(Received for publication, November 18, 1994; and in revised form, January 18, 1996)
From the
Transforming growth factor-1 (TGF-
1) is a potent
inhibitor of hematopoietic cell growth. Here we report that TGF-
1
signals inhibition of IL-3-dependent 32D-123 murine myeloid cell growth
by modulating the activities of cyclin E and cyclin-dependent kinase 2
(cdk2) proteins and their complex formation in the G
phase
of the cell cycle. Whereas the cyclin E protein was hyperphosphorylated
in TGF-
1-treated cells, TGF-
1 decreased both the
phosphorylation of cdk2 and the kinase activity of the cyclin E-cdk2
complex. Decreased cyclin E-cdk2 kinase activity correlated with
decreased phosphorylation of the retinoblastoma-related protein p107.
In support of these observations, transient overexpression of p107
inhibited the proliferation of the myeloid cells, and expression of
antisense oligodeoxynucleotides to p107 mRNA blocked TGF-
1
inhibition of myeloid cell growth. Furthermore, as reported previously,
in 32D-123 TGF-
1-treated cells, c-Myc protein expression was
decreased. TGF-
1 increased the binding of p107 to the
transcription factor E2F, leading to decreased c-Myc protein levels.
p107 inhibited E2F transactivation activity and was also found to bind
the c-Myc protein, suggesting p107 negative regulation of c-Myc protein
function. These studies demonstrate the modulation of p107 function by
TGF-
1 and suggest a novel mechanism by which TGF-
1 blocks
cell cycle progression in myeloid cells.
Cell cycle progression in eukaryotic cells is controlled at the
G/S and the G
/M phase transitions. In mammalian
cells, a variety of kinases, which control the crucial transitions
through the cell cycle, have been
identified(1, 2, 3, 4) . These
kinases have been termed cyclin-dependent kinases (cdk) (
)since their activity is regulated by their association
with cyclins. The activity of cyclin-cdk complexes is known to
fluctuate dramatically during the cell cycle(4, 5) .
In general, D type cyclins appear earlier in G
than cyclin
E. Cyclin D expression oscillates minimally throughout the cell cycle.
Cdk4, which is thought to act at the G
/S restriction point,
is associated exclusively with cyclin
D(6, 7, 8) . The expression of cyclin E and
its associated kinase, cdk2, reaches a peak late in the G
phase(9, 10, 11) . The S phase is
controlled by the expression of cyclin A, which associates with both
cdc2 and cdk2, whereas the G
/M transition is controlled by
cyclin B and cdc2(2, 3, 12, 13) .
Cdc2, cdk2, and their activating cyclins also interact with and/or
phosphorylate other proteins believed to be important in cell cycle
regulation and DNA synthesis, including the tumor suppressor gene
product RB, the RB-related protein p107, and the transcription factor
E2F(14, 15, 16, 17) . The RB protein
negatively regulates E2F. E2F is known to play a role in the activation
of genes required for cell cycle progression. Genes such as
c-myc, c-myb, thymidine kinase, and cdc2 contain E2F
binding sites in their promoters (18, 19) . The
biological activity of the RB protein is regulated by its
phosphorylation state throughout the cell cycle. Hypophosphorylated RB
(G/G
phase) has been shown to bind and
sequester the E2F protein, thereby inhibiting E2F
transactivation(20, 21) . The cyclin E-cdk2 and cyclin
D-cdk4 complexes are known to phosphorylate and inactivate RB at the
G
/S boundary, thus allowing cell cycle
progression(7, 10, 22) . The c-myc proto-oncogene, whose expression is transactivated by E2F, is also
known to play an important role during the G
/S transition
of the cell cycle(23, 24) .
The p107 tumor
suppressor protein shares many structural and biochemical features with
RB. Similar to RB, p107 is a potent inhibitor of E2F-mediated
transactivation, and overexpression of p107 can inhibit the
proliferation of certain cell types by arresting the cells in
G(17, 25) . More recent studies have
provided evidence for p107 binding to the c-Myc protein, suggesting for
the first time a possible role for p107 in the regulation of c-Myc
function(26) . p107 is usually underphosphorylated during
G
and is phosphorylated in the S, G
, and M
phases(27) . However, the regulation of p107 function during
growth suppression remains to be elucidated.
TGF-1 is a
multifunctional regulator of cell growth and differentiation in many
cell types, including epithelial, endothelial, and hematopoietic
cells(28, 29) . In epithelial cells, it has been shown
that the TGF-
1-induced proliferative block occurs in late G
and thus prevents DNA
synthesis(30, 31, 32, 33) .
TGF-
1 inhibition of keratinocyte proliferation involves the
suppression of c-myc transcription via hypophosphorylation of
RB. It has been shown that one of the steps by which TGF-
1 exerts
its inhibitory effects on cell cycle progression is by preventing the
hyperphosphorylation of RB(34, 35) . This
TGF-
1-mediated block in RB hyperphosphorylation also prevents the
transactivation of the c-myc gene, which causes c-Myc protein
levels to decline rapidly(36) . In addition, in Mv1Lu cells
TGF-
1 also has been shown to disturb the assembly of cyclin E-cdk2
complexes and cyclin E-associated kinase activity(32) .
The
growth of interleukin 3 (IL-3)-dependent myeloid leukemic cell lines is
inhibited by TGF-1(37, 38) . The molecular
mechanisms involved in TGF-
1-induced growth inhibition and cell
cycle progression in myeloid cells remain largely unknown and may be
different from those found in keratinocytes and other epithelial cells.
Because TGF-
1 plays a critical role in hematopoiesis, the
molecular mechanisms by which TGF-
1 inhibits myeloid cell
proliferation, specifically at the G
/S transition, were
studied. These results indicate that in myeloid cells, TGF-
1
modestly inhibits the association of the cyclin E-cdk2 complexes and
markedly inhibits the activity of the complex, as well as markedly
stimulating phosphorylation of cyclin E and dephosphorylation of cdk2.
However, phosphorylation of RB was unchanged in synchronized
IL-3-dependent 32D-123 myeloid cells treated with TGF-
1. In
contrast, we found that in response to TGF-
1 treatment of the
32D-123 myeloid cells, p107 was hypophosphorylated, and p107 binding to
E2F and to c-Myc was increased. This modulation of p107 by TGF-
1
together with the ability of antisense to p107 to block TGF-
1
inhibition of growth suggests a novel mechanism by which TGF-
1
blocks cell cycle progression in myeloid cells.
GAL4-E2F was constructed by inserting a polymerase chain reaction-generated fragment that contains murine E2F sequences into the SaII and XbaI sites in the polylinker of pBluescript SK+(42) . The GAL4-E2F construction used in these experiments contains the complete 440 amino acids of the E2F protein fused to the DNA binding domain of GAL4 (amino acids 1-147). The reporter construction, G1B, contains one GAL4 binding site upstream of the adenovirus E1b box driving CAT(41) .
Figure 1:
Cell synchronization and TGF-1
treatment. 32D-123 murine myeloid cells were synchronized at the
G
/S boundary by means of an aphidicolin block (panel
A) or during G
phase by IL-3 starvation (panel
B). Cell aliquots (5
10
) were taken at the
indicated times after release from the block. The synchrony of the
successive populations was observed by subjecting the samples to flow
cytometric analysis after staining of nuclear DNA with propidium
iodide. Data are from a single experiment that is representative of
three experiments. Aphidicolin-treated 32D-123 (panel C) and
IL-3-deprived (panel D) cells were treated with TGF-
1 (10
ng/ml) at 2-h intervals as indicated. Proliferation was assayed 24 h
after TGF-
1 treatment using [
H]thymidine
incorporation. Cumulative incorporations after TGF-
1 treatment at
the indicated times are plotted as a percentage of control
(unsynchronized cells treated with TGF-
1 for 24 h). Data are the
means of triplicates of a representative experiment. Analysis of
variance and Duncan's test showed the TGF-
1 treatment to be
statistically significant (p <
0.0001).
To
determine the optimal time point for TGF-1 treatment (maximal
growth inhibition) and because 32D-123 cells require 12 h to complete
the cell cycle following aphidicolin treatment (Fig. 1A), at 2-h intervals (0, 2, 4, 6, 8, 10, and 12
h) after aphidicolin treatment cells were treated with TGF-
1 (10
ng/ml), and proliferation was analyzed by
[
H]thymidine incorporation. As shown in Fig. 1C using aphidicolin-treated cells maximal
inhibition of [
H]thymidine incorporation (45%)
was observed by 8 h. Table 1shows the FACS analysis of cells
treated in the presence and absence of TGF-
1 at 8 h after
aphidicolin treatment. By 24 h after TGF-
1 treatment 64% of the
cells arrested in the G
phase (control 39.6%, Table 1). Longer incubation times in the presence of TGF-
1
increased the number of cells arrested in G
(data not
shown). In Fig. 1C time zero refers to the addition of
aphidicolin. There was no significant inhibition of 32D-123 cell
proliferation when TGF-
1 was added after 12 h of aphidicolin
treatment because the cells are at the G
/S restriction
point (as indicated in Fig. 1A). 32D-123 cell growth
inhibition was seen at 8 h, when a significant number of cells are
found in the G
phase (a point of the cell cycle when they
are most sensitive to TGF-
1) moving toward the restriction
point(34) . At earlier time points low levels of inhibition are
seen because of the lack of synchronization of the cells. Therefore,
the following TGF-
1 studies were done with cells treated with
aphidicolin for 12 h. TGF-
1 was added to the cells 8 h after the
initial treatment with aphidicolin, for a total of 24 h. Aphidicolin
and TGF-
1 treatment overlapped only for a period of 4 h.
IL-3-deprived cells were treated with IL-3 and serum followed by
TGF-1 treatment (for a total of 24 h) at 2-h intervals (0, 2, 4,
6, 8, and 10 h). Maximal inhibition of
[
H]thymidine incorporation (76%) was observed
when 32D-123 cells were treated with TGF-
1 at 6 h after IL-3
addition (Fig. 1D). Lower levels of 32D-123 growth
inhibition by TGF-
1 treatment at 2 and 4 h following IL-3 addition
were not unexpected. Most, if not all growth factors up-regulate
TGF-
cell surface receptors(45, 47) , and
previous studies by our laboratory have indicated the expression of a
low number of TGF-
receptors in the 32D-123 cells(45) .
Specifically, TGF-
ligand binding studies previously indicated the
absence of TGF-
1 receptors in IL-3-deprived cells (data not
shown). However, whether low or high number of receptors are bound by
TGF-
1 ligand followed by internalization, the reappearance of the
TGF-
receptors in the outer cell membrane only occurs 72 h
later(45) . Consequently, since inhibition of proliferation by
TGF-
1 occurs only after threshold levels of the intracellular
regulators are reached, 6 h after IL-3 addition is when maximal
expression/internalization of receptor-ligand complexes of TGF-
takes place. The TGF-
1-induced maximum growth inhibition of
IL-3-deprived cells at 6 h indicated optimal expression of cell surface
TGF-
receptors. FACS analysis of these cells identified that by 14
h, 64.3% of the cells were growth arrested at the G
phase (Table 2). This FACS analysis confirms our earlier observation
that aphidicolin synchronization defines the phases of the cell cycle
more clearly than IL-3 deprivation ( Table 1and Table 2).
Figure 2:
Expression of cyclins and effect of
TGF-1 treatment on the levels of G
cyclins in 32D-123
cells. Panel A, cell aliquots were taken at the indicated
times after release from an aphidicolin block, and cell lysates (150
µg of protein) were subjected to immunoblot analysis. Blots were
probed with antibodies to cyclin A, B, D, E, cdc2, cdk2, cdk4, or cdk5
as indicated. Panel B, cells were aphidicolin synchronized for
a total of 12 h. Before the end of the aphidicolin treatment TGF-
1
was added to the cells for a total of 24 h (aphidicolin and TGF-
1
treatment overlap for 4 h). Cell aliquots were taken at the
G
/S boundary (24 h after TGF-
1 treatment), lysed, and
protein samples (150 µg) were subjected to immunoblot analysis.
Blots were probed with antibodies to cyclins D, E, and A and cdks 2, 4,
and 5 as indicated.
Figure 3:
TGF-1 modulation of cyclin E protein
levels and associated protein kinase activity in lysates from 32D-123
myeloid cells synchronized at the G
/S phase (24 h after
TGF-
1 treatment). 32D-123 cells were synchronized and treated as
described in the legend to Fig. 2. Panel A, immunoblot
analysis of anti-cyclin E immune complexes. Immunoblots of proteins
immunoprecipitated with anti-cyclin E were probed with antibodies to
cyclin E and cdk2. Panels B and C, TGF-
1
modulation of cyclin E and cdk2, respectively, as measured by in
vivo phosphorylation. Cells were labeled with
[
P]orthophosphate for 3.5 h, lysed, and cyclin E
or cdk2 proteins were analyzed by immunoprecipitation and SDS-PAGE (12%
for cyclin E and 15% for cdk2) followed by autoradiography. Panel
D, histone H1 kinase assay was carried out using anti-cyclin E and
B immune complexes prepared from 32D-123 cells treated with TGF-
1.
The immune complexes were incubated under the appropriate conditions in
the presence of [
-
P]ATP and histone H1.
This was followed by SDS-PAGE and
autoradiography.
Figure 4:
TGF-1 modulation of RB and p107
protein and associated kinase activity. Western blot analyses of RB (panel A) and p107 (panel B) proteins in
IL-3-deprived myeloid cells treated with IL-3 and serum for 6 h
followed by treatment in the presence and absence of TGF-
1 for the
time points indicated below. Four hundred (for RB; panel A) or
150 (for p107; panel B) µg of protein obtained after
TGF-
1 treatment, at G
(0 h, lane 1),
mid-G
(6 h, lane 2), late G
(10 h, lanes 3 and 4), and G
/S (14 h, lanes
5 and 6) were separated by SDS-PAGE (7.5%), transferred
to Immobilon membrane, and probed with antibodies to RB or p107. Panel C, analysis of histone H1 kinase activity associated
with RB (lanes 1 and 2) and p107 (lanes 3 and 4) was carried out using anti-RB and p107 immune
complexes prepared from 32D-123 cells treated with TGF-
1. The
complexes were incubated under the appropriate conditions in the
presence of [
-
P]ATP and histone H1,
followed by SDS-PAGE and autoradiography. Data are representative of
three experiments.
Figure 5:
Characterization of p107 phosphorylation.
TGF-1 modulation of p107 phosphorylation. Panel A,
exponentially growing 32D-123 cells treated with and without TGF-
1
for 14 h. Cells were labeled with
[
P]orthophosphate for 3.5 h, lysed, and
immunoprecipitated using anti-p107 (lanes 1 and 2)
and anti-RB (lanes 3 and 4) antibodies.
Immunoprecipitates were analyzed by SDS-PAGE (4-15%) and
autoradiography. Panel B, IL-3-deprived myeloid cells treated
with IL-3 and serum for 24 h in the presence and absence of TGF-
.
[
P]Orthophosphate-labeled cell lysate were
immunoprecipitated with anti-p107 antibodies (lanes
1-4), and dephosphorylation of
P-labeled p107
immunoprecipitates was carried out using protein phosphatase 1A (pp1A; lanes 3 and 4). An immunoblot of
P-labeled immunoprecipitates from the above (lanes
1-4) was analyzed by Western blotting with an anti-p107
antibody. Data are representative of three independent
experiments.
Expression
(transfection) of pMAM-p107 AS and pMAM-p107 S had little effect on
32D-123 cell proliferation (89.8 and 101.9% proliferation,
respectively) compared with mock transfected cells (Table 3). In
addition, TGF-1 treatment of pMAM-p107 AS transfected cells did
not inhibit cell proliferation compared with untreated pMAM-p107 AS
transfected cells (82.6 and 89.8% proliferation, respectively, Table 3). However, TGF-
1 treatment of pMAM-p107 S
transfected cells resulted in an inhibition of proliferation similar to
that of the control (Table 3). The ability of antisense to p107
mRNA to reverse the TGF-
1 inhibitory effect is evidence that p107
is a mediator of TGF-
1 inhibition of myeloid cell proliferation,
suggesting a novel mechanism that requires p107 regulation for
TGF-
1 function.
Figure 6: p107 represses GAL4-E2F transactivation activity. 32D-123 cells were cotransfected with 10 µg of G1B reporter plasmid and 10 µg of GAL4-E2F, or 1, 5, and 10 µg of p107 or RB expression vector or pBR322 control. CAT activity was measured 24 h later, and values were normalized as described under ``Experimental Procedures.'' The CAT activity of cells transfected with GAL4-E2F and without the p107 expression vector was set equal to 1.0 (control). The relative CAT activity expressed here represents the averages from at least three individual transfections. Standard deviations were approximately 10% of the mean values obtained.
Figure 7:
Effect of TGF-1 on the association
between p107 and the transcription factors E2F and c-Myc. Panel
A, 32D-123 cell lysates were treated as described in the legend to Fig. 5and were immunoprecipitated with antibodies to normal
mouse serum (lane 2) and a monoclonal antibody to E2F (lanes 3 and 4). Immunoblots were probed with a
polyclonal antibody to p107. The p107 control (lane 1; 150
µg) obtained from the above described lysates was analyzed by
Western blotting with the anti-p107 antibody (lane 1). Panel B, the TGF-
1-treated (lane 1) and
untreated (lane 2) lysates described in Fig. 5were
analyzed by Western blotting with anti-c-Myc antibody and by
immunoprecipitation (panel C) with an anti-p107 antibody
followed by Western blotting with anti-c-Myc
antibody.
Previous studies have indicated that TGF-1 inhibits cell
proliferation by affecting cell cycle
progression(10, 32, 34, 56) . As in
the case of fibroblasts and lymphocytes, we found TGF-
1 to
suppress myeloid cell growth in the G
phase, therefore our
studies focused on the molecular mechanisms affected by TGF-
1
during the G
and G
/S transition of the myeloid
cell cycle. In 32D-123 murine cells and in human TF-1 (data not shown)
TGF-
1 markedly inhibits the activity of the cyclin E-cdk2 complex
by increasing both the phosphorylation of cyclin E and the
dephosphorylation of cdk2. As a result of the decrease in cyclin E-cdk2
kinase activity by TGF-
1, the p107 protein but not the RB protein
is dephosphorylated, and the binding of p107 to E2F and c-Myc proteins
is increased, thus regulating c-Myc protein expression and function.
The TGF-
1 modulation of p107 function was confirmed further when
transient overexpression of p107 inhibited the proliferation of the
myeloid cells, and expression of antisense oligodeoxynucleotides to
p107 mRNA blocked TGF-
1 inhibition of myeloid cell growth.
First, we examined the effect of TGF-1 on the cyclins and cdks
in the G
phase. Recent observations of fibroblasts have
shown that TGF-
1 inhibits the association of cyclin E-cdk2
complexes(30) . This study prompted us to examine the
regulation of cyclins and cdk2 by TGF-
1 in myeloid cells. Results
indicated that TGF-
1 targets the regulation of the G
cyclin-cdk complex, cyclin E-cdk2. In 32D-123 cells, TGF-
1
treatment increased the levels of the 48-kDa form and decreased the
levels of the 45-kDa form of cyclin E and its associated cdk2 partner,
resulting in a modest decrease in cyclin E-cdk2 complex formation. The
immunoprecipitation studies suggest that the 45-kDa form of cyclin E
has the highest affinity for cdk2, whereas the 48-kDa form has lower
affinity. It is not known whether these two forms of cyclin E have
different functions.
Studies using in vivoP
labeling have demonstrated that markedly increased phosphorylation of
both forms of cyclin E occurs in response to TGF-
1 treatment. The
kinase activity of cdk2, which is determined by its phosphorylation
state, has been shown to vary during the different phases of the cell
cycle(30, 57) . TGF-
1 markedly decreased the
active phosphorylated form of cdk2 in 32D-123 cells. Previous studies
have shown that TGF-
1 prevents phosphorylation of p33
in epithelial cells, thus maintaining cdk2 in its inactive
form(30, 58) . In vitro analysis of cyclin
E-cdk2 kinase activity in 32D-123 lysates from TGF-
1-treated cells
indicated a reduction in cyclin E-associated kinase activity compared
with the untreated control. Thus, in 32D-123 cells, TGF-
1 alters
the phosphorylation state of cyclin E and cdk2, decreases the
association of the complex, and decreases the kinase activity of the
complexes formed. More recent studies have shown that stimulation of
C3H fibroblastic cell growth by TGF-
1 results in increased
phosphorylation of cyclin E. (
)Therefore, the role of cyclin
E phosphorylation and dephosphorylation during TGF-
1 signal
transduction is not clear. The phosphorylation of cyclin E could be an
epiphenomenon and may not be involved directly in cell cycle control by
TGF-
1.
Because regulation of cdk activity mediates
cytokine-induced RB regulation (i.e. RB-related proteins can
bind cyclins and cdks to establish potential regulatory feedback
loops(17, 59, 60) ), we investigated the
effect of TGF-1 on RB and RB-related proteins. TGF-
1
treatment of 32D-123 cells decreases p107 phosphorylation and increases
p107-E2F complex formation. p107 dephosphorylation following TGF-
1
treatment results from inactivation of cyclin E-cdk2 complex. This
complex (p107-E2F) represses c-myc expression at the protein
levels, consequently repressing c-Myc function.
Our studies suggest
that the cyclin E-cdk2 complex is less stable in TGF-1-treated
cells as a result of the changes in the phosphorylation states of their
components. The kinases involved in the phosphorylation of the cyclins,
particularly cyclin E, have not yet been identified. Thus far only the
tyrosine phosphatase 25 (cdc25) and protein phosphatases 1 and 2A have
been implicated in the dephosphorylation of
cdc2(61, 62, 63) . In 32D-123 cells, the
down-regulation of cdk2 phosphorylation in TGF-
1-treated cells
results from increased phosphatase activity. TGF-
1 treatment of
the 32D-123 cells may render cdk2 intrinsically incompetent for cyclin
E activation. This regulation may also occur via a post-translational
modification or a block imposed by the binding of a third protein to
cdk2, like the association that has been reported for cyclin D-cdk4 and
the p27 protein(64) .
In 32D-123 myeloid cells, TGF-1
did not significantly affect the phosphorylation state of RB during the
G
/S phase. Other studies support this observation; for
example, TGF-
1 treatment of murine B-cell lymphomas did not affect
the phosphorylation state of RB in growth-arrested cells(65) ,
and overexpression of RB did not affect the inhibition of cell
proliferation in cervical carcinoma cells(14, 25) . A
more defined role for RB is found in proliferating Mv1Lu cells, where
TGF-
1 has been shown to prevent RB phosphorylation during
G
, thus retaining RB in a hypophosphorylated active state
that may suppress cell progression into the S
phase(35, 60, 66) . Studies by Clarke et
al. (67) with RB -/- knockout mice indicated
abnormal differentiation or a failure to differentiate, particularly in
the hematopoietic and neuronal
lineages(67, 68, 69) , suggesting that the
role of RB may be lineage-specific and may also be related to the
differentiation state of these cells.
Because p107 has been shown to
be phosphorylated by cyclin E-cdk2 in a manner similar to RB, we next
investigated whether p107 was a target for TGF-1
regulation(27) . TGF-
1 was found to decrease the
phosphorylation of p107 at the G
/S boundary. Furthermore,
TGF-
1 treatment decreased histone H1 kinase activity in p107
immunoprecipitates. p107 is known to be physically associated with
cyclin E and cdk2 in a variety of human cell
lines(17, 70) . Our studies suggest that decreased
p107 phosphorylation in response to TGF-
1 treatment is a result of
decreased cyclin E-cdk2 activity.
Binding of the transcription
factor E2F to the c-myc promoter is required for
transcriptional activity to occur(71, 72) ; this
transcriptional activity is modulated by other proteins that bind E2F,
such as RB and p107(14, 15, 17) . In the
32D-123 cells, p107 was found to be associated with E2F, and TGF-1
treatment increased the E2F-associated p107 protein, resulting in
decreased c-Myc protein expression. In support of this, transient
transfection assays with NIH-3T3 cells have indicated that p107
inhibits E2F-dependent transcription, and free E2F accumulates as cells
leave G
and enter S phase(15, 18) . In
addition, recent studies have identified the growth-inhibitory block of
TGF-
to be located close to the G
/S border in the cell
cycle in human embryonic lung fibroblasts(73) .
The
functional relevance of p107 mediating TGF-1 regulatory effects in
the 32D-123 cells was established in several ways. Overexpression of
p107 led to suppression of cell proliferation, presumably by blocking
cell cycle progression. In addition, by using GAL4-E2F chimeric
constructions, p107 was shown to inhibit directly E2F transactivation
activity. More recent studies have identified the regulation of the
c-Myc protein function by the binding of p107 to the c-Myc
transactivation domain. The binding of c-Myc by p107 resulted in
inhibition of c-Myc transactivation activity(26) . In our
studies, the steady-state levels of c-Myc in 32D-123 cells were
significantly decreased by TGF-
1 treatment, whereas the levels of
c-Myc bound to p107 were increased. Moreover, the ability of the p107
antisense to reverse the TGF-
1 inhibition of 32D-123 cell
proliferation confirms the direct role of p107 as a mediator of
TGF-
1 function.
Our studies indicate that some of the
inhibitory effects of TGF-1 on cell cycle progression in myeloid
cells occur via regulation of the p107 protein. p107 was demonstrated
to be important for inhibition of cell proliferation and
TGF-
1-induced p107 activation (dephosphorylation), allowing the
p107-E2F complexes to form. TGF-
1 also modulated cyclin E and cdk2
at the post-translational level (phosphorylation). As a result, the
G
/S cyclin E-cdk2 transitional complex activity was
decreased, p107 was activated, and cell cycle progression was blocked.
These studies provide a greater understanding of the molecular
mechanisms by which TGF-
1 regulates cell cycle progression in
myeloid cells.