From the Division of Molecular Oncology, Institute
for Genetic Medicine, ¶ Department of Medicine II, School of
Medicine, Hokkaido University, Kita-ku, Sapporo 060, and the
§ Department of Viral Oncology, Cancer Institute, Japanese
Foundation for Cancer Research, Toshima-ku, Tokyo 170, Japan
Received for publication, October 31, 2000, and in revised form, February 6, 2001
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ABSTRACT |
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pRB family pocket proteins consisting of pRB,
p107, and p130 are thought to act as a set of growth regulators that
inhibit the cell cycle transition from G1 to S phases
by virtue of their interaction with E2F transcription factors. When
cells are committed to progressing through the cell cycle at the late
G1 restriction point, they are hyperphosphorylated by
G1 cyclin-cyclin-dependent kinase and
are functionally inactivated. Consistent with such a G1
regulatory role, pRB and p130 are abundantly expressed in quiescent
cells. In contrast, p107 is present at low levels in the
hypophosphorylated form in quiescent cells. As cells progress toward
late G1 to S phases, the levels of p107 increase, and the majority become hyperphosphorylated, suggesting a possible role of p107
in post-G1 cell cycle regulation. In this study, we have demonstrated that a nonphosphorylatable and thus constitutively active
p107 has the potential to inhibit S phase progression. The levels of
the phosphorylation-resistant p107 required for the S phase inhibition
are significantly less than those of endogenous p107. We further show
herein that the exposure of cells to the DNA-damaging agent, cisplatin,
provokes S phase arrest, which is concomitantly associated with the
accumulation of hypophosphorylated p107. Furthermore, the S phase
inhibitory response to cisplatin is augmented by the ectopic expression
of wild type p107, although it is diminished by the adenovirus E1A
oncoprotein, which counteracts the pocket protein functions.
Because p107 is a major pRB family protein expressed in S phase cells,
our results indicate that p107 participates in an inhibition of cell
cycle progression in response to DNA damage in S phase cells.
The decision to commit to cell division takes place at a late
G1 point, termed the restriction point, that precedes the
onset of DNA synthesis in the S phase. The passage through the
restriction point is primarily controlled by the retinoblastoma protein
(pRB) family, which consists of pRB, p107, and p130 (1-4). The pRB family proteins, acting as negative growth regulators, share a structure termed the "pocket domain" and, through the domain, interact with multiple cellular proteins, most notably the E2F family
of transcriptional factors (4-6). E2Fs transactivate a set of genes
whose products are critical in S phase entry and progression (5-8).
Upon complex formation, pRB family proteins neutralize the
transcriptional activities of E2Fs (4, 6, 9, 10). Furthermore, the pRB
family-E2F complexes appear to also act as E2F site-specific
transcriptional repressors (6, 11-13). Hence, by actively repressing
E2F-dependent gene expression, the pRB family proteins
prevent cell cycle progression from G1 to S.
The pocket function of the pRB family proteins is considered to be
negatively regulated by phosphorylation (14-22). Upon mitogenic stimulation, G1 cyclin-cyclin-dependent kinase
(CDK)1 complexes are
activated and collaboratively phosphorylate the pRB family proteins
(19-29). Through extensive phosphorylation, the pRB family proteins
lose their ability to form complexes with E2Fs, and the released E2Fs
initiate S phase entry and subsequent progression (4, 8, 14, 30).
Accordingly, inactivation of the pRB family proteins appears to be an
essential prerequisite for traversing the restriction point.
Ectopic expression of pRB family proteins in a variety of cell types
gives rise to G1 cell cycle arrest (31-35), supporting their crucial roles in preventing S phase entry. Consistently, pRB and
p130 are abundantly expressed in G0/G1-arrested
cells in their active, hypophosphorylated forms (4, 19, 20, 36). In
contrast, the p107 protein levels are modulated in an opposing manner.
In quiescent cells, p107 protein levels are very low, which is at least
in part due to E2F-dependent transcriptional repression of
the p107 gene, most probably through the pRB-E2F and/or
p130-E2F repressor complex (4, 37). As cells progress toward
mid-to-late G1, the levels of p107 increase, and in S phase cells p107 becomes a predominant pocket protein, although the majority
are hyperphosphorylated and hence inactivated (4, 19, 37). These
results indicate that low levels of p107 present during mid-to-late
G1 may play an important role in traversing the restriction
point in conjunction with other pocket proteins. Alternatively, p107
may play a unique role among the pocket proteins in cells that have
already passed the G1 restriction point.
We have previously shown that in certain hematopoietic cells, including
BaF3 and 32D cells, ectopically expressed p130 inhibits the cell cycle
in G1, whereas pRB, even in its phosphorylation-resistant form, fails to do so (34, 38). Given this observation, we wished to
know the role of p107, which is structurally closer to p130 than pRB
(4), in hematopoietic cell cycle regulation. We therefore generated
BaF3 lymphoid cells in which wild type or a nonphosphorylatable and
thus constitutively active p107 was inducibly expressed. Here we
demonstrate that p107 is capable of inhibiting the S phase cell cycle
progression. We further provide evidence that p107 is involved in
inhibiting the cell cycle in S phase cells with DNA damage.
Construction of Plasmids--
A p107 mutant, p107 Cell Culture--
COS-7 and SAOS-2 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum. The 6-1 cell is a BaF3-derived mouse pro-B cell line that stably
co-express tTA and LacI (34, 40, 41). Cells were cultured in RPMI 1640 medium containing 10% fetal calf serum and 20% WEHI-3B-conditioned medium (20% WEHI) as a source of interleukin 3 (IL-3). Stable transfectants that conditionally express p107HA, p107 Immunoprecipitation and Immunoblot Analysis--
COS-7 cells in
a 100-mm plate were transfected with 10 µg of each expression plasmid
using the DEAE-dextran method. SAOS-2 cells in a 100-mm plate were
transfected with 20 µg of each expression plasmid using the
calcium-phosphate precipitation method. The transfected cells were
harvested 2 days after transfection and lysed in E1A lysis buffer (250 mM NaCl, 5 mM EDTA, 50 mM HEPES, pH
7.0, 0.5% Nonidet P-40, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml trypsine inhibitor, 0.5 µM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride (32). Cell lysates were
then immunoprecipitated with anti-HA mouse monoclonal antibody (12CA5)
or anti-adenovirus E1A mouse monoclonal antibody (PharMingen,
PM-14161A). Immunoprecipitates were recovered on protein A-Sepharose
beads, washed four times with E1A lysis buffer, and eluted by boiling
in SDS-containing sample buffer. Immunoprecipitates and lysates were
resolved by electrophoresis on a 7.5, 10 or 12% SDS-polyacrylamide
gel. Gels were transferred to polyvinyldenedifluoride membrane filters
(Millipore) as described (42) and subjected to immunoblot analysis
using anti-HA, anti-p107 (Santa Cruz Biotechnology, sc-318), anti-p130 (sc-317), anti-p53 (sc-1314), anti-p21WAF1 (Oncogene
Research Products, OP79), anti-cyclin A (sc-751), anti-E2F4 (sc-512),
or anti-adenovirus E1A, followed by anti-mouse IgG (Amersham Pharmacia
Biotech), anti-rabbit IgG (Amersham Pharmacia Biotech), or anti-goat
IgG (sc-2020) secondary antibody conjugated to horseradish peroxidase.
Proteins were visualized using the ECL detection system (PerkinElmer
Life Sciences).
Cell Cycle Analysis--
6-1 cells and their stable
transfectants were washed three times with phosphate-buffered saline
(PBS) and resuspended at a density of 4×105/ml with RPMI
1640 medium containing 10% fetal calf serum for cytokine starvation.
Cells were then divided into two and cultured with IL-3-depleted medium
in the presence of 1 µg/ml Tc or 5 mM IPTG. Following
24 h of culture in the absence of cytokine, cells were
restimulated with IL-3 at a final concentration of 200 pg/ml. At 8 h after IL-3 restimulation, cisplatin was added to the culture at a
final concentration of 8 µg/ml. Cells were harvested at appropriate time points.
For cell cycle synchronization at early S phase, the 6-1-derived
transfectant cells were first starved for cytokine and restimulated with IL-3 in the presence of 1 µg/ml Tc as above described. After 4 h of IL-3 restimulation, they were washed twice with PBS,
divided into two, and cultured with IL-3-containing medium in the
presence of 1 µg/ml Tc or 5 mM IPTG. These cells were
respectively treated with hydroxyurea for additional 16 h at a
final concentration of 5 mM. The cells were then washed
twice with PBS to remove HU and cultured with IL-3-containing medium in
the presence of 200 ng/ml nocodazole and 1 µg/ml Tc or 5 mM IPTG.
For flow cytometric analysis, harvested cells were washed in PBS and
fixed in 80% ethanol on ice. The cells were washed again and
resuspended in PBS containing 500 µg/ml RNase A for 20 min at
37 °C. The samples were incubated for another 15 min at 4 ° with
propidium iodide solution (100 µg/ml propidium iodide, 0.1% sodium
citrate) prior to flow cytometric analysis with a Becton Dickinson FACS
Calibur. Cell cycle profiles were determined by use of the CELL Quest
and ModFit cell cycle analysis software.
Effect of the Phosphorylation-resistant p107 on the Hematopoietic
Cell Cycle--
6-1 cell is a BaF3-derived mouse lymphoid cell line
whose growth is totally dependent on IL-3, with the depletion of IL-3 24 h from the start of culture inducing
G0/G1 cell cycle arrest (Fig.
1A, 0 h). In these
growth-arrested 6-1 cells, a member of pRB family, p130, was abundantly
expressed, whereas expression of p107 was very low (Fig. 1B,
lane 1). We have previously shown that the
IL-3-dependent growth of the 6-1 cell is inhibited by ectopic expression of p130 but not by pRB, indicating that the cell is
actively halted in G0/G1 by the accumulated
p130 in the absence of IL-3 and that inactivation of p130 is an
essential prerequisite to traversing the G1 restriction
point in this cell (34).
Restimulation of the growth-arrested 6-1 cells with IL-3 gave rise to
synchronized cell cycle reentry and progression from G1 to
S phases (Fig. 1A). At 8 h after IL-3 stimulation, most of the p130 became hyperphosphorylated (Fig. 1B, lane
2), indicating that by this time G1 cyclin-CDKs were
activated sufficiently to phosphorylate and inactivate the pRB family
pocket proteins (4, 19, 20, 22, 36, 37). This in turn suggests that
most if not all cells passed through the G1 restriction
point by 8 h after the IL-3 stimulation. After 16 h, ~80%
of cells entered S phase (Fig. 1A). By this time point, p130
became barely detectable, whereas p107 was potently induced and
abundantly expressed but was hyperphosphorylated (Fig. 1B,
lane 3). 24 h after IL-3 stimulation, the cell cycle
profile returned to the pattern that is typical of asynchronously
growing cells (Fig. 1A).
Using this cell cycle re-entry/progression protocol, we wished to
address the possible role of p107 in cell cycle regulation, particularly in S phase progression. As an initial approach, we generated 6-1-derived stable transfectants, p107-13 and p107-16, that
conditionally express wild type p107 (Fig.
2A) with the use of the
tetracycline/IPTG dual-regulated inducible system, in which expression
of the cDNA is strongly inhibited by tetracycline and is
potently activated by IPTG (34, 40). However, despite ectopic p107
expression (Fig. 2B), we were not able to see any changes in
the cell cycle entry and progression in response to elevated p107 (Fig.
2C). This may be at least in part due to insufficient expression of ectopic p107, because actively cycling 6-1 cells already
express high levels of endogenous p107 (Fig. 1B). Indeed, we
could only increase the p107 levels by at most 2-fold in relation to
endogenous levels (Fig. 2B, compare the expression of total p107 between Tc- and IPTG-treated lanes at the corresponding times). Furthermore, the massive phosphorylation of p107 in response to IL-3
restimulation (Fig. 2B) indicates that under such
circumstances cells possess p107 kinase activity that is sufficient to
inactivate both endogenous and exogenous p107.
Accordingly, we next generated a phosphorylation-resistant p107 mutant
by replacing all of the serine and threonine residues that constitute
possible cyclin-CDK phosphorylation motifs (either serine-proline or
threonine-proline) with nonphosphorylatable alanine residues (Fig.
3A) (22). Upon its transient
expression in COS-7 cells, the phosphorylation-resistant p107,
p107
The cDNA encoding p107
To investigate the S phase inhibitory activity of the
phosphorylation-resistant p107 more directly, we examined the effect of
p107
Although the phosphorylation-resistant p107 is capable of inhibiting S
phase progression, it remains possible that the S phase inhibitory
effect is due to supraphysiologic levels of the protein expressed. To
address this possibility, we examined the expression levels of the p107
mutant. Because p107 Inhibition of S Phase Progression by Genotoxic Stress--
To
pursue the biological relevance of the p107-mediated S phase
inhibition, we next addressed whether the hypophosphorylated p107 could
be detected in cells that have passed the G1 restriction point. To do so, we arrested 6-1 cells in G0/G1
by IL-3 starvation. The growth-arrested cells were then stimulated with
IL-3, and at 8 h after IL-3 stimulation, by which time a majority
of cells have passed through the restriction point as judged by the
phosphorylation status of pRB family proteins (Fig. 1B,
lane 2), a DNA damaging agent cisplatin was added to the
culture. Cell cycle analysis revealed that the cisplatin treatment
provoked a delay in S phase entry that was followed by a severe
inhibition of S phase cell cycle progression (Fig.
6A), a change that is
reminiscent of that observed with the cells expressing the
phosphorylation-resistant p107 (Fig. 4C and 5C).
These results indicate that genotoxic stress such as DNA damage can
inhibit S phase cell cycle progression. We also note that p53 and
p21 proteins were up-regulated within several hours after cisplatin
treatment as reported previously (Fig. 6B) (39).
We also examined the phosphorylation status of p107 in cells treated
with cisplatin. Whereas p107 was mostly hyperphosphorylated when
cisplatin was added to the culture (8 h after IL-3 stimulation), the
hypophosphorylated form of p107 reappeared at 16 h after IL-3 stimulation in the cisplatin-treated cells, but not in untreated cells,
and increased thereafter (Fig. 6B, lanes 3 and
6-8). This observation suggests that cell cycle inhibition
in S phase cells is concomitantly associated with the reappearance and
accumulation of hypophosphorylated p107.
The Effect of Ectopic p107 on Cellular Responsiveness to
Cisplatin--
To address the role of p107 in S phase cell cycle
inhibition in DNA damaged cells more directly, we employed the p107-16
cells that conditionally express HA-tagged, wild type p107 under the control of the tetracycline/IPTG dual regulation system (Fig. 2A). The p107-16 cells were growth-arrested by IL-3
deprivation and then restimulated with IL-3. During IL-3 starvation,
p107 was induced by IPTG (Fig. 2B). As previously
demonstrated in Fig. 2C, a simple induction of ectopic p107
did not significantly alter the cell cycle profile of the 6-1 cells.
However, following treatment with cisplatin at 8 h after IL-3
stimulation, p107-induced cells exhibited a stronger inhibition of S
phase progression than that observed in p107-uninduced cells (Fig.
7A). These results indicate that the cell cycle inhibitory activity of cisplatin is proportional to
the p107 levels. Intriguingly, in these cisplatin-treated cells, ectopic p107 existed most exclusively in its hypophosphorylated form as
late as 24 h after IL-3 stimulation (Fig. 7B,
lane 10). Because the HA-tagged p107 is constitutively
produced under the TcIP promoter in the transfectants, our results
indicate that phosphorylation of p107 may be inhibited in the
DNA-damaged S phase cells.
Effect of E1A Oncoprotein on the S Phase Inhibitory Effects of
Cisplatin--
Adenovirus E1A protein is known to physically interact
with and functionally inactivate pRB family pocket proteins (43-46), including p107 (33, 47, 48). Accordingly, if p107 is actively involved
in the inhibition of S phase cell cycle progression in response to DNA
damage, then E1A would be expected to counteract the cell cycle
inhibitory activity of cisplatin. To address this, we generated a
6-1-derived, stable transfectant, E1A15-1, that ectopically expresses
the E1A 12 S product (Fig.
8A). Expectedly, in the E1A
transfectant cell, the E1A protein formed physical complexes with
endogenous p107 (Fig. 8B). However, a simple overexpression of E1A did not provoke any significant change in
IL-3-dependent growth in 6-1 cells.2 The
parental and the E1A-expressing cells were then growth-arrested by IL-3
deprivation and were subjected to cisplatin treatment at 8 h after
IL-3 restimulation. In cells expressing E1A, the S phase inhibitory
activity of cisplatin was significantly reduced, and a relatively large
fraction of cells was capable of exiting from S phase within 32 h
(Fig. 8, C and D). These results provide additional evidence that p107 is actively involved in the cell cycle
inhibition induced by DNA damage in S phase cells.
In mammalian cells, the point of decision regarding the move
toward quiescence or proliferation occurs at the G1
restriction point (49). A series of works have highlighted the critical role of pRB family proteins in traversing the G1
restriction point (1, 2), raising the possibility that pRB family
proteins may be dispensable in cell cycle regulation beyond the
G1 restriction point. Consistent with this idea, once cells
pass through the restriction point, they are systemically inactivated
by phosphorylation and are kept inactive throughout subsequent cell
cycle phases. When cells enter the next G1 phase, they are
converted into their hypophosphorylated form by phosphatases (50, 51).
This scenario may well be applicable to cells that continuously
proliferate. However, factors such as DNA damage can activate otherwise
concealed cell cycle checkpoints and arrest cells in
post-G1 (52). It is therefore possible that, under certain
cellular settings, the pRB family pocket proteins may play an active
role in post G1 cell cycle regulation.
The expression levels of pRB are relatively constant throughout
the cell cycle (4, 19). In contrast, the levels of 107 and p130
proteins change dramatically as cells progress through the cell cycle.
p130 is abundantly present in quiescent cells, and, upon mitogenic
stimulation, it is hyperphosphorylated in mid-to-late G1
phase. The p130 protein levels are then progressively down-regulated as
cells move into S phase (4, 20, 36). In contrast, the levels of p107
are regulated in a manner opposite to that observed for p130. In
quiescent cells, p107 expression is very low. As the cell cycle
progresses from the early to the mid/late G1 phases, p107
begins to accumulate dramatically (4, 19). This p107 expression is
primarily regulated at the transcriptional level (8). In particular,
reciprocal expression between p107 and p130 can be explained at least
in part by the E2F sites present in the promoter of the p107 gene (37).
It has been suggested that the p107 promoter is under the
negative control of p130-E2F and/or pRB-E2F complex present in the
quiescent cells. Increases in p107 levels as the cell cycle progresses
from the quiescent state again suggests that the pocket protein might
be able to regulate post-G1 cells rather than simply
preventing S phase entry.
We have previously shown that growth of hematopoietic cells such as
BaF3 and 32D is strongly inhibited by ectopic expression of p130, but
not by pRB, even in its phosphorylation-resistant form (34, 38). In
these cells, p130 prevents S phase entry, at least in part, through
inhibiting the transcriptional activity of E2F-4 (34). Because p107 is
more similar to p130 than pRB among the pRB family pocket proteins (4,
53), we wished to determine the role of p107 in the hematopoietic cell
cycle. As an initial approach, we generated 6-1 transfectants that
conditionally express wild type p107 through the use of the Tc/IPTG
dual-regulated, inducible promoter system (34, 40). Because p107 is a
relatively abundant protein in cycling cells, we could only increase
the cellular p107 levels at most by 2-fold upon induction. Under this condition, we were not able to observe any changes in
IL-3-dependent cell cycle entry and progression by the
elevated p107. Possibly, the amounts of ectopic p107 expressed were not
sufficient to exert biological activities. Alternatively, p107 might be
much more sensitive to G1 cyclin-CDK than pRB and p130 and
may be instantly inactivated during G1 progression. If the
latter is correct, then we can conclude that p107 does not play a major
role in G1 cell cycle regulation.
To pursue further the possible role of p107 in cell growth regulation,
we generated in this work a novel p107 molecule that is completely
resistant to cyclin-CDK-mediated phosphorylation/inactivation. Upon its
inducible expression in the BaF3-derived cells, we observed two
distinct effects of the phosphorylation-resistant p107 in cell cycle
regulation. First, the phosphorylation-resistant p107 is capable of
retarding the G1-to-S transition. Because p107
preferentially binds E2F-4 and E2F-5 (5, 54, 55), it is reasonable that the phosphorylation-resistant p107 would inhibit G1 cell
cycle progression when ectopically expressed in G1 cells,
as is the case with p130 (33, 37). Second, the p107 mutant was found to
strongly inhibit S phase progression. Indeed, in the presence of the
phosphorylation-resistant p107, virtually no cells exited from S phase
by as late as 48 h after IL-3 stimulation. Because the levels of
phosphorylation-resistant p107 required to induce S phase inhibition
were significantly less than those of endogenous p107, we conclude that
the p107-mediated S phase inhibition indeed takes place when a certain
fraction of the endogenous p107 is converted from the
hyperphosphorylated form to the hypophosphorylated form. The
observation that only the hypophosphorylated p107 is capable of
inhibiting S phase progression indicates that the activity is pocket
structure-dependent but not spacer
domain-dependent.
The biological relevance of the S phase inhibitory activity of p107 was
provided by the observation that the DNA-damaging agent cisplatin is
capable of inhibiting an S phase progression that is concomitantly
associated with the accumulation of the hypophosphorylated form of
endogenous p107. This finding suggests that conditions exist that
result in the appearance of active p107 beyond the restriction point in
late G1. Furthermore, cisplatin was found to inhibit S
phase cell cycle progression more effectively as cells expressed higher
levels of p107. Conversely, the inhibition of p107 activity by the
overexpression of the adenovirus E1A 12S product, which binds and
inactivates pRB family proteins (33, 43-48), at least partially
restored the S phase inhibitory activity of cisplatin. Together with
p107 being a predominantly expressed pRB family protein in S phase
cells (4, 19), our results indicate that p107 is involved in the
inhibition of S phase progression in response to DNA damage.
Although the mechanism through which hypophosphorylated p107
accumulates in cisplatin-treated cells needs further investigation, our
observation implies that a p53-p21 pathway (56-58) may be involved. Upon cisplatin treatment, p53 and p21 proteins are up-regulated in the
6-1 cells as has been previously demonstrated (39). Because p21 acts as
a universal inhibitor of cyclin-CDK (59, 60), p107 phosphorylation may
be inhibited in the presence of p21 in the cisplatin-treated cells.
Consequently, de novo synthesized p107 would remain
hypophosphorylated because of the reduced p107 kinase activity. This
possibility is supported by our finding that in cisplatin-treated
cells, the newly synthesized p107 molecules, which can be marked by an
HA epitope tag, are detectable almost exclusively in their
hypophosphorylated form. It is also possible that p107 phosphatases are
induced in the cisplatin-treated cells, converting hyperphosphorylated
p107 to less phosphorylated forms. In this regard, p107 was shown
to be rapidly dephosphorylated by protein phosphatase 2A upon UV
irradiation (61).
Recent studies have shown that overexpression of
phosphorylation-resistant pRB mutants in fibroblasts is capable of
inhibiting S phase progression (62-64). This S phase inhibitory
activity is not overcome by E2F or cyclin A/E (62, 63), indicating that pRB is capable of regulating the S phase cell cycle through the mechanisms independent of E2F. Taken together, the two pRB family pocket proteins, pRB and p107, may function in a combinatorial way as S
phase cell cycle brakes that are turned on in response to DNA damage in
cells that have passed the G1 restriction point. Probably,
the relative contributions of these two proteins in S phase response
may vary among cell types, because pRB even in its constitutively
active form is incapable of effectively inhibiting cell cycle
progression in BaF3 cells (34).
The molecular mechanisms through which p107 or pRB inhibits S phase
progression is currently unknown. In the case of pRB, the S phase
response is suggested to involve origin activation machineries (65). As
is the case of pRB, the early S phase-synchronized cells were capable
of initiating some degree of DNA synthesis but were incapable of full
genome replication in the presence of phosphorylation-resistant p107.
This finding is consistent with the idea that p107 inhibits the origin
activation process rather than the DNA elongation process (66). In this
regard, it has been recently reported that pRB family proteins (pRB,
p107, and p130) can bind MCM7, a component of prereplication complex, and inhibit in vitro DNA replication in an
MCM7-dependent manner (65). It is therefore intriguing to
speculate that hypophosphorylated p107 is capable of inhibiting origin
activation by directly interacting with MCM or the related
prereplication complex components. Indeed, pRB family members are
associated with early S phase replication sites (67). Alternatively,
p107 may inhibit S phase progression through transcriptional control
because ORC1 and CDC6 genes, whose products are also essential
components of prereplication complex, are transcriptionally activated
by E2F, the critical target of the pRB family proteins (68, 69). The
involvement of p107 in S phase cell cycle arrest in response to DNA
damage indicates that its functional loss may provoke abnormal cell
cycle progression that is concomitantly associated with genetic instability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S/T-P, that
lacks all of the potential phosphorylation sites by cyclin-CDK was
generated from human p107 cDNA by multiple rounds of
oligonucleotide-mediated mutagenesis with use of Chameleon
site-directed mutagenesis system (Stratagene) according to the
manufacturer's instructions (22). In this mutant, threonines 332, 340, 369, 385, 915, and 997 and serines 368, 515, 615, 640, 650, 749, 762, 964, 975, 988, 1009, and 1041 were substituted with alanine residues.
Wild type and the phosphorylation-resistant p107 were tagged with
influenza hemagglutinin (HA) epitope at the carboxyl terminus (p107HA
and p107
S/T-P-HA). cDNAs encoding p107HA and p107
S/T-P-HA
were inserted into a mammalian expression vector, pSP65SR
2 (39).
pOPTET-BSD is an inducible cDNA expression vector having the TcIP
promoter and the blasticidin-resistance gene as a drug-selection marker
(34, 40). A cDNA encoding p107HA, p107
S/T-P-HA, or adenovirus
E1A 12S (E1A) was subcloned into the pOPTET-BSD vector.
S/T-P-HA, or
E1A were created by transfecting pOPTET-BSD-p107HA,
pOPTET-BSD-p107
S/T-P-HA, or pOPTET-BSD-E1A into 6-1 cells by
electroporation as described (34, 38). Expression of the
cDNA-directed proteins in these transfectants was repressed in
medium containing 1 µg/ml tetracycline (Tc) and was induced in medium
containing 5 mM isopropyl
-D-thiogalactopyranoside (IPTG) for 24 h in the
absence of Tc.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of pRB family proteins during cell
cycle entry and progression of 6-1 cells. BaF3-derived 6-1 cells
were growth-arrested by IL-3 starvation for 24 h and then
restimulated with IL-3. A, cell cycle kinetics of the
IL-3-restimulated 6-1 cell. Cells were harvested at the indicated time
points after IL-3 restimulation and stained with propidium iodide, and
DNA contents were measured by flow cytometry. B, expression
and phosphorylation of p107 and p130 in the IL-3-stimulated 6-1 cells.
Whole cell lysates were prepared at the indicated time points after
IL-3 restimulation, resolved on 7.5% SDS-PAGE, and analyzed by
immunoblotting with anti-p107 (upper panel) or anti-p130
(lower panel). pp indicates a hyperphosphorylated
form of p107 or p130, whereas p indicates a
hypophosphorylated form of p107 or p130.
View larger version (38K):
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Fig. 2.
Effect of ectopic p107 expression on the cell
cycle entry and progression of 6-1 cells. A, induction
of the HA-tagged, wild type p107 in the 6-1-derived stable transfectant
clones, p107-13 and p107-16. The stable transfectants were cultured in
IL-3-containing medium in the presence of tetracycline (Tc; uninduced
condition) or IPTG (induced condition) for 24 h. Whole cell
lysates were prepared, resolved on 7.5% SDS-PAGE, and analyzed by
immunoblotting with anti-HA. The asterisk indicates a
nonspecific band. B, induced expression of ectopic p107
during cell cycle entry and progression. The p107-16 cells were
growth-arrested by IL-3 starvation for 24 h in the presence of Tc
or IPTG and restimulated with IL-3. Whole cell lysates were prepared at
the indicated time points after IL-3 restimulation, resolved on 7.5%
SDS-PAGE, and analyzed by immunoblotting with anti-HA, which
specifically detects ectopic p107 (upper panel), or
anti-p107, which recognizes both endogenous and ectopic p107 molecules
with the same affinity (lower panel). pp
indicates a hyperphosphorylated form, whereas p indicates a
hypophosphorylated form. The asterisk shows a nonspecific
band. C, cell cycle entry and progression in the presence or
absence of ectopic p107. The p107-16 cells were growth-arrested by IL-3
starvation for 24 h in the presence of Tc or IPTG and then
restimulated with IL-3, harvested at the indicated time points after
IL-3 restimulation, and stained with propidium iodide, and DNA contents
were measured by flow cytometry.
S/T-P-HA, was detected exclusively in its quickly migrating,
hypophosphorylated form (Fig. 3B). Furthermore, it formed
physical complexes with endogenous E2F4 and cyclin A in both of COS-7
cells and SAOS-2 osteosarcoma cells (Fig. 3C), indicating
that the mutant is biologically active despite multiple point mutations
(22).
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Fig. 3.
Biological properties of the
phosphorylation-resistant p107 mutant,
p107 S/T-P-HA. A, schematic
view of p107
S/T-P-HA that lacks all of the potential cyclin-CDK
phosphorylation sites. Boxes A and B are
conserved regions among pRB family proteins. The
phosphorylation-resistant p107 was tagged with the HA epitope at the
COOH terminus. B, expression of p107
S/T-P-HA in COS-7
cells. Whole cell lysates of COS-7 cells transiently transfected with
an empty vector, pSP65SR
2 (lane 1), an expression
vector for the HA- tagged, wild type p107, pSP65SR
2-p107HA
(lane 2), or a p107
S/T-P-HA expression vector,
pSP65SR
2-p107
S/T-P-HA (lane 3), were analyzed by
immunoblotting with anti-HA. pp indicates a
hyperphosphorylated form, whereas p indicates a
hypophosphorylated form. C, physical interaction of
p107
S/T-P-HA with cyclin A and E2F4 in vivo. Whole
cell lysates were prepared from COS-7 cells (left panel) or
SAOS-2 cells (right panel) transiently transfected with
pSP65SR
2 (lanes 1 and 4), pSP65SR
2-p107HA
(lanes 2 and 5), or pSP65SR
2-p107
S/T-P-HA
(lanes 3 and 6). The lysates were
immunoprecipitated with anti-HA (lanes 4-6), and the
immunoprecipitates were analyzed on immunoblotting with anti-cyclin A
(upper panel, lanes 4-6) or anti-E2F4
(lower panel, lanes 4-6). Anti-cyclin A and
anti-E2F4 immunoblottings of the whole cell lysates are shown in
lanes 1-3.
S/T-P-HA was introduced into the 6-1 cells
and was inducibly expressed in the stable transfectants, PRp107-14 and
PRp107-24, with the use of the tetracycline/IPTG dual regulation system
(Fig. 4A). The transfectant
clones were first growth-arrested in G0/G1 by
IL-3 deprivation for 24 h. During IL-3 starvation, cells were also
treated with Tc or IPTG. Upon IPTG treatment, the
phosphorylation-resistant p107
S/T-P-HA was expressed in the
growth-arrested cells (Fig. 4B, lane 3), whereas it was undetectable in the same transfectant that was treated with Tc
(Fig. 4B, lane 1). Restimulation of the
G0/G1 arrest cells with IL-3 in the presence of
Tc (i.e. the uninduced condition) gave rise to a cell cycle
progression that is basically indistinguishable from that observed with
the parental 6-1 cells (Fig. 4C, Tc; see also
Fig. 1A). In striking contrast, the same cells inducibly expressing p107
S/T-P-HA exhibited a severe delay of S phase entry and progression, despite the continued presence of IL-3 for 48 h
(Fig. 4, C and D).
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Fig. 4.
Inhibition of S phase entry and
progression by the ectopic expression of
p107 S/T-P-HA. A, induction
of p107
S/T-P-HA in the 6-1-derived stable transfectant
clones, PRp107-16 and PRp107-24. Cells were cultured in IL-3-containing
medium in the presence of Tc (uninduced condition) or IPTG (induced
condition) for 24 h. Whole cell lysates were prepared, resolved on
7.5% SDS-PAGE, and analyzed on anti-HA immunoblotting. p
indicates a hypophosphorylated form of p107. B, inducible
expression of p107
S/T-P-HA during cell cycle entry and
progression. The PRp107-24 cells were growth-arrested by IL-3
starvation for 24 h in the presence of Tc or IPTG and restimulated
with IL-3. Whole cell lysates were prepared at the indicated time
points after IL-3 restimulation, resolved on 7.5% SDS-PAGE, and
analyzed on immunoblotting with anti-HA, which specifically detects
p107
S/T-P-HA (upper panel), or anti-p107, which
recognizes both endogenous and ectopic p107 molecules with the same
affinity (lower panel). p indicates a
hypophosphorylated form. C, effect of p107
S/T-P-HA on the
cell cycle entry and progression. The PRp107-24 cells were
growth-arrested by IL-3 starvation for 24 h in the presence of Tc
or IPTG and restimulated with IL-3, harvested at the indicated time
points after IL-3 restimulation, and stained with propidium
iodide, and DNA contents were measured by flow cytometry. DNA
histograms shown are representative of three independent experiments.
Essentially the same result was obtained with another stable
transfectant clone, PRp107-14. D, inhibition of S phase
progression by p107
S/T-P-HA. The percentage of cells in each cell
cycle phase in the presence or absence of p107
S/T-P-HA was
calculated from the flow cytometric data shown in C with the
use of ModFit cell cycle analysis software. Data are representative of
three independent experiments.
S/T-P-HA in cells synchronized in the early S phase (Fig. 5A).
G0/G1-arrested PRp107-24 cells were
restimulated with IL-3 and, at 4 h after the IL-3 restimulation,
the DNA synthesis inhibitor HU was added to the culture at a final
concentration of 5 mM. At this time, IPTG was also added to
the culture to induce p107
S/T-P-HA. Because these cells pass through
the G1 restriction point by 8 h after the onset of
IL-3 restimulation and 4 h of IPTG induction is too short to
induce p107
S/T-P-HA protein in
cells,2 the HU-treated cells
were expected to traverse from G1 to S without receiving
the effect of the phosphorylation-resistant p107. The cells were
treated with HU and IPTG for 16 h to induce sufficient amounts of
p107
S/T-P-HA while arrested in the early S phase. As expected, in
these HU-blocked cells, endogenous p107 molecules were totally
hyperphosphorylated (Fig. 5B, lanes 2 and
4). Furthermore, the cells expressed p107
S/T-P-HA, whose
levels were not increased by further incubation with IPTG (Fig.
5B, lanes 4 and 5). The early S
phase-synchronized cells were then released from the HU block, and
subsequent cell cycle progression was monitored by flow cytometry in
the presence of 200 ng/ml of the tubulin inhibitor nocodazole to
prevent entry into the next cell cycle. In the control cells in which
expression of p107
S/T-P-HA was not induced, DNA synthesis was
rapidly initiated upon HU release, and completion of full genome
replication occurred within 18 h after the release (Fig.
5C). In striking contrast, in the presence of
p107
S/T-P-HA, DNA synthesis was severely impaired, and most cells
failed to achieve replication of full genome as late as 24 h (Fig.
5C). These results clearly demonstrate that the
phosphorylation-resistant p107 is capable of inhibiting progression of
the S phase. It should be noted, however, that the cells were obviously
capable of initiating some degree of DNA synthesis in the presence of
p107
S/T-P-HA, although they failed to complete genome replication
(Fig. 5C). This may indicate that chain elongation can still
take place in the presence of the phosphorylation-resistant p107.
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Fig. 5.
Cell cycle effect of the
phosphorylation-resistant p107 expressed in early S phase
cells. A, S phase cell cycle synchronization protocol.
The PRp107-24 cells were growth arrested in the early S phase by HU
treatment. The phosphorylation-resistant p107 was inducibly expressed
in the S phase arrested cells by IPTG treatment. The HU block was then
canceled by eliminating HU from the culture, and the subsequent cell
cycle progression was examined in the presence of nocodazole, which
prevents next cell cycle entry. B, inducible expression of
p107 S/T-P-HA in cells specifically synchronized in the early S
phase. Whole cell lysates were prepared at the appropriate time points
shown in A, resolved on 7.5% SDS-PAGE, and analyzed on
immunoblotting with anti-HA, which specifically detects p107
S/T-P-HA
(upper panel), or anti-p107, which recognizes both
endogenous and ectopic p107 molecules with the same affinity
(lower panel). pp indicates a hyperphosphorylated
form, whereas p indicates a hypophosphorylated form of p107.
C, effect of p107
S/T-P-HA on S phase cell cycle
progression. The same PRp107-24 cells used in B were stained with
propidium iodide. DNA contents were measured by flow cytometry. DNA
histograms shown are representative of three independent experiments.
Essentially the same result was obtained with another stable
transfectant clone, PRp107-14.
S/T-P-HA is detectable exclusively in its
hypophosphorylated form, it is possible to compare expression levels
between endogenous p107 and p107
S/T-P-HA. In the IPTG-treated
PRp107-24 cells wherein p107
S/T-P-HA was inducibly expressed (Figs.
4B, lanes 3-6, and 5B, lanes
4 and 5) and cell cycle progression was inhibited
(Figs. 4, C and D, and 5C), the
increase in the levels of the hypophosphorylated form of p107 was
marginal, and the overall p107 levels elevated only slightly (Figs.
4B and 5B, compare the expression of total p107
between Tc- and IPTG-treated lanes at the corresponding times) as
measured by immunoblotting with the use of anti-p107 that recognizes COOH-terminal 18 amino acids that are perfectly identical between human
and mouse p107. These results indicate that there is significantly less
expression of the phosphorylation-resistant p107 than that of
endogenous p107.
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Fig. 6.
Effect of DNA-damaging agent, cisplatin, on S
phase cell cycle progression. The 6-1 cells were growth-arrested
by IL-3 starvation for 24 h and then restimulated with IL-3. At
8 h after IL-3 restimulation, cells were treated with cisplatin at
a final concentration of 8 mg/ml. The cisplatin-treated and untreated
cells were harvested at the indicated time points after IL-3
stimulation. A, effect of cisplatin on the cell cycle entry
and progression. The cells were stained with propidium iodide, and DNA
contents were measured by flow cytometry. B, effect of
cisplatin on the expression of cell cycle regulatory molecules. Whole
cell lysates prepared at the each time point were resolved on 7.5%
(for p107 and p130) or 12% (for p53 and p21) SDS-PAGE and analyzed by
immunoblotting with anti-p107, anti-p130, anti-p53, or anti-p21.
pp indicates a hyperphosphorylated form of p107 or p130,
whereas p indicates a hypophosphorylated form of p107 or
p130.
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Fig. 7.
Effect of ectopic p107 expression on cellular
responsiveness to cisplatin. A p107HA transfectant clone, p107-16,
was growth-arrested by IL-3 starvation for 24 h in the presence of
Tc or IPTG and then restimulated with IL-3. Following 8 h of IL-3
stimulation, cells were treated with 8 mg/ml cisplatin for 2 h and
cultured in the IL-3-containing medium for the indicated periods.
A, cell cycle profiles of the cisplatin-treated cells in the
presence or absence of ectopic p107. The p107-16 cells were harvested
at the indicated time points after IL-3 restimulation and stained with
propidium iodide, and DNA contents were measured by flow cytometry.
B, inducible expression of ectopic p107 during cell cycle
entry and progression of the p107-16 cells in the presence or absence
of cisplatin. Whole cell lysates were prepared from the
cisplatin-treated and untreated cells at the indicated time points
after IL-3 restimulation, resolved on 7.5% (for p107HA) or 12% (for
p53 and p21) SDS-PAGE, and analyzed by immunoblotting with anti-HA,
which specifically detects ectopic p107, anti-p53 or anti-p21.
pp indicates a hyperphosphorylated form of p107, whereas
p indicates a hypophosphorylated form of p107.
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Fig. 8.
Effect of E1A oncoprotein on the S phase
inhibitory activity of cisplatin. The parental 6-1 and a 6-1 transfectant, E1A15-1, that inducibly expresses adenovirus E1A
oncoprotein were growth-arrested by IL-3 deprivation in the presence of
IPTG. After 24 h of IL-3 starvation, cells were restimulated with
IL-3. At 8 h after IL-3 restimulation, they were treated with
cisplatin at a final concentration of 8 mg/ml. The cisplatin-treated
and untreated cells were harvested at the indicated time points.
A, whole cell lysates were prepared from the
cisplatin-treated and untreated cells, resolved on 7.5% (for p107) or
10% (for p53 and E1A) SDS-PAGE, and analyzed by immunoblotting with
anti-p107, anti-p53, or anti-E1A. pp indicates a
hyperphosphorylated form of p107, whereas p indicates a
hypophosphorylated form of p107. B, complex formation
between E1A and p107 in cells. whole cell lysates of the parental 6-1 and the E1A-expressing cells harvested at 24 h were subjected to
immunoprecipitation with anti-E1A monoclonal antibody. The E1A
immunoprecipitates were then analyzed on immunoblotting with anti-p107.
C, cell cycle profiles of cisplatin-treated cells in the
presence or absence of E1A. The parental and the E1A expressing
transfectant cells were harvested at the indicated time points after
IL-3 restimulation and stained with propidium iodide, and DNA contents
were measured by flow cytometry. D, S phase inhibitory
activity of cisplatin was reduced in cells expressing E1A. The
percentage of cells in each cell cycle phase was calculated from the
flow cytometric data shown in C with the use of ModFit cell
cycle analysis software. Data are representative of three independent
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Liang Zhu for p107 cDNA, Dr. Tetsuro Takebayashi for assistance, and members of the Hatakeyama laboratory for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by a Grant-in Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Research Grant from the Human Frontier Science Program Organization, and a Research Grant from the Nippon Boehringer Ingelheim Co., Ltd.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.
To whom correspondence should be addressed: Div. of Molecular
Oncology, Inst. for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. Tel./Fax:
81-11-709-6482; E-mail: mhata@imm.hokudai.ac.jp.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M009911200
2 T. Kondo, and M. Hatakeyama, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
CDK, cyclin-dependent kinase;
HA, hemagglutinin;
PAGE, polyacrylamide gel electrophoresis;
S/T-P motif, serine-proline or
threonine-proline motif;
Tc, tetracycline;
IPTG, isopropyl
-D-thiogalactopyranoside;
HU, hydroxyurea;
IL, interleukin;
PBS, phosphate-buffered saline.
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REFERENCES |
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