT6 cells along with the c-Myc expression plasmid, the
reporter plasmid pODC (228-281) (51) and the control plasmid
pRL-CH110. After 2 days, the cell were harvested and assayed for
firefly luciferase activity using the Dual Luciferase System (Promega).
The luciferase activities were normalized to Renilla
luciferase activity. Shown here are the activation fold obtained by
setting the value of the empty vector control as 1.0. C and
D, mycRX and max2RX cloned in pOPRSVI-Puro/MCS vector were
transfected into 32D/c-myb cells together with empty vector
(pOPRSVI-Puro/MCS) as negative control. Northern blot analysis of total
RNA extracted from different transfected cell lines using full-length
mycRX and max2RX constructs as probes. mRNA expression of mycRX
transcript (lower band in C) in
32D/pMT-c-myb/pOPRSVI/MycRX/1 and 32D/pMT-c-myb/pOPRSVI/MycRX/2 cell
lines and that of max2RX transcript (lower band in
D) in 32D/pMT-c-myb/pOPRSVI/Max2RX/1 and
32D/pMT-c-myb/pOPRSVI/Max2RX/2 cell lines are shown. As an internal
control for RNA loading, gels were stained with ethidium bromide to
compare levels of 18 S/28 S ribosomal RNAs across lanes (shown below).
E, expression of MycRX protein by Western blot analysis in
the presence of IL-3 in mock-transfected cells
(32D/pMT-c-myb/pOPRSVI/MCS/1 and 32D/pMT-c-myb/pOPRSVI/MCS/2) and
mycRX-transfected cells (32D/pMT-c-myb/pOPRSVI/MycRX/1 and
32D/pMT-c-myb/pOPRSVI/MycRX/2). F, expression of Max2RX
protein by Western blot analysis in the presence of IL-3 in
mock-transfected cells (32D/pMT-c-myb/pOPRSVI/MCS/1 and
32D/pMT-c-myb/pOPRSVI/MCS/2) and max2RX-transfected cells
(32D/pMT-c-myb/pOPRSVI/Max2RX/1 and 32D/pMT-c-myb/pOPRSVI/Max2RX/2).
The primary antibodies used are described under "Materials and
Methods."
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Fig. 7.
Growth and differentiation of 32Dcl3 cells
overexpressing wild-type c-Myb and c-Myc dominant-negative MycRX and
Max2RX in the presence of GCSF. 32Dcl3/c-myb cells transfected
with empty vector (32D/pMT-c-myb/pOPRSVI/MCS/1 and
32D/pMT-c-myb/pOPRSVI/MCS/2) and c-myc dominant-negative
(32D/pMT-c-myb/pOPRSVI/MycRX/1, 32D/pMT-c-myb/pOPRSVI/MycRX/2,
32D/pMT-c-myb/pOPRSVI/MaxRX/1, and 32D/pMT-c-myb/pOPRSVI/MaxRX/2)
constructs were analyzed for their growth in the presence of GCSF.
Cells were washed twice in IL-3-free medium and cultured in the medium
containing GCSF and were split 1:4 with complete medium containing GCSF
when the cells reached a density of 5-7 × 105/ml. On
each indicated day, the number of cells were determined by trypan blue
exclusion. Growth curves of 32Dcl3 cells transfected with empty vector
pOPRSVI/MCS and mycRX (A) and empty vector pOPRSVI/MCS and
maxRX (B) are shown.
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Fig. 8.
Effect of c-Myc dominant-negative MycRX and
Max2RX on GCSF-induced granulocyte differentiation of 32Dcl3 cells with
ectopic expression of c-Myb. 32Dcl3/c-myb cells overexpressing
mycRX (32D/pMT-c-myb/pOPRSVI/MycRX/1 and 32D/pMT-c-myb/pOPRSVI/MycRX/2)
and max2RX (32D/pMT-c-myb/pOPRSVI/MaxRX/1 and
32D/pMT-c-myb/pOPRSVI/MaxRX/2) and mock-transfected cells
(32D/pMT-c-myb/pOPRSVI/MCS/1 and 32D/pMT-c-myb/pOPRSVI/MCS/2) were
induced to differentiate by the addition of GCSF. Following incubation
for 0, 5, and 10 days in the presence of GCSF (Day 0, Day 5, Day 10),
aliquots of cells were cytospun and stained with May-Grunwald-Geimsa.
Representative samples of cytospin slides of 32D/c-myb cells
overexpressing the two c-myc dominant-negative constructs
and mock-transfected cells at different time points are shown.
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Fig. 9.
Effect of c-Myc dominant-negative MycRX and
Max2RX on IL-6-induced differentiation of M1 cells with ectopic
expression of c-Myb. Morphology of infected cells. Wild-type M1
cells and retroviral infected M1 cells expressing V/c-Myb, MycRX/c-Myb,
and Max2RX/c-Myb were treated with IL-6 for 4 days. Cytospin smears of
those cells were stained with Wright-Giemsa stain. Representative
samples of cytospin slides are shown. Blasts contain scant cytoplasm
and round or oval nuclei; while mature cells have large cytoplasm
containing vacuoles.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-arabinofuranosylcytosine (50).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Bruno Amati for kindly providing the mycRX and max2RX constructs. We also thank Dr. Dan Liebermann for kindly providing LK444A and LK444A/c-myc constructs. The core facilities used in the study were supported by a grant from NCI, National Institutes of Health (NIH R24 CA88261).
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FOOTNOTES |
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* This work was supported by Grant CA79086 from the NCI, National Institutes of Health.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.
Both authors contributed equally to this work.
** To whom correspondence should be addressed: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-4307; Fax: 215-707-1454; E-mail: reddy@unix.temple.edu.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M300080200
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ABBREVIATIONS |
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The abbreviations used are: IL, interleukin; LIF, leukemia inhibitory factor; CAPS, 3-(cyclohexylamino)propanesulfonic acid; bHLH-LZ, basic helix-loop-helix leucine zipper; GCSF, granulocyte colony stimulating factor.
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