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Molecular and Cellular Biology, November 1998, p. 6605-6615, Vol. 18, No. 11
The Dana-Farber Cancer Institute and the
Harvard Medical School, Boston, Massachusetts 02115
Received 30 April 1998/Returned for modification 17 June
1998/Accepted 18 August 1998
The synthesis of cyclin D1 and its assembly with cyclin-dependent
kinase 4 (CDK4) to form an active complex is a rate-limiting step in
progression through the G1 phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), we show that this kinase plays a significant role in positively
regulating the expression of cyclin D1. This was found both in
quiescent serum-starved cells and in cells expressing dominant-negative
Ras. Despite the observation that cyclin D1 is a target of MEK1, in
cycling cells, activated MEK1, but not cyclin D1, is capable of
overcoming a G1 arrest induced by Ras inactivation. Either
wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in
reversing the G1 arrest induced by inhibition of Ras
activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin
D1 or activated MEK1, cyclin D1 is able to efficiently associate with
CDK4; however, the complex is inactive. A significant percentage of the
cyclin D1-CDK4 complexes are associated with p27 in serum-starved
activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by
expression of antisense p27 allows for S-phase entry from quiescence in
NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells.
Both positive and negative
extracellular growth factors exert their influence upon proliferation
during the G1 phase of the cell cycle. At a point in late
G1, termed the restriction point (R), cells become largely
refractory to these factors and once past R are committed to completing
the mitotic cycle (62, 87, 97). The action of serum growth
factors is mediated, in part, through their activation of receptor
tyrosine kinases. Ras, an inner plasma membrane-bound GTPase, plays a
significant role in receiving and transducing extracellular signals by
functioning as a downstream mediator of several membrane-bound receptor
and nonreceptor tyrosine kinases (5, 45, 46, 69). Ras
influences, and is required for, both the G0/G1
transition and passage through G1 to a point temporally
coincident with the restriction point. In certain situations, Ras has
been shown to be sufficient for progression from G0 to S
phase, as demonstrated by the ability of microinjected activated or
wild-type Ras to induce DNA synthesis from quiescence in the absence of
serum factors (19, 84). Furthermore, microinjection of
murine fibroblasts with anti-Ras neutralizing antibodies prevents
efficient serum-induced S-phase entry from a G0 state
(16, 52), and expression of a dominant-negative Ras protein
(RasN17) inhibits the proliferation of NIH 3T3 and 32D
myeloid cells, resulting in a G1 arrest (8, 18,
59).
In terms of nuclear events influencing progression through the
G1 phase of the cell cycle, a key role is played by
G1 cyclins and their catalytic subunits, the
cyclin-dependent kinases (CDKs). The three D-type cyclins associate
with and activate CDK4 and CDK6. Cyclin E is required for the
activation of CDK2. The synthesis of D-type cyclins, whose expression
is rate limiting for G1 progression, is regulated by
various mitogens (79, 80). The assembly of D cyclins with
their catalytic partners is also a mitogen-regulated process occurring
in early to mid-G1 (48, 49). Cyclin D-CDK4/CDK6 complexes promote G1 progression, at least in part, by
phosphorylating the retinoblastoma protein, Rb, thereby inactivating
its ability to act as a transcriptional repressor in a complex with E2F
(74, 80). In turn, the expression of cyclin E, which is also
rate limiting for G1 progression, is controlled in part by
Rb (22, 26, 57). Cyclin E-CDK2 complexes then participate in
the phosphorylation and inactivation of Rb but are also thought to have
other critical substrates whose phosphorylation is required for DNA
synthesis (11, 34, 44, 58, 71, 78, 98). The phosphorylation of Rb roughly coincides with the passage of cells from serum dependence to serum independence and, like Ras, has been linked to restriction point control (26, 92).
The activity of G1 cyclin-CDK complexes is regulated, in
part, by the CDK inhibitors p21 and p27. These CDK inhibitors can directly inhibit the activity of both cyclin D-CDK4/CDK6 and cyclin E-CDK2 holoenzymes or prevent the activation of these kinases by
cyclin-dependent kinase-activating kinase (CAK) (28, 32, 66,
82). Indeed, the accumulation of p27 is required for cells to
efficiently exit from the cell cycle and enter a quiescent state
(12, 72). Conversely, p21 and p27 may have a positive role
in G1 progression. p21, and to a lesser extent p27, can
promote the assembly and nuclear localization of cyclin D-CDK4
complexes (35). In many but not all cell systems described
elsewhere, p27 levels are high and p21 levels are low during
quiescence, with mitogenic stimulation leading to a reciprocal
reduction in p27 levels and elevation in p21 levels (21, 24, 39,
55, 56). These observations indicate the physiological importance of the connection between the influence of mitogens on the expression of CDK inhibitors and their actions during G1.
The influence of Ras on G1 progression has recently been
shown to be mediated through direct effects on the cell cycle
machinery. Ras transduces extracellular signals through multiple
downstream pathways, and activation of more than one effector pathway
is required for efficient mitogenesis (27). In this context,
the Ras effector pathway involving the Raf1, mitogen-activated protein kinase kinase 1 (MEK1), and mitogen-activated protein kinase (p42mapk and p44mapk [also known as ERK1 and ERK2, respectively, and hereafter referred to as MAPK]) cascade is best characterized. Each element of
this pathway has been shown to participate in G1
progression (7, 14, 33, 61) and to positively regulate
cyclin D1 expression (2, 20, 25, 36, 40, 94). The importance of this link between Ras and cyclin D1 is highlighted by a number of
observations: (i) the activity of cyclin D1-CDK4 appears to be required
only in cells expressing functional Rb (81); (ii) the
CDK4/CDK6-specific inhibitor p16 inhibits Ras-induced mitogenesis (75); (iii) the expression of dominant-negative Ras can
inhibit the expression of cyclin D1 (2, 65); and (iv)
inactivation of Ras activity leading to a G1 arrest is Rb
dependent (38, 50, 65). In addition, Ras-mediated signaling
has also been suggested to be involved in regulating the expression of
CDK inhibitors. Both Ras-dependent and -independent pathways have been
suggested to have a role in downregulating the expression of p27 during G1 progression (1, 86, 93). With Raf-nuclear
receptor fusions, the acute induction of Raf kinase activity has been
shown to induce the expression of p21 (76, 77, 96).
Here we have reassessed the relative contribution of cyclin D1
expression to Ras-induced G1 progression, with particular
focus on the Raf/MEK/MAPK pathway. This is motivated by a number of observations. Previously, we have shown that expression of cyclin D1
plus CDK4, achieved by transient transfection, can reverse the
G1 cell cycle arrest induced by dominant-negative Ras
(65). This result, together with the observation that the
Ras/Raf/MEK/MAPK pathway regulates the expression of cyclin D1, might
be taken to suggest that this cyclin is the sole critical target of Ras during mitogenic stimulation. However, recent findings question the
physiological relevance of such overexpression studies. For example,
although p16 can inhibit Ras-induced mitogenesis, Ras plus Myc can
drive cells from G0 to S phase in the absence of the
activation of cyclin D1-CDK4 and without inducing Rb phosphorylation (38). In addition, microinjection of active cyclin D-CDK4
complexes, or overexpression of cyclin E, can also promote
G1 progression in the absence of Rb phosphorylation
(37, 43). We have thus also addressed the potential
importance of the coordinate regulation of cyclin D1 and p27. This is
motivated by the realization that both downregulation of cyclin D1 and
elevated expression of p27, two events which can be brought about
through the inhibition of Ras activity, are characteristic of quiescent
cells. Together with the finding that p27 can inhibit the activity of,
yet paradoxically possibly promote the assembly of, cyclin D-CDK4, we
have asked whether regulation of the levels of cyclin D1 and p27 is
required to maintain a quiescent state.
Plasmids and antibodies.
The retroviral plasmid encoding
activated MEK1 (MEK-E217/E221) (14) was kindly provided by
C. Marshall. pCMV5-asp27 (72) for expression of antisense
p27 was kindly provided by N. Rivard and J. Pouysségur.
pMT-RasN17 (and its empty vector counterpart, pMT- Cell lines.
NIH 3T3 derivatives expressing activated MEK1
(MEK1-E217/E221, MEK-EE) were made with retroviral supernatants
prepared from the packaging cell line Bosc23 (64)
transfected with pBABE-puro-MEK-EE or pBABE-puro (control). Pooled
populations of infected NIH 3T3 cells were generated by selection in
puromycin (2 µg/ml; Sigma) for 4 to 6 days. Cyclin D1 derivatives of
NIH 3T3 cells and the parental line (70) were kindly
provided by M. Roussel and C. Sherr. NIH 3T3 cells and their
derivatives were maintained in Dulbecco's modified Eagle medium (DMEM)
supplemented with 5% bovine calf serum (BCS; HyClone), glutamine,
penicillin, and streptomycin. Cell lines were rendered quiescent by
culture in DMEM-0.25% BCS for 96 h. Rb Immunoprecipitations and Western blot analysis.
Cells
(2 × 105) were washed twice with phosphate-buffered
saline (PBS) and then lysed in DIP buffer (50 mM HEPES [pH 7.2], 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween 20 and 1%
Triton X-100 containing 1 mM NaF, 0.5 mM phenylmethylsulfonyl fluoride,
0.5 mM sodium orthovanadate, 10 mM CDK4 kinase assays.
CDK4 kinase assays were performed as
described elsewhere (48, 54). In time course assays,
aliquots of lysates containing equivalent amounts of total protein, 100 µg, determined by the Bradford assay (Bio-Rad), were used for kinase
assays. Magnetic sorting was performed by transfecting cells with a
plasmid encoding the CD20 surface marker alone with the other indicated
plasmids. Forty-two hours after transfection, cells were trypsinized
and incubated with monoclonal antibody to CD20 as described elsewhere (65), followed by incubation with magnetic beads covalently attached to sheep anti-mouse immunoglobulin G antibody (Dynabeads; Dynal, Inc.). The CD20-positive transfected cells were then
magnetically separated from the untransfected cell population. The
sorted cells were then used to perform CDK4 kinase assays.
Cell cycle analysis.
Cell cycle phase distribution analysis
of transfected cells was performed as described elsewhere (17,
65). When these assays are performed, titrations with both
RasN17- and p16-encoding plasmids are carried out. The data
shown are for the plasmid concentrations which result in a maximal
increase in the G1 population for a given cell line.
Microinjection.
Microinjection experiments were performed as
described previously (65) with the following modifications.
Cells to be microinjected were plated on coverslips and made quiescent
by serum starvation. The indicated plasmids were injected into the
nuclei of starved cells. Approximately 100 to 150 cells were
microinjected per experiment. To monitor injected cells, a plasmid,
pcDNA3-GFP, encoding GFP was coinjected. At 24 h after injection,
the cells were incubated for 12 to 14 h with 5-bromodeoxyuridine
(BrdU) at 10 µM. Cells were fixed in 3% paraformaldehyde-2%
glucose in PBS and then washed with PBS. A PBS solution containing
0.2% Triton X-100 was used to permeabilize the cells. After being
washed, cells were blocked with 0.1% Triton X-100-5% fetal bovine
serum in PBS with subsequent washing in 5 mM MgSO4 in PBS.
Cells were then incubated for 1 h in 5 mM MgSO4 in PBS
containing 100 U of DNase (Boehringer Mannheim) per ml and anti-BrdU
monoclonal antibody (unconjugated; Becton Dickinson) at a 1/10 dilution
in a humidified chamber at room temperature. Cells were then washed in
0.1% Triton X-100 in PBS and incubated with rhodamine-conjugated goat
anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc.) at a
1/200 dilution for 15 min at room temperature. After being washed with
0.1% Triton X-100 in PBS, nuclei were stained with DAPI
(4',6-diamidino-2-phenylindole), washed, and mounted. Cells were
examined by fluorescence microscopy.
Neither ectopic expression of cyclin D1 nor that of activated MEK1
is sufficient to activate CDK4 in quiescent cells.
NIH 3T3 cells
expressing ectopic cyclin D1 or constitutively active MEK1
(MEK1-E217/E221 [14], hereafter referred to as MEK-EE)
were characterized for the expression of these exogenously expressed
proteins (Fig. 1A). As previously
reported (70), compared to parental NIH 3T3 cells the cyclin
D1 lines expressed approximately five times more cyclin D1 (Fig. 1A;
see also Fig. 5A). MEK-EE was expressed at a level two to three times
that of the endogenous protein. These lines were used to investigate
the early mitogenic activation of cyclin D1-CDK4 complexes.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Regulation of Exit from Quiescence by p27 and
Cyclin D1-CDK4
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Bam),
pDCR-HRasV12, pCMV-Rb, pRc/CMV-cyclin D1, and pcDNA3-p16
plasmids encoding dominant-negative Ras, oncogenic Ras, Rb, murine
cyclin D1, and p16, respectively, have been described previously
(65). pBABE-puro (51) was kindly provided by J. Morgenstern and H. Land. pCMV-CDK4 (91) encoding human CDK4
was kindly provided by E. Harlow. pRc/CMV-CDK4K35M
(30) encoding inactive murine CDK4 was kindly provided by C. Sherr. pcDNA3-GFP encoding green fluorescent protein (GFP) was kindly
provided by P. Silver. Polyclonal antibody to cyclin D1 was from
Upstate Biotechnology Inc. Polyclonal antibody to CDK4 (C-22) was from
Santa Cruz Biotechnology Inc. Monoclonal antibodies to p27 (catalog no.
K25020) and MEK1 (catalog no. M17020) were from Transduction
Laboratories. Monoclonal antibodies to cyclin D2 (DCS-5.2) and cyclin
D3 (DCS-22) were from NeoMarkers.
/ 3T3
cells were maintained as described previously (65).
-glycerophosphate, 1 µg of
aprotinin per ml, 1 µg of leupeptin per ml) for 30 min on ice. The
samples were centrifuged at 16,000 × g, and
supernatants were precipitated with appropriate antibodies as indicated
in the figure legends. Immune complexes were collected with protein A-Sepharose beads, then washed three times with DIP buffer containing inhibitors, and then suspended in sample buffer. Boiled samples were
electrophoresed through a sodium dodecyl sulfate-10 or 12% polyacrylamide gel electrophoresis gel. The gels were then transferred onto polyvinylidene difluoride membranes. After the transfer, the
filter was blocked with 5% nonfat dry milk in Tris-buffered saline
containing 0.05% Tween 20 (TBST), and the filter was incubated at room
temperature for 60 min with primary antibodies in TBST. After three
washes with TBST, 60 min of incubation with horseradish peroxidase-conjugated secondary antibody (anti-mouse or anti-rabbit), and three more washes with TBST, detection was performed with enhanced
chemiluminescence (Amersham). Signal was detected with XAR5 film
(Kodak).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (27K):
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FIG. 1.
Expression of activated MEK1 (MEK-EE) and cyclin D1 in
NIH 3T3 cells. (A) Parental NIH 3T3 cells and their cyclin D1 and
MEK-EE derivatives were rendered quiescent by serum starvation or
maintained in a cycling asynchronous state. Under these two conditions,
the levels of endogenous and exogenous MEK1, cyclin D1, and CDK4 were
determined by Western blot analysis of whole-cell lysates. (B) Same as
panel A, except that the cell cycle distribution of the cells was
monitored by fluorescence-activated cell sorting analysis. (C) Lysates
were prepared from serum-starved and asynchronous cultures of parental
NIH 3T3 cells and the cyclin D1 and MEK-EE derivatives.
Immunoprecipitation (IP) with antibody to CDK4 were performed. Immune
complexes were assayed for CDK4-associated kinase activity with
recombinant glutathione S-transferase (GST)-Rb as
substrate. NRS, normal rabbit serum.
Expression of activated MEK1 or cyclin D1 and CDK4, but not cyclin D1 alone, can overcome G1 arrest induced by dominant-negative Ras. In cells stimulated out of a quiescent state, either serum withdrawal or inhibition of Ras activity in the presence of serum, prior to the restriction point, prevents S-phase entry. Likewise, in an asynchronous population Ras inhibition leads to a G1 arrest. Given that neither the serum-starved cyclin D1 line nor the MEK-EE line showed detectable amounts of CDK4 activity, a characteristic of mid- to late G1 cells, they have likely not progressed beyond the G0/G1 transition under these conditions. This motivated us to next determine if ectopic expression of cyclin D1 or MEK-EE has similar effects on the cell cycle in an asynchronous population of cycling cells with respect to Ras-mediated signaling. Specifically, we compared the effects of serum withdrawal (noted above) and inhibition of Ras activity. We also reinvestigated the requirements of cyclin D1 and CDK4 with respect to their participation in the override of a G1 arrest induced by the expression of dominant-negative Ras.
We have previously demonstrated that expression of dominant-negative Ras, RasN17, results in an Rb-dependent G1 arrest that is mediated, at least in part, by the downregulation of cyclin D1 expression (65). This mutant of Ras has a preferential affinity for GDP and is thought to function by sequestering guanine nucleotide exchange factors, thereby inhibiting endogenous Ras (8, 18, 83, 85), and blocking the activation of MAPKs in response to receptor tyrosine kinases (15, 73, 89, 95). Furthermore, we had shown that transient transfection of both cyclin D1 and CDK4 could override a G1 arrest caused by inhibition of Ras activity (65). Given that transient overexpression studies can sometimes lead to aphysiological results, we determined whether stable expression of cyclin D1 could override a G1 arrest induced by RasN17 and whether there was a requirement for the coexpression of CDK4. This would also allow us to more accurately compare the outcomes when analyzing the cyclin D1 and MEK-EE lines, which, as opposed to transient transfection of cyclin D1, express roughly the same amount of cyclin D1. Either stable or transient expression of cyclin D1 alone only partially overrode a G1 arrest induced by RasN17 (Fig. 2A), by the same experimental approach we had previously employed (65). Ectopic expression of CDK4 caused a significant reversal in the G1 arrest induced by dominant-negative Ras in the cyclin D1 lines but not in the parental line, consistent with our previous results. Given that the expression of cyclin D1 and not that of CDK4 is thought to be rate limiting for G1 progression and that the expression of the former but not of the latter is inhibited by expression of RasN17, we also investigated the requirement for CDK4 activity in these override experiments. Expression of a catalytically inactive allele of CDK4, CDK4K35M (30), also allowed the cyclin D1 line, but not parental NIH 3T3 cells, to override the RasN17-induced G1 arrest (Fig. 2A). Thus, expression of cyclin D1 alone is not sufficient to override a cell cycle arrest mediated by the inhibition of Ras activity. That there is a requirement for either wild-type or catalytically inactive CDK4 suggests the possibility that titration of CDK inhibitors, an established function of cyclin D-CDK4 complexes (10, 66), participates in the override.
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Cyclin D1 and CDK4 complex formation in cell lines that constitutively express cyclin D1 during G1 progression. The above results suggest that cyclin D1 and activated MEK1 differ in their abilities to overcome Ras inactivation in cycling cells, while both the cyclin D1 and activated MEK1 lines responded similarly to serum deprivation and subsequently to quiescence; both lines could be arrested in G0/G1 with CDK4 inactive. As a first step in determining why CDK4 might be inactive in the cyclin D1 and MEK-EE lines, we assayed the ability of cyclin D1 and CDK4 to form complexes in serum-starved cells. Cyclin D1 immunoprecipitates were found to contain CDK4, and conversely, CDK4 immune complexes were found to contain cyclin D1. The degree to which complexes between cyclin D1 and CDK4 could be detected in serum-starved cells was comparable to that observed in mid- to late G1 or in cycling cells (Fig. 5). The parental line, used as a control, did not show complex formation between cyclin D1 and CDK4 in starved and early G1 cells, simply due to the lack of cyclin D1 expression under these conditions. Cyclin D1 was detected at 6 to 9 h after serum stimulation of quiescent parental NIH 3T3 cells. At this time in mid- to late G1, complexes could be detected between cyclin D1 and CDK4 (Fig. 5) (48), commensurate with the appearance of kinase activity (see below) (48). The results suggest that the inability to detect CDK4-associated kinase activity in starved cyclin D1 and MEK-EE lines was not due to the inability of cyclin D1 to associate with CDK4.
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p27 is bound to inactive CDK4 in quiescent cyclin D1 and MEK-EE lines. We next sought to determine why CDK4 was inactive in the serum-starved NIH 3T3 cells expressing cyclin D1 and MEK-EE. Three possibilities were considered. First, under conditions of serum starvation, CDK4 might be rendered inactive due to tyrosine phosphorylation at position 17 (88). Second, cyclin D1 when expressed in serum-starved cells might fail to localize to the nucleus. In some cell systems, cyclin D1 has been reported to be localized to the cytoplasm in starved cells (4). Third, one or more CDK inhibitors might be responsible for maintaining cyclin D1-CDK4 in an inactive state. In terms of the first possibility, we were unable to detect the presence of tyrosine phosphate in CDK4 either in the parental NIH 3T3 cells or in the cyclin D1 or MEK-EE derivatives, with an antiphosphotyrosine antibody. Similarly, we did not detect significant amounts of cyclin D1 in the cytoplasm of starved cyclin D1 or MEK-EE lines by indirect immunofluorescence.
We next investigated the possibility that CDK inhibitors might be involved in the inhibition of CDK4 in the cyclin D1 and MEK-EE lines under conditions of serum starvation. The CDK inhibitor p27 is present at comparatively high levels in starved cells, and the levels decrease upon mitogenic stimulation (56, 82). p27 has the ability to inhibit active cyclin D-CDK4 complexes (67, 90). In addition, an approximately twofold elevation in p27 levels has been reported to inhibit CAK-dependent activation of CDK4 in murine macrophages (28). CAK is active in serum-starved cells (29, 47); thus, p27 association with cyclin D1-CDK4 complexes might prevent the access of CAK to CDK4. Together, these observations suggested that p27 might participate in the inhibition of CDK4 in starved cyclin D1 and MEK-EE lines. In the parental line, the levels of p27 were found to be highest in starved cells and decreased during passage through G1 (Fig. 7A). In the cyclin D1 and MEK-EE lines, the levels of p27 were approximately twofold higher than that in the parental line, as determined by densitometric scanning, under conditions of serum starvation. For these lines, the levels of p27 decreased during G1 progression, although not as dramatically as in the parental cells (Fig. 7A).
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Antisense p27 cooperates with ectopic cyclin D1 to promote progression from quiescence to S phase. The above results suggest the possibility that increased expression of p27 found in serum-starved cyclin D1 and MEK-EE lines may be responsible for the lack of detectable CDK4-associated kinase activity under these conditions. Theoretically, lowering p27 levels might lead to the activation of cyclin D1-CDK4 complexes. To test this possibility indirectly, we determined whether microinjection of a plasmid expressing antisense p27 into the cyclin D1 lines, and two clonal derivatives, would allow these cells to progress through G1 and enter S phase.
The p27 plasmid construct used in these studies has previously been shown to significantly reduce the levels of p27 in transfected cells (72). DNA replication was monitored by incorporation of BrdU. The MEK-EE lines failed to adhere sufficiently well to the coverslips to be microinjected; thus, only the cyclin D1 and parental lines were used. Microinjection of antisense p27 plasmid led to a significant increase in DNA synthesis in NIH 3T3 cells expressing exogenous cyclin D1 but not in the parental NIH 3T3 cells (Fig. 8).
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Progression from G0 through G1 and into S phase is thought to require the integration of extracellular signals that coordinately regulate the cell cycle machinery. Two major pathways identified that participate in this process are the assembly and activation of cyclin D-dependent kinase, and the Ras/Raf/MEK/MAPK kinase cascade. The data presented by our group and by others suggest that these pathways are, in part, linked in that cyclin D1 expression is a downstream effect of MEK1 activation. The expression of the CDK inhibitor p27 is also regulated by extracellular signaling. p27 plays a critical role in entrance into G0, and the data presented here suggest that p27 has a role in maintaining a G0 state. Furthermore, our data suggest that elevated expression of cyclin D1 and reduced expression of p27 may cooperate to deregulate the G0/G1 transition. Activating mutations in Ras, amplification or overexpression of cyclin D1, and low levels of p27 are found in a significant percentage of human cancers (6, 23, 41, 68). These data are consistent with the observation that deregulation of the signal transduction pathways that control progression through G1 is a major target in the development of cancer (63).
We show here that ectopic expression of cyclin D1 alone is insufficient to override a G1 arrest induced by expression of dominant-negative Ras in cycling cells in the presence of serum. Wild-type or catalytically inactive CDK4 is able to cooperate with cyclin D1 to reverse the arrest caused by RasN17 expression. In contrast, cyclin D1 alone can override the G1 arrest induced by Rb. Rb accumulates in its unphosphorylated growth-suppressive form when reintroduced into Rb-negative cells, and ectopic expression of cyclin D1 alone can reverse this effect (data not shown). Likewise, we and others had previously shown that expression of RasN17 also results in the accumulation of unphosphorylated Rb (38, 65). Together, these results suggest that ectopic expression of cyclin D1 can promote the activation of CDK4 in the setting of Rb overexpression in Rb-negative cells but not in cells expressing dominant-negative Ras. Indeed, RasN17 expression in the cyclin D1 derivatives of NIH 3T3 cells results in a significant inhibition of CDK4 activity (Fig. 3). These results suggest that the Ras inactivation brings about other events in addition to the inhibition of cyclin D1 synthesis. Our observation that catalytically inactive CDK4 can cooperate with cyclin D1 to reverse the G1 arrest brought about by Ras inactivation suggests that one or more CDK inhibitors may be involved. Thus far, we have not been able to demonstrate that the inhibition of CDK4 in the cyclin D1 lines is caused by p27 due to our inability to accurately assess the degree to which this or other CDK inhibitors are associated with CDK4 in transfected and sorted cells. Consistent with the idea that CDK inhibitors are involved is the observation that expression of RasN17 in NIH 3T3 cells during mitogenic stimulation out of a quiescent state prevents the downregulation of p27 normally seen during G1 (1, 86).
In contrast to ectopic cyclin D1, expression of an activated form of MEK1 effectively overcame the G1 arrest induced by inactivation of Ras (Fig. 2). Our data suggest that MEK-EE overrides a G1 arrest induced by expression of RasN17 in part by positively regulating cyclin D1 expression (Fig. 4) but also through another pathway(s) that remains to be identified. It has been shown that p27 can be phosphorylated in vitro by MAPK and that this phosphorylation event may play a role in p27 degradation and/or regulate its ability to bind CDK2 (3, 31). These data are consistent with our interpretation that catalytically inactive CDK4 cooperates with cyclin D1 to override a G1 arrest induced by Ras inactivation by titrating CDK inhibitors. Furthermore, the data support our finding that CDK4 is activated earlier after mitogenic stimulation in the MEK-EE lines than in the cyclin D1 lines, although our data do not allow us to directly suggest the involvement of p27.
The results presented here suggest that, in serum-starved derivatives of NIH 3T3 cells, under conditions where cyclin D1 is constitutively expressed, cyclin D1 is associated with CDK4 (Fig. 5). This contrasts with previous observations indicating that ectopic cyclin D3 and CDK4 fail to assemble in serum-starved NIH 3T3 cells (48). NIH 3T3 cells from a variety of sources yielded similar results (data not shown). The results are consistent with a number of observations. First, we have noted that, in serum-starved cells that express cyclin D1, there are elevated p27 levels compared to those in parental cells and significant amounts of cyclin D1 and CDK4 are found associated with p27. Recently, it has been demonstrated that members of the p21 family of CDK inhibitors can function as cyclin D-CDK4 assembly factors (35). Thus, the presence of elevated levels of p27 in the quiescent cyclin D1 and MEK-EE derivatives of NIH 3T3 cells, compared to those in the parental line, may be responsible for promoting the assembly of cyclin D1 and CDK4 in this setting. Second, our results obtained with microinjection of antisense p27 plasmid indirectly support the observation that cyclin D1 and CDK4 can assemble in vivo in serum-starved cells. Expression of antisense p27 was effective in driving the cyclin D1 lines, but not parental NIH 3T3 cells, into S phase. Assuming that the reduction in p27 levels allows CDK4 to become active and in turn promote S-phase entry suggests that cyclin D1 and CDK4 were assembled in serum-starved cells. We do not rule out the possibility that reduction of p27 levels also promotes S-phase entry, in part, through activation of cyclin E-CDK2.
During the completion of this work, Cheng et al. (10) reported that MEK1 plays a significant role in both the synthesis of cyclin D1 and its assembly with CDK4. By contrast, we find that cyclin D1 and CDK4 assemble in the presence or absence of MEK1 activation, although these complexes are inactive in the absence of mitogenic stimulation. There are a number of differences between our experimental approach and those of Cheng et al. that may account for the different experimental outcomes. The most significant difference is that they used cell clones harboring an inducible activated MEK1, whereas we utilized pooled populations that constitutively express activated MEK1. The Ras/Raf/MEK/MAPK pathway is known to be subject to a negative feedback loop limiting the amount of total activated MAPK. In this regard, it has been noted that in NIH 3T3 cells expressing activated MEK1 (the same retroviral vector used in this study) there is only a modest increase in overall MAPK activity (14). It is likely that in an inducible system the degree of transient activation of MAPK is greater than that in a constitutive system. Second, the antibodies used in this study to detect cyclin D1-CDK4 complexes are different from those used by Cheng et al., possibly accounting for a relative difference in the ability to detect complexes. Lastly and independent of studying MEK1, we compared the assembly of cyclin D1 and CDK4 in cyclin D1 lines and MEK-EE lines. Cheng et al. utilized cell lines overexpressing both cyclin D1 and CDK4. There is the possibility that the relative levels of cyclin D1 and CDK4 may influence the ability of CDK inhibitors such as p27 to facilitate their assembly. It is also noteworthy that a requirement for MAPK in the assembly of active CDK4 may not be universal. Work with breast epithelial cells suggests that MAPK activation is not required for the activation of cyclin D1-CDK4 (42).
Our finding that, in serum-starved MEK-EE lines, CDK4 kinase is inactive despite the formation of cyclin D1-CDK4 complexes (Fig. 6 and 7) suggests that an additional mitogenic event is required to activate CDK4 which is not provided by activated MEK1. Given that in the MEK-EE lines the timing of CDK4 activation appeared to precede a significant reduction in p27 associated with cyclin D1 and CDK4, this additional event probably does not solely involve p27 binding. Furthermore, our results suggest that mitogens, possibly operating in a Ras-independent manner, may contribute to CDK4 activation. This follows from the observation that, in the MEK-EE line, CDK4 is inactive following serum starvation but remains active upon introduction of RasN17. Consistent with this interpretation, recently it has been demonstrated that the effect of activated Ras or MEK1 on the cell cycle machinery and G1 progression can be altered by extracellular signaling (60).
In human diploid fibroblasts, expression of antisense p21 has been reported to allow cells to enter S phase from a quiescent state (53). Consistent with the possible requirement for cyclin D1 to achieve this effect, in this study cyclin D1 was reported to be present in quiescent human fibroblasts, in contrast to the situation in quiescent NIH 3T3 cells. Similar to our results obtained with p27, in the cyclin D1 and MEK-EE lines, both cyclin D1 and CDK4 were found associated with p21 in serum-starved cells (data not shown). In the parental line, p21 was found associated with cyclin D1 only at times when complexes between cyclin D1 and CDK4 could be detected (data not shown). Whether expression of antisense p21 will induce S-phase entry in NIH 3T3 cells expressing ectopic cyclin D1 remains to be determined. Together, these data suggest that both p27 and p21 play an active role in maintaining a quiescent state. In addition, our data suggest that downregulation of cyclin D1 expression participates in maintaining a state of quiescence.
CDK4 normally becomes activated in mid-G1 and is thought to promote DNA synthesis by phosphorylating and inactivating Rb at this time. Our data suggest, but do not prove, that activation of cyclin D1-CDK4 in serum-starved cells, by lowering p27 levels, is sufficient to allow for entrance into and progression through G1 with subsequent S-phase entry. This is consistent with the recent demonstration that microinjection of active cyclin D1-CDK4 complexes, prepared from insect cells, is sufficient to initiate DNA synthesis from a quiescent state in human diploid fibroblasts (13).
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ACKNOWLEDGMENTS |
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We thank Martine Roussel and Charles Sherr for the cyclin D1 lines, Christopher Marshall for the activated MEK1 retroviral vector, Jacques Pouysségur and Natalie Rivard for the antisense p27 plasmid, Geoffrey Cooper for the RasN17 plasmid, Pamela Silver for the GFP plasmid, Warren Pear and David Baltimore for the Bosc23 packaging line, and Edward Harlow for the CDK4 plasmid. We thank Othon Iliopoulos, Christine McMahon, Justin Lamb, Martine Roussel, and Peter Adams for critical review of the manuscript.
This work was supported by National Institutes of Health grant CA65842 to M.E.E. M.E.E. is a Scholar for the Leukemia Society of America.
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FOOTNOTES |
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* Corresponding author. Mailing address: Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Phone: (617) 632-2206. E-mail: mark_ewen{at}dfci.harvard.edu.
Present address: Cytomatrix, Inc., Cambridge, MA 02139.
Present address: Department of Surgery, Brigham and Women's
Hospital and Harvard Medical School, Boston, MA 02115.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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