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Molecular and Cellular Biology, July 2000, p. 4745-4753, Vol. 20, No. 13
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Bcl-2 Retards Cell Cycle Entry through
p27Kip1, pRB Relative p130, and Altered E2F
Regulation
Gino
Vairo,1,
Timothy J.
Soos,2
Todd M.
Upton,3
Juan
Zalvide,3
James A.
DeCaprio,3
Mark E.
Ewen,3
Andrew
Koff,2 and
Jerry M.
Adams1,*
The Walter and Eliza Hall Institute of
Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia1; Memorial Sloan-Kettering
Cancer Center, New York, New York 100212; and
Dana-Farber Cancer Institute and Harvard Medical School,
Boston, Massachusetts 021153
Received 28 December 1999/Returned for modification 24 February
2000/Accepted 13 April 2000
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ABSTRACT |
Independent of its antiapoptotic function, Bcl-2 can, through an
undetermined mechanism, retard entry into the cell cycle. Cell cycle
progression requires the phosphorylation by cyclin-dependent kinases
(Cdks) of retinoblastoma protein (pRB) family members to free E2F
transcription factors. We have explored whether retarded cycle entry is
mediated by the Cdk inhibitor p27 or the pRB family. In quiescent
fibroblasts, enforced Bcl-2 expression elevated levels of both p27 and
the pRB relative p130. Bcl-2 still slowed G1 progression in
cells deficient in pRB but not in those lacking p27 or p130. Hence, pRB
is not required, but both p27 and p130 are essential mediators. The
ability of p130 to form repressive complexes with E2F4 is implicated,
because the retardation by Bcl-2 was accentuated by coexpressed E2F4. A
plausible relevant target of p130/E2F4 is the E2F1 gene, because Bcl-2
expression delayed E2F1 accumulation during G1 progression
and overexpression of E2F1 overrode the Bcl-2 inhibition. Hence, Bcl-2
appears to retard cell cycle entry by increasing p27 and p130 levels
and maintaining repressive complexes of p130 with E2F4, perhaps to
delay E2F1 expression.
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INTRODUCTION |
In addition to its well-established
function in controlling cell survival, the Bcl-2 family (1)
has been found to influence the cell cycle. Although Bcl-2 and its
prosurvival relatives do not affect the growth rate in proliferating
cultures, they can both accelerate withdrawal from the cycle
(46) and retard reentry (4, 29, 30, 35).
Conversely, a shortened G1 is found in lymphocytes that
lack Bcl-2 (29) or express the Bcl-2 antagonist Bax
(4). Thus, Bcl-2 appears to have a physiological role in influencing the transition between the quiescent and cycling states. That this ability is separate from its role in cell survival
(46) is most clearly shown by mutations of Bcl-2 that
eliminate its cell cycle activity but spare its antiapoptotic function
(17, 45).
The cell cycle control function of Bcl-2 has ramifications for cellular
homeostasis. Cycling cells are often more vulnerable to apoptosis,
perhaps because, under conditions unfavorable for proliferation,
certain cell cycle effectors promote apoptosis (11). Hence,
promoting quiescence under conditions of stress may provide Bcl-2 with
an additional, albeit indirect, means to enhance cell survival
(30, 46). Interference with Bcl-2's cell cycle effect may
also augment its oncogenic role (see Discussion).
Progression through the cell cycle requires the action of
cyclin-dependent kinases (Cdks) (38, 39). As cells enter the cycle, newly synthesized D-type cyclins associate with and activate their Cdk-4/6 catalytic partners in mid- to late G1 phase,
while cyclin E appears later in G1 and activates its Cdk-2
kinase subunit near the G1/S boundary. Opposing their
activity are Cdk inhibitors (Cki) of two classes: INK4 proteins, such
as p16, specifically inhibit D-cyclin kinases, whereas Cip/Kip
proteins, such as p21 and p27, also inhibit Cdk-2 (38, 39).
Other key negative regulators include the best-known Cdk
substrates, the retinoblastoma (RB) family of nuclear "pocket"
proteins: pRB itself, p130, and p107 (10, 34). They are
thought to act by forming repressive complexes with E2F transcription
factors, which control the expression of genes essential for cell cycle
progression. Phosphorylation of the pocket protein by Cdks frees the
E2F and thereby derepresses or activates E2F target genes.
The G1-S-phase transition is thought to be controlled by
pRB, which binds E2Fs 1 to 3. Hence, p107 or p130, both of which bind
E2F4 or E2F5 and probably regulate a distinct subset of E2F target
genes (18), might govern the poorly understood exit from G0. p107 is unlikely, because its expression is restricted
to cycling cells and it associates with E2F4 only late in
G1, but p130 is prominent during quiescence, and p130-E2F4
complexes appear almost exclusively during G0 (20, 32,
47). Hence, regulation of E2F4 activity by p130 has been
implicated in control of the G0/G1 transition,
but no antiproliferative pathway is yet known to rely on p130.
How Bcl-2 favors the quiescent state is largely unknown. Neither p53
nor p16 can be essential, because Bcl-2 promoted quiescence in cells
lacking those genes (35, 46). In bcl-2 transgenic cells, delayed cycle entry correlated with increased expression of p27
(4, 29), hypophosphorylated pRB (30), and
increased p130 (28), but those alterations might simply be
an indirect consequence of the increased proportion of noncycling cells.
To establish the negative regulators through which Bcl-2 acts, we have
used cells derived from mice deficient in pRB, p130, and p27. We report
that pRB is dispensable for the inhibitory effect but that both p27 and
p130 are essential, and we present evidence that p130 may act through
E2F4 to control the level of E2F1. These findings thus establish the
framework through which Bcl-2 impacts the cell cycle. They also
identify the first antiproliferative pathway requiring p130 and provide
the first functional evidence that p130 plays an essential role in a
pathway controlling G0 exit.
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MATERIALS AND METHODS |
Cell lines, expression vectors, transfection, and DNA
analysis.
The RB
/ fibroblast cell
lines, immortalized by the 3T3 protocol, have been described
(50), and fibroblast lines were generated similarly (T. M. Upton and M. E. Ewen, unpublished) from embryonic fibroblasts
of p130/ and wild-type mice (18).
The NIH 3T3 lines stably transfected with bcl-2 (cl.AH2) or
its Y28A mutant (cl.FF3) were characterized previously (17).
The expression vectors pCDNA1-HAp130 (47) and
pEFhBcl-2pGKpuro and the parent vector pEFpGKpuro
(35) have been described previously. pGFP-E2F1 and pGFP-E2F4
were constructed by inserting the human E2F1 and E2F4 cDNAs
(15), respectively, into pEGFP-C1 (Clontech).
Exponentially growing cultures of the fibroblast lines (10
6
cells) were electroporated (500 V and 25 µF capacitance) with 15
µg
of linearized expression vector using a Gene Pulser (Bio-Rad).
The
cells were then resuspended in ~44 ml of Dulbecco's modified
Eagle's medium (DME) with 10% fetal bovine serum (FBS) and dispensed
into 10-cm dishes, and puromycin (3 µg/ml; Sigma) was added 2
days
later. Puromycin-resistant clones, picked from separate dishes
to
ensure independent derivation, that had closely matched levels
of
hBcl-2, determined by immunoblot analysis or by flow cytometric
analysis using a monoclonal antibody specific for human Bcl-2
(
35,
46), were chosen for study. For transient transfection,
exponential cultures of control or Bcl-2-expressing fibroblasts
in
10-cm dishes were lipofected with 1 µg of pEGFP-C1 together
with 8 µg of pCDNA1-HAp130 or empty pCDNA1 vector or with 8 µg
of
pGFP-E2F4, pGFP-E2F1, or parent GFP vector. DNA premixed with
20 µl
(2 mg/ml) of lipofectAMINE (Gibco-BRL) was added to the
cells in
antibiotic-free DME for ~6 h according to the manufacturer's
instructions. The medium was then replaced with DME-10% FBS. After
incubation overnight, the cells were harvested by trypsinization
and
resuspended in 0.5 ml of DME-2% FBS containing 0.5 µg of propidium
iodide per ml for sorting. Viable (propidium iodide excluding)
cells
that displayed similar levels of GFP-E2F were isolated using
a FACStar
Plus cell sorter (Becton Dickinson). Recovered cells
were resuspended
in DME-10% FBS and seeded into six-well plates
(Falcon) at 5 × 10
4 cells/well. After 2 days they were washed and
resuspended in
DME-0.1% FBS and cultured for a further 5 days with
medium changes
on days 3 and 4 to render them quiescent. The cells were
stimulated
to reenter the cell cycle by replacing the medium with
DME-10%
FBS, and the entire contents of the well were collected after
~20 h. Concurrent cell cycle and apoptosis analyses were performed
as
described previously (
46).
Immunoblots and antibodies.
Immunoblotting was performed
essentially as described (47) except that fibroblasts were
harvested and lysed at 107 cells/ml in
radioimmunoprecipitation (RIPA) buffer (150 mM NaCl, 1% NP-40, 0.5%
deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mM Tris [pH
7.5], 1 mM dithiothreitol, 50 mM NaF) containing protease inhibitors.
Samples containing equivalent amounts of protein were subjected to
SDS-polyacrylamide gel electrophoresis and transferred to Immobolin-P
membranes (Millipore). Bands were visualized by enhanced
chemiluminescence (Amersham). Where indicated, membranes were stripped
by incubation in 2% SDS-100 mM 
-mercaptoethanol-62.5 mM Tris-HCl
(pH 6.8) at 50°C for 30 to 60 min, washed, and reprobed.
Rabbit polyclonal antibodies against p130 (C-20), p107 (C-18), p27
(C-19), p27 (N-20), Cdk-2 (M-2), cyclin E (M-20), E2F1
(C-18), and E2F4
(C-18) came from Santa Cruz Biotechnology. Mouse
monoclonal antibody to
HSP70 (SPA820) was from StressGen
Technologies.
Transgenic mice and lymphocytes.
Mice carrying mutant p27
alleles (22) were crossed with Eµ-bcl-2
transgenic mice (strain bcl-2-36) (42). We used strains backcrossed onto a C57BL/6 genetic background. Mice were genotyped for
p27 status and the bcl-2 transgene by PCR. The
bcl-2 transgene did not alter the ratio of CD4 to CD8
single-positive lymphocytes in either the wild-type (wt) or
p27/ background (data not shown). Single-cell
suspensions from spleens of 8- to 12-week-old mice were cleared of red
blood cells. Incubation over nylon wool and passage through a murine
T-cell isolation column (Pierce) yielded >90% pure CD4 and CD8
single-positive T cells. For cell proliferation assays, the purified T
cells were seeded into 96-well plates at 2 × 106
cells/ml (0.2 ml/well) and treated with soluble anti-CD3e antibody (1.5 µg/ml) (Pharmingen, LE 01080 D) and recombinant human interleukin-2 (IL-2; 100 U/ml; Stem Cell Technologies). The cells were pulsed with
[3H]thymidine (1 µCi/well) for the last 6 h prior
to harvest, and thymidine incorporation was determined by liquid
scintillation counting. For cyclin E-associated kinase assays, lysates
were prepared from the activated lymphocytes, protein content was
determined, and equivalent amounts of protein (30 to 50 µg) were
immunoprecipitated as described previously (22), using 2 µg of anti-cyclin E or a control antibody.
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RESULTS |
Cell cycle retardation by Bcl-2 is independent of pRB.
To
explore whether pRB is required for the Bcl-2 cell cycle effect, 3T3
fibroblast cell lines derived from wt or pRB-null mice (50)
were transfected with either an empty vector or one directing Bcl-2
expression, and independent clonal lines were isolated. After their
synchronization in G0 by serum deprivation, the cells were
stimulated with serum to reenter the cycle. As reported (17,
35), Bcl-2-overexpressing wt fibroblasts entered S phase more
slowly than the control cells (Fig. 1A),
although their rates of proliferation once in cycle were equivalent
(data not shown; see also Fig. 2). In the
RB/ fibroblasts, Bcl-2 retained its ability
to retard cell cycle entry (Fig. 1B). Thus, Bcl-2 can retard
G0-to-S-phase progression independently of pRB. Although
the experiment in Fig. 1B suggests a more pronounced effect of Bcl-2 in
the RB-null cells than the wt cells, in other experiments Bcl-2 had
equivalent effects in the two cell types.
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FIG. 1.
Retardation of cell cycle reentry by Bcl-2 is
independent of pRB. Quiescent (A) wt or (B)
RB/ fibroblasts were stimulated to reenter
the cell cycle with 10% FBS, and their cell cycle distribution was
analyzed at the indicated times by DNA content. The data represent the
percentage of cells in S and G2/M phases relative to all
cells with  2N DNA content for the untransfected parental
cell line and independent clonal derivatives stably transfected with
the empty vector (puro) or the bcl-2 expression vector. The
results are representative of three similar experiments performed for
each genotype.
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Bcl-2 augments p130 levels but decreases p107.
To address
whether Bcl-2 might instead act through p130, we first assessed whether
it affected the level of the protein (Fig. 2, top panel). As expected (10,
34), in the control cells the p130 level was highest in
G0 and gradually fell as cells went into cycle, becoming
almost undetectable by 16 h (early to mid-S phase) (lane 7). In
the presence of Bcl-2, however, p130 was elevated during quiescence
(Fig. 2, top panel, c.f. lanes 1 and 2) and persisted for at least
24 h (lane 12). The elevation in p130 levels is unlikely to be an
indirect consequence of retarded entry into cycle because it was
observed even in the quiescent cells and the p130 level remained higher
in the Bcl-2 line even when its proportion of cycling cells was similar
to the control (e.g., compare 24-h Bcl-2 cells in lane 12 with 16-h
control cells in lane 7). Thus, p130 is likely to lie on the pathway
regulated by Bcl-2.
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FIG. 2.
Reciprocal regulation of p130 and p107 protein levels by
Bcl-2. Wt fibroblast lines, stably transfected with the control vector
(odd-numbered lanes) or a vector expressing Bcl-2 (even-numbered
lanes), were stimulated for the indicated times with 10% FBS to
reenter the cell cycle or growing asynchronously (asyn). Immunoblot
analysis was performed on lysates (50 µg of protein per lane) using
an 8% polyacrylamide gel. The membrane was probed with p130 antibody,
then stripped and reprobed for p107 and finally for HSP70 as a control
for loading and protein integrity. (Note that HSP70 levels reproducibly
increased as quiescent cells entered the cycle.) The proportion of
cells in the S and G2/M phases, determined from replicate
cultures, is also shown. Lanes 1 to 8 and lanes 9 to 14 were from
separate gels run simultaneously in the same tank. The p130 C-terminal
peptide antibody used cross-reacts with murine p107 (open arrow).
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The p107 levels were also influenced by Bcl-2, but in the opposite
manner (Fig.
2, middle panel). As expected (
10,
34),
p107
levels in the control cells were lowest in quiescence and
gradually
rose. The resting cells expressing Bcl-2 displayed less
p107 than the
controls (compare lanes 1 and 2), and the increase
was delayed (lanes 7 and 8), although their p107 level approached
that of the controls by S
phase (lanes 11 and 12) and remained
comparable in asynchronous
cultures (lanes 13 and 14). Reprobing
for the housekeeping protein
HSP70 (lower panel) confirmed that
the differences did not reflect
variations in loading or transfer.
The concomitant rise in p130 and
fall in p107 is consistent with
the ability of p130 to regulate
p107 transcription (see
Discussion).
p130 is essential for the retardation of cycle reentry by
Bcl-2.
To test whether p130 was an essential mediator of the
retardation by Bcl-2, we investigated whether Bcl-2 retarded cycle
entry in fibroblast lines derived from mice lacking p130
(8). Notably, Bcl-2 did not significantly affect their cycle
entry (Fig. 3A). This result did not
reflect an inadequate Bcl-2 level, because the levels in these lines
were comparable to those in the transfectants in Fig. 1A (data not
shown), and the transgene prevented apoptosis in serum-deprived
cultures of the p130/ fibroblasts (Fig. 3B).
Furthermore, the inability of Bcl-2 to retard cell cycle entry of the
p130-deficient 3T3 fibroblasts was specifically due to loss of the p130
gene and not some other mutation arising during immortalization,
because reintroduction of p130 restored this effect of Bcl-2 (Fig. 3C,
compare subpanels C and D). The ectopic p130 had little effect on the
cycling of the control p130/ cells (compare
subpanels A and B), even though its level was similar in the two lines
(data not shown). We conclude that p130 is essential for the
retardation of cell cycle entry by Bcl-2 and hence must have a function
in G0-S-phase control that cannot be met by p107 or pRB.
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FIG. 3.
Bcl-2 fails to retard cell cycle reentry of
p130/ fibroblasts. (A) Kinetics of cell
cycle reentry of p130/ fibroblast lines.
Quiescent p130/ fibroblasts were stimulated
by addition of 10% FBS, and their cell cycle distribution was
followed. The results, shown for two independent control cell lines
(puro) and four independent Bcl-2-expressing lines, are representative
of four similar experiments. (B) Bcl-2 enhances survival of
p130/ fibroblasts deprived of serum. The
p130/ fibroblasts were incubated in medium
without any FBS, and cell death was monitored (see Materials and
Methods). The results, shown for one of the control cell lines and the
four Bcl-2 lines used in A, depict the averages ± SEM of
triplicate cultures and are representative of two separate experiments.
(C) Reintroduction of p130 restores the cell cycle-inhibitory activity
of Bcl-2 in p130/ fibroblasts. The
p130/ fibroblasts stably expressing control puro vector
(subpanels A and B) or the Bcl-2 vector (subpanels C and D) were
transiently cotransfected with GFP and the p130 or empty vector
(control) as indicated. Viable GFP-expressing cells were isolated,
cultured for another 2 days, and rendered quiescent by serum
deprivation (see Materials and Methods). DNA content was determined
20 h after serum addition. Boxed numbers are the percentages of
the live cells that were in the S and G2/M phases.
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Bcl-2 retards E2F1 accumulation.
The ability of p130 to
restrain cell cycle progression probably involves its complexes with
E2F4 (47), which can repress the gene for E2F1, a critical
inducer of cell cycle entry (see Discussion). Since the rise in E2F1
protein during G0-S-phase progression largely reflects
increased transcription (10, 34), we reasoned that if Bcl-2
acted through p130-E2F4 complexes, it might retard the accumulation of
E2F1. Indeed, Figure 4A shows that to be
the case. As expected (15), E2F1 levels in the control fibroblasts peaked at 16 h (lane 7) and then declined rapidly. In
the Bcl-2-expressing cells, E2F1 appeared later (compare 12-h data in
lanes 5 and 6) and reached a plateau at 20 h (lane 10). Bcl-2 did
not affect the level of E2F1 in asynchronously growing fibroblasts
(lanes 13 and 14) or retard the serum-induced appearance of E2F1 in
p130/ fibroblasts (data not shown). Nor did Bcl-2
affect the E2F4 level (Fig. 4A), which is low in quiescent cells and
increases with entry into cycle (15, 47). Thus, the retarded
accumulation of E2F1 provoked by Bcl-2 paralleled the retarded cell
cycle entry.
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FIG. 4.
E2F4 accentuates but E2F1 overrides the retardation by
Bcl-2. (A) Bcl-2 retards expression of E2F1 but not E2F4 during
G0-S-phase progression. The wt fibroblast lines stably
transfected with the control vector (odd-numbered lanes) or that
expressing Bcl-2 (even-numbered lanes) were stimulated for the
indicated times with 10% FBS or growing asynchronously (asyn).
Immunoblot analysis was then performed on lysates containing 50 µg of
protein using a 7.5% polyacrylamide gel. The membrane was probed with
E2F1 antibody, stripped and reprobed for E2F4, and finally for HSP70.
(B) Cell cycle inhibition by Bcl-2 is enhanced by E2F4 but overcome by
E2F1. A wt fibroblast line bearing the control puro vector (subpanels A
to C) or one that expresses Bcl-2 (subpanels D to F) was transiently
transfected with the GFP vector GFP-E2F-4 or GFP-E2F-1, as indicated.
Viable GFP-expressing cells were isolated, cultured for another 2 days,
and rendered quiescent by serum deprivation (see Materials and
Methods). DNA content was determined 20 h after serum addition.
The boxed number in each subpanel is the percentage of cells in S and
G2/M phases relative to all cells with 2N DNA
content (NA, not applicable); the other number in each panel is the
percentage of dead (i.e., subdiploid) cells to the left of the dashed
line that arose during the serum treatment. The data were reproduced on
four separate occasions.
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Bcl-2 effect was augmented by E2F4 but overridden by E2F1.
Our
evidence that Bcl-2 retards the elevation of E2F1 but not E2F4 is
consistent with a model in which Bcl-2, by promoting formation of
p130-E2F4 complexes, represses E2F1 gene expression (see Discussion).
To explore the functional consequences of this model, we tested the
effects of coexpressing E2F1 or E2F4 with Bcl-2 in transiently
transfected fibroblasts. To facilitate isolation of transfectants and
to ensure that transfectant pools contained comparable E2F levels, some
experiments used E2Fs having a green fluorescent protein (GFP) tag, but
non-GFP-tagged E2Fs gave similar results. Figure 4B shows the cell
cycle analysis of the transfectants rendered quiescent by serum
starvation for 5 days and restimulated with serum for 20 h.
E2F4 alone had little effect on the proportion of cells residing in the
S and G
2/M phases (~70%) (Fig.
4B, compare subpanels
A
and B), but its coexpression with Bcl-2 profoundly inhibited
cycling:
only ~15% of cells expressing both were in those phases
(subpanel
E), versus ~55% of those expressing Bcl-2 alone (subpanel
D). This
result reflects retention in G
0/G
1 rather than
accelerated
entry into the next G
1 phase, because cells
prevented from exiting
mitosis by nocodazole behaved similarly (data
not shown). Thus,
E2F4 enhanced the retardation by Bcl-2. In striking
contrast,
coexpression of E2F1 with Bcl-2 increased the cycling
population
to the levels in control cells without Bcl-2 (compare
subpanels
F and A). This result may implicate the E2F1 gene as a target
for inhibition by Bcl-2 (see
Discussion).
Ectopic E2F1 but not E2F4 induces apoptosis (
9,
43).
Accordingly, almost no cells expressing E2F4 alone were subdiploid,
i.e., apoptotic (Fig.
4B, subpanel B), whereas few E2F1 transfectants
lacking the
bcl-2 transgene remained at harvest, and a
substantial
fraction of those (29%) were apoptotic (subpanel C)
the
scatter
reflects the low cell numbers remaining for analysis. The
decreased
cell number and accumulation of apoptotic cells by E2F1 was
completely
blocked by coexpression of Bcl-2 (subpanel
F).
To account for the synergy between E2F4 and Bcl-2 in retarding entry
into cycle, we suggest that the ectopic E2F4 together
with the
increased p130 levels produced by Bcl-2 (see Fig.
2)
generate higher
levels of repressive p130-E2F4 complexes (see
Discussion). Accordingly,
the amount of endogenous E2F4 that coprecipitated
with p130 was ~40%
higher in quiescent Bcl-2-expressing fibroblasts
than in quiescent
control cells (data not shown), even though
the total E2F4 level was
the same (Fig.
4A). That the introduced
E2F4 cooperates with Bcl-2
rather than overcoming its cell cycle
effect, as might have been
expected, is probably due to the relatively
modest levels of ectopic
E2Fs achieved under the serum-deprived
conditions employed (data not
shown). Although the marked difference
between the effects of E2F4 and
E2F1 is consistent with evidence
that E2F1 is a much more potent
promoter of cycle progression
(
9), to our knowledge this is
the first evidence that ectopic
E2F4 can under certain conditions act
to restrain G
0-S-phase
transit.
Enhanced p27 accumulation requires a critical Bcl-2 N-terminal
residue.
Since Bcl-2-overexpressing lymphocytes have elevated p27
levels (4, 29), we examined whether Bcl-2 also affected p27 levels in fibroblasts entering the cell cycle. As expected
(39), the p27 level in the control fibroblasts was maximal
in quiescence and almost indiscernible by 12 h (Fig.
5A), as they began to enter S phase (see
cell cycle data in Fig. 2). Although Bcl-2 did not affect the p27 level
during asynchronous growth, its level was augmented in quiescent cells
(compare Fig. 5A, lanes 1 and 2) and remained elevated for 16 to
20 h. Like the elevation in p130, the increase in p27 in the
quiescent cells is compatible with a direct effect of Bcl-2 rather than
an indirect consequence of slower cell cycle progression. As in other
cell types (4, 29), Bcl-2 did not alter the level of the
closely related Cki p21 (data not shown). As expected from the ability
of p27 to inhibit Cdk-2 activity, Bcl-2-expressing cells emerging from
quiescence displayed slower Cdk-2 activation than control fibroblasts
(data not shown, but Fig. 7B provides data for lymphocytes).
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FIG. 5.
Accumulation of p27 induced by Bcl-2 requires a critical
N-terminal residue. (A) Bcl-2 enhances p27 levels in wt fibroblasts. Wt
fibroblast lines stably transfected with the control vector
(odd-numbered lanes) or that expressing Bcl-2 (even-numbered lanes)
were stimulated for the indicated times with 10% FBS to reenter the
cycle or growing asynchronously (asyn). The cells were collected at the
indicated times, lysates were prepared, and p27 and HSP70 levels were
determined. The immunoblots used a 12% polyacrylamide gel with 50 µg
of protein per lane. (B) Tyrosine 28 of Bcl-2 is required for increased
p27 levels. Quiescent NIH 3T3 fibroblasts which overexpressed either wt
Bcl-2 or the Y28A mutant were either left untreated or stimulated with
10% FBS for 12 h prior to collection for immunoblot analysis.
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The cell cycle effects of Bcl-2 depend upon a region near its N
terminus: a Bcl-2 mutant with alanine replacing tyrosine 28
(Y28A)
cannot retard cycle entry of fibroblasts but retains full
survival
activity (
17). To determine whether tyrosine 28 was
required
for the p27 accumulation, we compared p27 levels in NIH
3T3 cell lines
that stably overexpress comparable amounts of either
wt Bcl-2 or the
Y28A mutant (
17), both during quiescence and
12 h after
readdition of serum. The p27 levels fell considerably
more in the cells
expressing the Y28A mutant than in those expressing
wt Bcl-2 (Fig.
5B).
Hence, maintenance of elevated p27 levels
requires a residue essential
for the cell cycle effects of Bcl-2.
Bcl-2 elevates p27 independently of cell cycle retardation.
Could the elevated p27 levels be a consequence rather than a cause of
the retarded cell cycle entry? The failure of Bcl-2 to affect cycle
entry for cells deficient in p130 (Fig. 3) prompted us to use those
cells to determine whether p27 levels also rose when cell cycle entry
was unperturbed (Fig. 6). As in wt
fibroblasts (see Fig. 5A), p27 levels were augmented by Bcl-2 in the
quiescent cells and declined more slowly. Hence, the rise in p27 does
not merely reflect the slower cell cycle kinetics normally elicited by
Bcl-2. This finding also establishes that the inhibitory signals still
emanate from Bcl-2 in the p130/ cell line. The
increased p27 is likely to be functional, because Cdk-2 kinase activity
in the p130/ cells was reduced by Bcl-2 (data not
shown).
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FIG. 6.
Bcl-2 directly enhances p27 levels, independently of
p130-mediated cell cycle effects. The p130/
fibroblast-transfected lines were either growing asynchronously (asyn)
or had been stimulated for the indicated times with 10% FBS to reenter
the cell cycle. The immunoblots (50 µg of protein per lane) involved
a 4 to 20% polyacrylamide gradient minigel (Novex). The line
separating the 16-h and 20-h lanes indicates that the data came from
different gels run simultaneously in the same tank. The less pronounced
effect of Bcl-2 at the 16-h time point compared to later times was not
seen in other experiments and reflects experimental variation.
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p27 is essential for cell cycle retardation by Bcl-2 in
lymphocytes.
We next wished to determine whether p27 was required
for the Bcl-2 effect. As immortal p27/
fibroblast lines were unavailable and the limited proliferative potential of primary p27/ fibroblasts
(7) renders them unsuitable for stable transfection, we
instead examined the entry of nullizygous T lymphocytes into cycle.
Splenic T cells from either wt or p27/ mice
that either did or did not express a bcl-2 transgene were stimulated with IL-2 and an agonistic antibody to the T-cell antigen receptor (anti-CD3). Figure 7A compares
the cell cycle entry, measured by [3H]thymidine
incorporation, for the four genotypes. As expected, the
bcl-2 transgene slowed entry of the
p27+/+ cells into cycle. Consistent with other
reports (7, 22), the rate of cycle entry was similar for the
wt and p27/ lymphocytes. Importantly, the
inhibition by Bcl-2 was completely reversed in lymphocytes lacking p27.
The elevated p27 levels must therefore be essential for the cell cycle
retardation by Bcl-2, and other Cki such as p21 cannot functionally
compensate.
View larger version (20K):
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|
FIG. 7.
Inhibition by Bcl-2 of S-phase entry and cyclin
E-associated kinase activity in T lymphocytes requires p27. (A) To
assess entry into cycle, splenic T cells isolated from littermates of
the four genotypes were stimulated with IL-2 and CD3 antibody. Six
hours before harvest, the cells were pulsed with
[3H]thymidine, and incorporation of label was determined.
The data represent the averages ± SEM of at least five replicate
wells; at least four mice of each genotype yielded similar results. (B)
To assay cyclin E-associated kinase activity, equivalent amounts of
protein from extracts of T cells stimulated as above were
immunoprecipitated with cyclin E antibody, and the associated histone
H1 kinase activity was measured (arbitrary units).
|
|
Because p27 is a major regulator of cyclin E and Cdk-2 activity in
lymphocytes (
7), we also followed cyclin E-associated
kinase
activity as cells progressed into S phase (Fig.
7B). As
previously
found, loss of p27 significantly elevated the kinase
activity. In the
p27+/+ cells, Bcl-2 expression markedly
suppressed kinase activity,
consistent with its ability to enhance p27
levels (see above).
However, Bcl-2 also significantly reduced kinase
activity in cells
that lacked p27, even though the activity remained
higher than
in the nontransgenic
p27+/+ cells
and was therefore unlikely to be limiting for cell cycle
progression,
as was observed. This unexpected result indicates
that Bcl-2 can also
inhibit cyclin E and Cdk-2 kinase activity
via a p27-independent
mechanism. Although normal mouse lymphocytes
usually have a relatively
low level of p130, the ability of Bcl-2
to enhance p130 levels might
nevertheless be involved, because
p130, under certain conditions, can
functionally substitute for
loss of p27 (
7).
| |
DISCUSSION |
Bcl-2 is functionally linked to negative regulators of the cell
cycle.
By exploiting cells lacking important cell cycle
inhibitors, we have established the framework by which Bcl-2 interacts
with the cell cycle machinery (Fig. 8).
Consistent with earlier evidence that Bcl-2 acts primarily during entry
into G1 (see Introduction), pRB, which controls
G1/S progression, proved dispensable for the Bcl-2 effect.
However, Bcl-2 increased the levels of its relative p130 and of the Cdk
inhibitor p27, both previously implicated in control of G0
exit. The increases in both inhibitors elicited by Bcl-2 in quiescent
cells argue for a direct effect rather than an indirect effect of the
cell cycle perturbation. Importantly, the results with cells lacking
each inhibitor showed that both p130 and p27 were essential for the
Bcl-2 cell cycle effect. Moreover, because the p27 increase did not
require p130, it cannot be merely a consequence of slower cycle entry
(Fig. 6). Hence, as would be expected (40), p27 appears to
act upstream of p130 or in parallel with it, probably by inhibiting
Cdk-2 activity. It is likely that p130 functions mainly via its ability
to complex with E2F4. Thus, the simplest interpretation of our results
is that, by enhancing the levels of p27 and p130, Bcl-2 produces
repressive p130-E2F4 complexes that inhibit cell cycle entry (Fig. 8).
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|
FIG. 8.
Model for the cell cycle-inhibitory effect of Bcl-2,
based on the findings presented here and current understanding of cell
cycle control (see text). In normal quiescent cells having only a low
Bcl-2 level, mitogenic stimulation inhibits p27 synthesis and provokes
destruction of both p130 and p27, facilitating cell cycle entry. The
enhanced Bcl-2 activity induced by certain environmental cues, however,
increases the levels of both p27 and p130. Bcl-2 presumably acts
through the binding of its N-terminal domain near tyrosine 28 to an
unknown protein (X here). The increased p27 levels lower the kinase
activity of Cdk-2, and one consequence is the maintenance of repressive
pocket protein-E2F4 complexes; for example, the phosphorylation of p130
and p27 by Cdk-2 can target them for destruction. The resulting
increase in p130 also promotes a higher level of p130-E2F4 complexes,
and they repress transcription of a gene (such as E2F1) encoding a
product that allows cell cycle entry. The p130-E2F4 complexes thus have
an essential role in regulating G0 exit. Key features of
the model therefore are that Bcl-2 acts via p27 and p130 to maintain
p130-E2F4 complexes, which then repress a gene, such as that for E2F1,
which is required for entry from quiescence into cycle.
|
|
Role of pocket proteins and E2Fs.
Previously, p130 was
implicated in control of G0 exit by its expression pattern,
timing of association with E2Fs, and the arrest elicited by its
overexpression (10, 34). Its inhibitory activity is ascribed
to its ability to bind to and negatively regulate E2F4, thereby
repressing E2F4 target genes required for cell cycle entry
(47). The finding that E2F4 but not E2F1 (see below)
potently cooperated with Bcl-2 to retard entry (Fig. 4B) supports our
suggestion that Bcl-2 acts by increasing the repressive complexes of
E2F4 with pocket proteins. Since E2F4 binds predominantly to p130
during quiescence (20, 32, 47), p130-E2F4 may govern the
G0/G1 transition, much as pRB-E2F1 (or E2F3)
does the G1/S transition (47). The evidence that
these partnerships regulate distinct subsets of genes is growing
(9, 18, 49). Our findings support such models, because the
Bcl-2 cell cycle effects are confined to the G0-S
transition (17, 35, 46). Thus, p130 has for the first time
been shown to have an essential role in a pathway controlling
G0 exit. It seems relevant that p130-E2F4 complexes have
been shown recently to accumulate in cells treated with the growth
inhibitors transforming growth factor beta (19) and alpha
interferon (14).
As indicated in Fig.
8, the E2F1 gene is a plausible candidate for a
relevant E2F4 target. Repression of E2F1 during G
0/early
G
1 requires the E2F sites within its promoter (
10,
34) and
involves p130 (
21). E2F4 is now thought to
control induction
of E2F1 expression during cycle entry
(
14). Importantly, E2F1
has the remarkable ability to push
quiescent cells into cycle
(
10). Moreover, E2F1 loss delays
G
0-S phase entry but is dispensable
in actively dividing
cells (
48), features that mirror the cell
cycle effects of
Bcl-2 in diverse cells (see Introduction). We
found that Bcl-2 retarded
the serum-mediated induction of E2F1
by several hours (Fig.
4A), in
good agreement with the G
0-S delay
caused by Bcl-2.
Furthermore, overexpression of E2F1 overcame
cell cycle inhibition by
Bcl-2 (Fig.
4B), as expected if Bcl-2
acted by negatively regulating
its level. Collectively, these
considerations implicate E2F1 as a
critical regulator affected
by Bcl-2 during exit from quiescence.
Although potential involvement
of E2Fs 2 and 3, which are also subject
to E2F-mediated repression
(
34), cannot be excluded, neither
is likely, because E2F2 protein
and DNA-binding activity are
essentially undetectable in quiescent
cells entering the cell cycle
(
26,
32) and E2F3 appears to
be needed mainly after cells
have begun cycling (
26). Other
potential targets for
p130-E2F4 repression include the genes for
cyclin E (
24) and
Cdc25A (
19), which are subject to E2F control
in early to
mid-G
1, but unlike E2F1, their cell cycle roles are
not
restricted to quiescent cells entering the cell
cycle.
The activity of E2F1 is controlled not only by its level but also by
its inhibitory interaction with pRB. However, loss of
pRB, which might
be expected to free E2F1 to induce its target
genes, did not overcome
the effect of Bcl-2 (Fig.
1B). This is
consistent with earlier findings
indicating that loss of pRB is
not necessarily functionally equivalent
to deregulated E2F1 expression.
For instance, although ectopic E2F1 is
sufficient to drive cells
into S phase (
10,
34), this
process remains mitogen dependent
in
RB-null cells (see
reference
18 for an example). This seeming
paradox
might be due to the known ability of other pocket proteins
such as p107
to functionally compensate for the absence of pRB
(
25,
37).
Interestingly, the activities of p130 and p107 appear to be coupled.
Their reciprocal expression has been noted previously,
with p130
prevalent in quiescent cells and p107 prevalent during
proliferation
(
10,
34). Bcl-2 expression during cell cycle
reentry
dramatically increased p130 and concomitantly reduced
p107 levels. The
reciprocal effects may well reflect the ability
of p130 to repress
transcription of the
p107 gene in an E2F-dependent
manner
(
34,
41,
51). Hence, the drop in p107 expression
is likely
to be secondary to enhanced p130 function. Our hypothesis
that Bcl-2
acts through inhibitory E2F4 complexes is supported
by the
demonstration that Bcl-2 reduces expression of both p107
and E2F1,
genes normally repressed during G
0/early G
1, in
an E2F-dependent
manner.
Role of p27.
The function of p27 is strongly linked to control
of G0-S progression. Its overexpression induces
G1 arrest, while ablation of its synthesis delays
withdrawal from the cell cycle. In normal cells, p27 is elevated during
quiescence and declines as cells enter the cycle, whereas the related
p21 is low during quiescence and increases in cycling cells (38,
39). As reported for lymphocytes (4, 29), Bcl-2
markedly enhanced p27 levels in quiescent fibroblasts, whereas no
effects on the p21 level have been observed by us or others. The
elevation in p27 required tyrosine 28 of Bcl-2, which is required
specifically for its cell cycle effects (17). The rise in
p27 cannot be merely an indirect consequence of delayed cell cycle
entry, because p27 also increased in fibroblasts lacking p130 (Fig. 6),
in which Bcl-2 did not influence the cell cycle. Hence, Bcl-2
influences p27 levels in a direct fashion, independent of p130.
It might seem puzzling that the increase in p27 caused by Bcl-2 was
insufficient to retard cell cycle entry in cells that
lack p130.
G
1 progression is thought to be determined, however,
by a
balance between the p27 level and Cdk-2 activity, the so-called
threshold model (
38,
39). Cells lacking p130 have elevated
Cdk-2 activity (
5; M. Lahda, G. Vairo, and M. E. Ewen, unpublished),
probably because p130 can bind to and inhibit
cyclin E/A-Cdk-2
kinases via a domain similar to those in p21 and p27
(see reference
5 and references therein).
Accordingly, the threshold model
predicts that elevated levels of p27
would be required to overcome
the increased Cdk-2 activity in
p130/ cells. Thus, in addition to repressing
E2F4 target genes, enhanced
p130 function apparently can also raise the
threshold needed for
Cdk-2
activity.
Consistent with this notion, in fibroblasts lacking p27 or p21, p130
can assume the role of negatively regulating Cdk-2 activity
(
7). In lymphocytes, however, our data suggest that p130
cannot
compensate for loss of p27, probably because lymphocytes have
a
much lower basal level of p130. Thus, the relative contribution
of p130
and p27 to the cell cycle-inhibitory effect of Bcl-2 may
well vary in
different cell types, particularly in those where
one of the two
regulators is present in limiting amounts. Nevertheless,
in cells such
as fibroblasts where we found that Bcl-2 increased
expression of both
p130 and p27, it seems very likely that the
increased p27, via Cdk-2
inhibition, enhances the cell cycle-inhibitory
activity of p130. That
hypothesis is consistent with evidence
that the ability of p130, like
pRB, to bind E2Fs and inhibit cell
cycle progression is regulated by
phosphorylation (
10,
34).
Cdk-2 is a likely kinase, because
the phosphorylation of p130
coincides with Cdk-2 activation and is
prevented by ectopic p21
(
41), while cyclins E and A can
overcome the inhibitory effects
of p130 (
6). In further
support of a functional role for p27
upstream of p130 and E2F, its
conditional expression provoked
accumulation of p130-E2F complexes and
inhibited E2F-dependent
transcription (
40). Moreover, E2F1
can override the arrest caused
by inhibition of Cdk-2 activity and must
therefore act downstream
of it (
10). Hence, we propose that
p27 mediates the cell cycle-inhibitory
effects of Bcl-2 at least in
part by inhibiting the phosphorylation
of p130 by Cdk-2, thereby
increasing the abundance of repressive
p130-E2F4
complexes.
Our evidence that p130 but not pRB is required for the Bcl-2 effect
implicates p130 as an important mediator of cell cycle
control by
Cip/Kip proteins and is consistent with their ability
to inhibit cell
cycle passage independently of pRB (
38,
39).
However, the
regulation of other proteins by Cdk-2 phosphorylation
presumably would
also be modulated by p27, and those unknown Cdk-2
substrates might also
contribute to the cell cycle-inhibitory
effects of Bcl-2.
How might Bcl-2 influence p27 and p130 levels?
Bcl-2 affected
p130 and p27 similarly: both were elevated during G0 and
persisted longer during cell cycle reentry. Their levels are governed
predominantly by posttranscriptional mechanisms that include regulated
destruction by the proteasome pathway (10, 34, 38, 39) and,
at least for p27, translational control (see below). A report that both
p27 and p21 are caspase targets (27) suggested that Bcl-2
might enhance p27 levels through inhibition of caspase activation, but
several considerations make that unlikely. First, despite the similar
putative caspase recognition site in p21, its levels increase as cells
reenter the cycle. Second, the proline in the putative caspase site of
human p27 (27) would make it a poor substrate for the known
caspases (44). Finally, immunoblots using an antibody to the
N terminus of p27 revealed no C-terminal processing of p27 during entry
of fibroblasts into cycle, even though p27 levels declined notably (G. Vairo, unpublished results).
Interference with proteasome-mediated destruction cannot readily
account for the p27 and p130 increases induced by Bcl-2 in
quiescent
cells, because that mechanism occurs only during S-phase
progression
after they have been targeted through phosphorylation
by cyclin E and
Cdk-2 (
34,
38,
39,
41). However, in G
0 cells,
there is growing evidence for translational control of
at least p27
(
2,
16,
31). Mitogenic and antimitogenic signals
determine
the frequency with which p27 mRNA is translated by controlling
factors
that bind to its untranslated regions (Millard et al.,
unpublished
results). Hence, we favor the notion that Bcl-2 increases
the p27 level
in quiescent cells by enhancing translation of p27
mRNA. The elevated
p27 levels in quiescent cells would prolong
the period during which p27
remained above the threshold required
to inhibit Cdk-2 activity and
accordingly prolong the G
0-S-phase
interval. The p130
increase might also involve a translational
mechanism, but because less
is established about regulation of
its levels, other mechanisms such as
protein stability might be
relevant.
Whatever the mechanism, the N-terminal region of Bcl-2 near tyrosine 28 is strongly implicated as the critical binding site
for a potential
regulator of its cell cycle effects (
17). Importantly,
that
residue was shown here to be required for the rise in p27
(Fig.
5). The
relevant Bcl-2-binding protein (X in Fig.
8) remains
unknown, but Raf-1
and calcineurin have been reported to bind
to that portion of Bcl-2
(see reference
1 for a
review).
Significance of Bcl-2 cell cycle activity for oncogenesis.
The
antiproliferative activity of Bcl-2 may have evolved to modulate the
oncogenic potential of excessive cell survival (1, 11, 35,
46). In accord with that notion, a recent study demonstrated a
tumor suppressor activity of Bcl-2 that correlated with its ability to
restrain proliferation (33). Conversely, in
T-antigen-dependent mammary tumor development, the ability of Bcl-2 to
accelerate tumor progression correlated with a selective loss of its
antiproliferative activity (13). Our data may provide a
molecular explanation for that result, because T-antigen transformation requires its ability to inactivate the pocket proteins, including p130
(10, 34), which we have shown to be essential for the proliferation-restraining effect of Bcl-2.
Evidence that the cell cycle-inhibitory activity of Bcl-2 may impact on
human cancers comes from findings that the translocated
bcl-2 gene in follicular lymphoma is often mutated in
regions
associated with its cell cycle activity (
1).
Moreover, elevated
Bcl-2 levels are associated with decreased
proliferation and sometimes
also a favorable prognosis in diverse
malignancies, including
breast and colorectal cancer (
3,
23)
and multiple myeloma
(
36). Our evidence that p27 is a key
downstream target of Bcl-2
seems relevant, because in many tumor types,
elevated p27 is strongly
associated with a favorable outcome
(
39). Our results suggest
that, as well as specific
mutations in Bcl-2, loss of p130 or
p27 function (or gain of E2F1
function) would diminish the Bcl-2
cycle-inhibitory activity and
potentially enhance its oncogenic
impact. Although no mutations of p130
in human tumors have yet
been reported (
10,
34), a disrupted
p27 allele can contribute
to tumorigenesis (
12). Thus,
lesions in any of the steps in
the cell cycle pathway extending from
Bcl-2 (Fig.
8) could contribute
to the development of several types of
tumors.
| |
ACKNOWLEDGMENTS |
We thank G. Lindeman, B. Warner, and E. Harlow for discussions;
T. Diem and L.-C. Zhang for expert technical assistance; M. Ladha for
performing some of the Cdk-2 assays; and J. Birtles and G. Filby for
secretarial assistance. We also thank N. Saunders, D. C. S. Huang, L. A. O'Reilly, A. Strasser, J. Nikolic-Zuglic, D. Ginsberg, W. Krek, D. Livingston, N. Dyson, T. Jacks, and E. Harlow for
their kind gifts of plasmids, antibodies, or cells and are grateful to
the WEHI flow cytometry facility for assistance with cell sorting.
This work was supported by grants from the National Health and Medical
Research Council of Australia (Reg. Key 973002) and the U.S. National
Institutes of Health (CA80188 and CA43540). G.V. was supported by a
Queen Elizabeth II Fellowship from the Australian Research Council and
by an AMRAD Corporation Postdoctoral Award.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: C/- The Walter
and Eliza Hall Institute of Medical Research, Post Office, Royal
Melbourne Hospital, Victoria 3050, Australia. Phone: 61 3 9345 2555. Fax: 61 3 9347 0852. E-mail: adams{at}wehi.edu.au.
Present address: ExGenix Operations Ltd, Richmond, Victoria 3121, Australia.
| |
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Molecular and Cellular Biology, July 2000, p. 4745-4753, Vol. 20, No. 13
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
