Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK
* Author for correspondence (e-mail: chrism{at}icr.ac.uk)
Accepted 4 September 2002
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Summary |
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Key words: Ras, MAP kinase, ERK, Retinoblastoma
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Introduction |
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In mammalian cells, entry into the cell cycle is controlled at the G1/S
boundary (reviewed in Sherr,
1996). The requirement for Ras in cell cycle regulation and
proliferation is now well established (Cai
et al., 1990
; Feig and Cooper,
1988
; Mulcahy et al.,
1985
; Stacey et al.,
1991
; Dobrowolski et al.,
1994
; Aktas et al.,
1997
). These studies also show that Ras activity is required at
multiple points in cell cycle entry. Following growth factor stimulation of
quiescent non-proliferating cells, Ras activity is required for cells to exit
G0 and pass through the G1/S transition of the cell cycle
(Dobrowolski et al., 1994
;
Feig and Cooper, 1988
;
Mulcahy et al., 1985
). In
contrast to growth factor stimulation of quiescent cells, studies in
exponentially growing cells indicate that Ras is required in the G2 phase of
the preceding cycle and not during G1
(Hitomi and Stacey, 2001
).
The key event that regulates the G1/S transition is inactivation of the
retinoblastoma (Rb) protein via phosphorylation mediated by cyclin dependent
kinases. In its hypo-phosphorylated state Rb is bound to the E2F transcription
factor (reviewed in Weinberg,
1995). This represses transcription of target genes that are
required for progression through S phase of the cell cycle. Phosphorylation of
Rb leads to its inactivation and de-repression of E2F facilitating DNA
replication. Inactivation of Rb by phosphorylation is mediated by the
sequential activation of the cyclin dependent kinases (cdks)
(Sherr, 1996
;
Weinberg, 1995
). The D-type
cyclins which bind cdk4 or cdk6 and cyclin E and cyclin A which bind cdk2 are
implicated in this process (Sherr,
1996
; Weinberg,
1995
).
Many studies link cyclin D1 expression to Ras signalling. The use of
inducible Ha-Ras expression constructs has demonstrated that overexpression of
Ha-Ras in the rat epithelial cell line IEC
(Filmus et al., 1994) and
BalbC-3T3 cells (Winston et al.,
1996
) leads to the induction of cyclin D1 expression. Conversely
expression of dominant negative Ras mutants has been demonstrated to inhibit
expression of cyclin D1 following serum stimulation
(Aktas et al., 1997
). An
acceleration of G1 progression and shorter cell doubling times in
asynchronously growing Ha-Ras transformed NIH3T3 cells has been shown to
correlate with overexpression of cyclin D1
(Albanese et al., 1995
;
Liu et al., 1995
). These
reports make a clear link between Ras expression and the induction of cyclin
D1 expression in cell cycle control. Activation of ERK-MAP kinase appears to
be a key event in mediating Ras-dependent regulation of cyclin D1 expression
as well as cdk complex formation (Lavoie
et al., 1996
; Weber et al.,
1997
; Cheng et al.,
1999
). PI 3-kinase and Ral-GEF have also been postulated to
regulate cyclin D1 expression and stability
(Diehl et al., 1997
;
Muise-Helmericks et al., 1998
;
Gille and Downward, 1999
;
Henry et al., 2000
).
Levels of the cdk inhibitor p27Kip1 are high in quiescent cells.
Re-entry into the cell cycle is accompanied by a decline in the levels of
p27Kip1 facilitating activation of cyclin E/cdk2 complexes
(Sherr and Roberts, 1999).
Expression of oncogenic Ras had also been shown to regulate the levels of
p27Kip1 (Aktas et al.,
1997
; Takuwa and Takuwa,
1997
). Studies indicate that regulation of p27Kip1
expression by the Raf-ERK pathway and PI 3-kinase is mediated by regulating
the rate of translation and protein stability
(Aktas et al., 1997
;
Kerkhoff and Rapp, 1997
;
Takuwa and Takuwa, 1997
). A
further contribution to the relief of p27Kip1 inhibition of cdk2
complexes is made by the Mek-ERK pathway facilitating cyclin D1/cdk4 complex
formation which results in sequestration of p27Kip1 onto this
complex and allows activation of cdk2 complexes
(Cheng et al., 1998
).
Microinjection studies using the neutralising Y13-259 antibody
(Furth et al., 1982) against
Ras show that the requirement for Ras signalling to promote cell cycle entry
is altered in Rb-/- cells
(Mittnacht et al., 1997
;
Peeper et al., 1997
).
Interestingly these experiments defined a difference between cells that are
asynchronously growing and cells that have been rendered quiescent and then
treated with growth factors to stimulate cell cycle entry. Regardless of
whether they express Rb or are Rb null, cells require Ras activation in order
to leave G0. In contrast microinjection of the Y13-259 antibody into
asynchronously growing cells revealed that in comparison to wild-type MEFs,
Rb-null MEFs have a reduced requirement for Ras activity
(Mittnacht et al., 1997
;
Peeper et al., 1997
). This
reveals two interesting points. Ras activity is critical for both exit from G0
and for cells to make the G1/S transition. However with the loss of Rb, cells
are released from the requirement for Ras for the G1/S transition. The
observation that proliferation of asynchronously growing Rb-/- MEFs
is much less affected by blocking Ras function raises the question whether
Rb-/- cells have a reduced requirement for Ras dependent signalling
pathways such as the ERK-MAP kinase signalling pathway. We find that
Rb-/- cells no longer depend on ERK signalling for re-entry into
the cell cycle from G0, or for asynchronous growth. Consistent with this
phenotype is the observation that the main role of ERK-MAP kinase pathway is
to regulate cyclin D1 expression and associated cdk activity rather than the
regulation of cyclin E and cyclin A expression.
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Materials and Methods |
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Cell cycle analysis
To monitor proliferation, following treatment with inhibitor(s) cells were
pulsed with bromodeoxyuridine (BrdU) (Amersham Pharmacia Biotech) at the time
of serum stimulation for 12-16 hours. Cells were fixed and stained for
immunofluorescence as follows. Cells were rinsed once in phosphate-buffered
saline (PBS) and fixed in 4% paraformaldehyde for 15 minutes at room
temperature. After rinsing twice in PBS cells were permeabilized in 0.2%
Triton X-100/0.1 mM glycine for 30 minutes at room temperature. Cells were
rinsed several times in PBS and then blocked by incubation in 5% FCS in PBS
(PBS/FCS) for 30 minutes at room temperature. The cells were then treated with
RNase free DNase I (Roche) at 1-2 units/µl for 60 minutes at 37°C.
After washing three times in PBS/FCS cells were incubated with anti-BrdU rat
monoclonal antibody (diluted to 5 µg/mL in PBS/BSA) for 1 hour at room
temperature. After washing cells three times in PBS/BSA cells were incubated
with fluorescent secondary antibody (Jackson Immunoresearch Laboratories) to
detect incorporated BrdU and DAPI (Sigma) to detect total nuclei in each
field. In addition a Texas-Red-X-conjugated phalloidin stain (Molecular
Probes) was used to detect cells. Secondary antibodies and phalloidin were
diluted at 1:200, DAPI was diluted to a working concentration of 1 µg/ml in
PBS/FCS. Cells were then incubated for 1 hour at room temperature in a
light-proof container. After rinsing three times in PBS and once in water,
coverslips were mounted onto glass slides by inverting onto 5-10 µl of
moviol mountant containing 0.1% para-phenylenediamine as an antiquenching
agent. Stained preparations were examined with a Bio-rad MRC confocal imaging
system in conjuction with a Nikon Diaphot epifluorescence microscope.
Antibodies
An ERK2 rabbit polyclonal antibody generated against a C-terminal ERK2
peptide (no. 122) was used for immunoprecipitation experiments
(Leevers and Marshall, 1992).
A phospho-specific antibody to ERK1 and 2 (clone MAP-YT) was purchased from
Sigma. The antibody that detects the hypo and hyper phosphorylated forms of
the Retinoblastoma protein (14001A), was purchased from PharMingen.
Anti-cyclin D1 (72-13G), cyclin E (M20) and cyclin A (H-432) antibodies were
purchased from Santa Cruz Biotechnology. An anti-p27Kip1 (clone 57)
antibody was purchased from Transduction Laboratories.
Preparation of cell extracts
Cells were washed twice in ice-cold PBS and lysed in ELB buffer (50 mM
NaCl, 100 mM HEPES pH 7.0, 10 mM EDTA and 0.2% Triton-X-100) containing 20 mM
ß-glycerol phosphate, 20 mM sodium fluoride, 10 mM sodium vanadate, 1 mM
DTT, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin and 10 µg/ml
leupeptin. Cell debris was cleared by centrifugation at 18,000
g for 10 minutes. Protein concentrations were determined using
the BCA protein assay reagent (Pierce).
Western blots
Immunoblotting was performed using 25-30 µg of cleared lysate. For
detection of the Rb protein 250 µg of cleared lysate was loaded. Prior to
loading Laemmli buffer was added to each sample, followed by heating to
100°C for 3-5 minutes. Samples were resolved by electrophoresis through
denaturing SDS-polyacrylamide gels. Proteins were transferred to
polyvinylidene difluoride (PVDF) (Millipore) membrane for western blotting.
Membranes were probed with an appropriate dilution of primary antibody for 1-2
hours at room temperature followed by incubation with a horseradish peroxidase
(HRP) conjugated secondary antibody (Bio-Rad) for 45 minutes at room
temperature. Proteins were visualised using the enhanced chemiluminescence
(ECL) reagent (Amersham).
ERK2 assay
Cells were lysed in lysis buffer as described above. For each sample 50
µg of cleared lysate was immuopprecipitated with the 122 antibody coupled
to protein-A-Sepharose beads for 90 minutes at 4°C. Immuno-complexes were
washed twice in lysis buffer and twice in kinase buffer (30 mM Tris pH 8.0, 20
mM MgCl2 and 2 mM MnCl2) containing 10 mM
ß-glycerol phosphate, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10
µg/ml aprotinin and 10 µg/ml leupeptin. Kinase reactions were conducted
in a final volume of 30 µl kinase buffer containing 10 µM ATP (Sigma), 5
µCi [-32P]ATP Amersham) and 0.25 mg of myelin basic
protein (Sigma) as the substrate per reaction. After a 30 minute incubation at
30°C reactions were stopped by the addition Laemmli buffer followed by
heating to 100°C for 3-5 minutes. Proteins were then transferred to PVDF
membrane and ERK2 activity was detected by PhosphoImager (Molecular
Dymamics).
Cell-cycle-associated kinase assays
Cells were lysed in lysis buffer as described above. For each sample 500
µg of cleared lysate was immuoprecipitated with antibodies coupled to
protein-A-Sepharose beads for 90 minutes at 4°C. Immuno-complexes were
washed twice in lysis buffer and twice in kinase buffer (50 mM HEPES pH 7.4,
10 mM MgCl2, 10 mM MnCl2) containing 10 mM
ß-glycerol phosphate, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10
µg/ml aprotinin, 10 µg/ml leupeptin and 1 mM protein kinase inhibitor
(Sigma). For cyclin D1/cdk4 associated kinase assays a GST-fusion construct
encoding a C-terminal fragment (amino acids 763-928) of Rb
(Zarkowska and Mittnacht,
1997) was used at 0.5 µg per reaction. For cyclin E/cdk2 kinase
assays histone H1 was used as the substrate (a gift from G. Goodwin, ICR) at 2
µg per reaction. Kinase reactions were conducted in a final volume of 25
µl kinase buffer containing 50 µM ATP and 5 µCi
[
-32P] ATP. After a 15 minute incubation at 30°C
reactions were stopped by the addition of SDS-PAGE sample buffer. Proteins
were then transferred to PVDF membrane and kinase activity was detected by
PhosphoImager.
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Results |
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Since the requirement for ERK activation for cell proliferation was abrogated in Rb-/- cells, we investigated whether cells lacking other negative regulators of cell cycle entry had altered signalling requirements. Fig. 2 shows that MEFs that do not express the cyclin dependent kinase inhibitor(s) (CDKi) p21Waf1/Cip1, p27Kip1 or p16Ink4 all required ERK activation for cell cycle entry. Thus it is only the loss of Rb that alleviates the requirement for ERK signalling.
|
Regulation of ERK activity is comparable in Wt and Rb-deficient
MEFs
Lee et al. have shown that Rb-/- cells have elevated levels of
Ras-GTP during the G1 phase of the cell cycle
(Lee et al., 1999). In
addition, Alessi et al. have shown that PD098059 does not block ERK activation
at high levels of growth factor stimulation
(Alessi et al., 1995
). Higher
levels of active Ras in Rb-/- cells might result in higher levels
of ERK activation that cannot be blocked by U0126. Hence the inability of
U0126 to block cell cycle entry in Rb-/- cells could be a
consequence of higher levels of ERK activation following growth factor
stimulation. We therefore investigated whether ERK activity was different in
Rb-/- compared with Wt MEFs. Analysis of ERK activation
(Fig. 3) by in vitro kinase
assays (Fig. 3a,c) and western
blotting for (active) phospho-ERK (Fig.
3b,d) revealed that the basal level of ERK activation was the same
in quiescent Rb-/- and Wt MEFs and that it was stimulated to the
same degree with similar duration in both Rb-/- and Wt MEFs.
Furthermore, Fig. 3 shows that
U0126 inhibited ERK activation to a similar degree in both cell lines. Thus
the failure of U0126 to block cell proliferation of Rb-/- cells is
a consequence of a reduced requirement for ERK signalling rather than the
inability of U0126 to block ERK activation in these cells.
|
Growth of Rb-null tumour cell lines is uncoupled from MAP kinase
signalling
Since we observed a reduced requirement for ERK signalling in Rb-null MEFs,
we were interested to investigate whether human tumour cell lines lacking Rb
had a reduced requirement for ERK signalling. We compared tumour cell lines,
which are deficient for Rb expression to cells lines of similar origin, which
are wild-type for Rb. To facilitate these experiments we used two cell lines
derived from glioma (SF295 Rb wild-type and SF539 Rb deficient) and two cell
lines derived from non-small cell lung carcinoma (H1299 Rb wild-type and H2009
Rb deficient). Analysis of SF295 and H1299 (Rb wild-type) revealed that
treatment with U0126 blocked proliferation of asynchronously growing cells
(Fig. 4A and B, respectively).
In contrast the Rb-deficient tumour cell lines, SF539 and H2009, continued to
proliferate in the presence of U0126. Western blotting for phospho-ERK
(Fig. 4C) showed that U0126
inhibited ERK activation in SF539 and H2009
(Fig. 4C). These data show that
human tumour cell lines that lack Rb, like MEFs that are deficient for Rb
expression, have a reduced requirement for ERK signalling.
|
Inactivation of retinoblastoma protein by phosphorylation is blocked
by U0126 and LY294002 in Wt MEFs
The G1/S transition is facilitated by the inactivation of the
retinoblastoma protein. At the molecular level, inactivation of
pRb105 correlates with the hyper-phosphorylation of a number of
Ser/Thr sites (Mittnacht,
1998; Weinberg,
1995
) mediated by the G1 cyclin dependent kinases (cdks)
(Sherr, 1996
;
Weinberg, 1995
). Using a pan
Rb antibody in a western blot we were able to probe for endogenous hypo
(active) and hyper (inactive) forms of pRb as judged by altered
electrophoretic mobility (Fig.
5). Upon serum deprivation only the active faster migrating
(hypo-phosphorylated) form of pRb105 was detected
(Fig. 5, lane 1) correlating
with the quiescent state of the cells. On serum stimulation the inactive
slower migrating (hyper-phosphorylated) form of retinoblastoma was also
detected (Fig. 5, lane 2). In
contrast, when cells were treated with U0126 prior to serum stimulation we did
not detect the hyper-phosphorylated of Rb
(Fig. 5, lanes 3). When the
cells were treated with either LY294002 or the combination of both inhibitors
we were also unable to detect the hyper-phosphorylated form of Rb
(Fig. 5, lanes 4,5).
|
Levels of cyclin D1 but not cyclins E or A are regulated by ERK
signalling
The G1/S boundary is marked by expression of cyclin D1 and cdk4 activity
(Sherr, 1996). Therefore we
examined expression of cyclin D1 and cdk4 activity in MEFs treated with U0126
(Fig. 6a, Fig. 7A). Serum starved cells
had low levels of cyclin D1 (Fig.
6a) that was induced upon serum stimulation
(Fig. 6a). Treatment of cells
with U0126 partially blocked induction of cyclin D1 expression in both the Wt
and Rb-deficient MEFs. To determine whether this decrease in cyclin D1
expression affected cdk4 activity, cdk4 was immunoprecipitated from MEF cell
lysates and assayed for in vitro kinase activity using a C-terminal fragment
of Rb (amino acids 763-928) fused to GST as substrate
(Zarkowska and Mittnacht,
1997
). Serum starved MEFs had low cdk4 kinase activity
(Fig. 7A), while serum
stimulation lead to an increase in cdk4 activity
(Fig. 7A). Treatment with U0126
resulted in a marked decrease in cdk4 kinase activity in both the Wt and
Rb-/- MEFs (Fig.
7A). In agreement with previous studies,
(Lavoie et al., 1996
;
Weber et al., 1997
;
Cheng et al., 1999
), these
results support the model that activation of ERK is required for cyclin D1
expression, cdk4 complex formation and kinase activity. We consistently
observed that treatment with U0126 did not completely abrogate serum
stimulated cyclin D1 expression or cdk4 activity although it did block entry
into S-phase of WT cells (Fig.
1). Since the PI 3-kinase pathway has also been shown to regulate
cyclin D1 expression at both the transcriptional
(Gille and Downward, 1999
),
and postranslational level (Diehl et al.,
1997
; Diehl et al.,
1998
) we investigated how inhibition of PI 3-kinase affected
cyclin D1 expression and cdk4 kinase activity. These experiments revealed that
inhibition of PI 3-kinase with LY294002 lead to a partial block of cyclin D1
expression (Fig. 6a) and cdk4
activity (Fig. 7A).
Interestingly inhibition of both the ERK and PI 3-kinase pathways by treatment
with U0126 and LY294002 completely ablated serum stimulated cyclin D1
expression (Fig. 6a) and cdk4
activity (Fig. 7A). These
results show that both the ERK and PI 3-kinase pathways contribute to cyclin
D1 expression and that complete inhibition of cyclin D1 expression and
associated cdk4 activity requires inhibition of ERK and PI 3-kinase
signalling.
|
|
The G1-S phase of the cell cycle is regulated by cyclin E and cyclin A
associated cdk2 activity (Sherr,
1996). We therefore investigated whether inhibition of ERK
activation using U0126 would inhibit the expression of cyclin E and cyclin A
(Fig. 6b,c). In Wt MEFs
expression of cyclin E and A was unaffected by inhibition of the ERK pathway,
whilst interestingly the level of cyclin E/cdk2 activity was low. In contrast
inhibition of PI 3-kinase blocked expression of cyclin E and A
(Fig. 6b,c) and activation of
cyclin E/cdk2 activity in Wt MEFs. The lack of cdk4 and cdk2 activity that we
observed in Wt MEFs upon treatment with either U0126 or LY294002 correlates
with the absence of the inactive hyperphosphorylated form of Rb and the growth
arrest that we observe. These data show that ERK signalling is required to
regulate expression of cyclin D1 but does not impinge on the expression of
cyclin E or cyclin A. This is in contrast to PI 3-kinase signalling which our
studies show impinges on all 3 G1/S regulating cyclins and associated cyclin
D1/cdk4 and cyclin E/cdk2 activity.
As previously described (Herrera et
al., 1996), Rb-/- cells have elevated levels of cyclin
E and cyclin A both in serum starved and serum stimulated conditions
(Fig. 6b,c). As was observed in
Wt MEFs, treatment with U0126 had no effect on the levels of cyclin E or
cyclin A in Rb-/- cells (Fig.
7B,C). Stimulation of Rb-/- cells with serum induces a
higher level of cyclin E/cdk2 activity in comparison to the Wt MEFs. Whilst
treatment with U0126 had no effect on cyclin E expression in Rb-/-
MEFs, analysis of cyclin E/cdk2 activity by immunoprecipitation of cyclin E
from cell lysates indicated that treatment with U0126 had a small but
consistent decrease in cyclin E associated kinase activity
(Fig. 7B). However, the
remaining kinase activity was greater than or equal to that observed in serum
stimulated Wt MEFs in the absence of inhibitors. This high residual cyclin
E/cdk2 activity in U0126-treated Rb-/- MEFs, coupled with the
knowledge that Rb-/- cells do not require cyclin D1 associated
kinase activity for cell cycle progression
(Lukas et al., 1995
) may
explain why Rb-/- cells can proliferate when the ERK pathway is
inhibited.
Analysis of PI 3-kinase signalling in Rb-/- MEFs revealed that unlike Wt cells inhibition of PI 3-kinase had no effect on cyclin E or cyclin A expression levels (Fig. 6b,c). The combination of U0126 and LY294002 lead to a small ihhibition in the level of both cyclin E and cyclin A. Interestingly, we did not observe complete inhibition in expression levels (Fig. 6b,c). Analysis of cyclin E/cdk2 activity revealed that despite a negligible effect on the levels of cyclin E, treatment with LY294002 and the combination of inhibitors lead to a marked inhibition of kinase activityin the Rb-/- MEFs (Fig. 7B).
Regulation of p27Kip1 is controlled by ERK and PI 3-kinase
signalling
The level of the expression of the cdk inhibitor p27Kip1 is
known to be important determinant for cell cycle entry at least in part as a
regulator of cdk2 activity (Sherr and
Roberts, 1999). In an effort to study the mechanism by which cdk2
activity is regulated as a consequence of ERK and PI 3-kinase signalling, we
examined how inhibition of these pathways would affect p27Kip1
expression levels. Consistent with previous studies
(Slingerland and Pagano, 2000
)
removal of growth factors led to an accumulation of p27Kip1
(Fig. 7C) that was
downregulated upon serum restimulation in both Wt and Rb-deficient cells
(Fig. 7C). Analysis of cells
treated with either U0126 or LY294002 revealed that following serum
stimulation, the levels of p27Kip1 were still downregulated in both
Wt and Rb-/- MEFs (Fig.
7C). However, in the presence of both U0126 and LY294002 serum
stimulation was unable lead to the downregulation of p27Kip1 in Wt
and Rb-/- MEFs (Fig.
7C). These results show that either ERK or PI 3-kinase signalling
can regulate the levels of p27Kip1 suggesting that redundancy
exists between these two signalling pathways for the regulation of
p27Kip1 levels.
Asynchronous proliferation of Wt MEFs but not Rb-/- MEFs
is reduced by inhibition of MEK
We have shown that Rb-/- MEFs are capable of cell cycle entry in
the presence of U0126 and LY294002 in a synchronised cell growth assay
(Fig. 1). While we observe
residual cyclin E/cdk2 activity in Rb-/- MEFs treated with U0126,
treatment with LY294002 results in complete loss of kinase activity. Due to
this surprising result we were interested to assess the capacity of
Rb-/- cells to proliferate in a long term growth assay in the
presence of these inhibitors (Fig.
8). As expected treatment of asynchronously growing Wt MEFs with
U0126 retarded proliferation over a 4 day period
(Fig. 8a). In contrast,
Rb-/- MEFs were capable of sustained growth over a 4 day growth
period in the presence of U0126 (Fig.
8a), which was comparable to the growth kinetics of untreated MEFs
(Fig. 8a,b). Quiescent
Rb-/- MEFs stimulated with serum in the presence of LY294002
entered DNA synthesis but after 24 hours entered apoptosis. Furthermore
treatment with LY294002 led to inhibition of proliferation through apoptosis
of asynchronous Rb-/- MEFs over a 4 day period
(Fig. 8b)
|
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Discussion |
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When quiescent serum starved Wt MEFs were treated with the Mek inhibitor
U0126, serum restimulation did not lead to S-phase entry. In contrast Rb-null
MEFS were able to exit the G0 state, enter the cell cycle and proliferate when
Mek activity is inhibited. This inability of U0126 to block cell cycle entry
in Rb-null cells is not a consequence of the inhibitor failing to block Mek1/2
and thereby ERK1/2 activation in these cells because ERK1/2 activation was
similarly inhibited in both Wt and Rb-null cells. U0126 and PD098059, another
Mek inhibitor, have been shown to also block Mek5 in the ERK5/BMK1 pathway
(Kamakura et al., 1999). Since
the ERK5/BMK1 pathway has been implicated in cell cycle control
(Kato et al., 1998
) these
results could imply that loss of Rb may also abrogate the requirement for ERK5
activation. However we have been unable to reproducibly measure ERK5 kinase
activity and therefore do not know whether it is activated in the cell system
we have used. These results show that while Rb-null cells still require Ras
signalling to exit G0 they do not require ERK1/2 activation. The role of the
ERK1/2 pathway is to bring about the inactivation of Rb and the G1-S
transition rather than the Ras dependent step in G0 exit. While oncogenes have
long been known to affect cell signalling pathways, these results show for the
first time that loss of a tumour suppressor gene can alter the requirement for
the MAP kinase signalling pathway. However the requirement for ERK signalling
was unaffected in MEFs null for p21Waf1/Cip1, p27Kip1 or
p16Ink4 which are also tumour suppressor genes linked to cell cycle
control. Thus it is only the loss of Rb that alleviates the requirement for
ERK signalling.
We were interested to try and understand the mechanism that abrogates the
requirement for ERK signalling in Rb-null cells. These cells do not depend on
cyclin D1/cdk activity cdk (Lukas et al.,
1995) arguing that they no longer depend on ERK signalling for
cyclin D1/cdk activity. However Rb-null cells are known to require growth
factor stimulation so they might require ERK signalling for other cell cycle
events (Herrera et al., 1996
).
Although U0126 treatment reduced cyclin E/CDK2 activity by approximately 50%
in Rb-/- MEFS (without affecting cyclin E expression), the serum
stimulated level of E/CDK2 activity after U0126 treatment in Rb-/-
MEFs was comparable to that in serum-stimulated untreated Wt MEFs. This
suggests that this level of E/CDK2 activity is sufficient for proliferation.
Rb-null human tumour cell lines unlike their Rb Wt counterparts
also proliferated in the presence of U0126 despite complete inhibition
of ERK activation. We conclude that Rb-null cells do not depend on ERK
signalling for proliferation because the requirement for cyclin D dependent
kinase activity is abrogated by loss of Rb and cells have elevated cyclin
E/Cdk2 activity. This increased activity probably results from deregulated
cyclin E transcription caused by the de-repression of E2F that is associated
with loss of Rb (Herrera et al.,
1996
).
Like the ERK pathway PI 3-kinase signalling has been shown to be required
for quiescent cells to enter the cell cycle
(Gille and Downward, 1999;
Jones et al., 1999
;
Jones and Kazlauskas, 2001
).
Furthermore PDGF dependent PI 3-kinase signalling is known to occur at G0/G1
and mid to late G1, interestingly however entry into S-phase depends on PI
3-kinase activity during the mid to late phase of G1
(Jones et al., 1999
;
Jones and Kazlauskas, 2001
).
Therefore we were interested to investigate the effect of inhibition of PI
3-kinase signalling on cell cycle progression in Rb-null cells. In contrast to
Wt MEFS, treatment of quiescent Rb-null cells with serum in the presence of
the PI 3-kinase inhibitor LY294002 did not inhibit S-phase entry. However
unlike inhibition of the ERK pathway, cells could not proliferate long term
when PI 3-kinase was inhibited and died through apoptosis. At the molecular
level treatment with LY294002 partially blocked induction of cyclin D1
expression, cdk4 activity but in contrast to U0126 also blocked expression of
cyclin E, A and cyclin E/cdk2 activity. Thus, cyclin D1 expression is
responsive to both ERK and PI 3-kinase dependent signalling, but cyclin E and
cyclin A only requires PI 3-kinase signalling. Together these data are
consistent with a role for PI 3-kinase in regulating the G1/S transtion that
is overcome in the absence of the Rb check-point. However Rb-null cells are
still dependent on PI 3-kinase signalling for survival.
Downregulation of the CDKI p27Kip1 is a key element for the
movement of cells out of G0 and into S-phase
(Malek et al., 2001;
Slingerland and Pagano, 2000
).
Our data show that in both Wt and Rb-null cells p27Kip1 degradation
occurs after serum stimulation in the presence of either U0126 to block ERK
signalling or LY294002 to block PI 3-kinase signalling but not in the presence
of both inhibitors. Similar results were found with human tumour cell lines
(data not shown). These results demonstrate that there is redundancy between
these signalling pathways for regulation of p27Kip1. Cell cycle
analysis of MEFS revealed that in the presence of both inhibitors, S-phase
entry is inhibited regardless of Rb status. Our data predicts that it is the
block of p27Kip1 degradation that occurs when both pathways are
inhibited that underlies the block in cell cycle entry that we observe. The
residual levels of cyclin D1 and CDK4 activity that persist after treatment
with either U0126 or LY294002 suggest that these low levels of the cyclin
D1/CDK4 complex may be important to sequester p27Kip1 and permit
cell cycle entry in Rb-/- cells
(Cheng et al., 1998
). The
mechanism which underlies the accumulation of p27Kip1 is still to
be elucidated. However is has been shown that Ras signalling pathways can
control expression of p27Kip1 by regulating the rate of translation
and protein stability (Aktas et al.,
1997
; Kerkhoff and Rapp,
1997
; Takuwa and Takuwa,
1997
).
Since the Ras protein is a critical transducer of proliferative signals, it
is not surprising that a high percentage of some human cancers contain
activating Ras gene mutations (Bos,
1989). Most oncogenic Ras containing tumours that have been
examined contain Wt Rb (Horowitz et al.,
1990
; Kashii et al.,
1994
) and would be predicted to be sensitive to Mek inhibitors.
However where Rb is lost for example in non-small-cell lung cancers our data
predict that inhibition of the Mek pathway will not have an anti-proliferative
effect. Thus effective therapeutic use of signal transduction inhibitors may
require knowledge of the genetic alterations to the cell cycle machinery for
different tumours.
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Acknowledgments |
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References |
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