From the Queensland Cancer Fund Research Unit,
Queensland Institute of Medical Research and Joint Experimental
Oncology Program, University of Queensland, Brisbane, Queensland 4029, Australia and the ¶ Cancer Research Program, Garvan Institute of
Medical Research, St. Vincent's Hospital, Sydney,
New South Wales 2010, Australia
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ABSTRACT |
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Cyclin D-Cdk4 complexes have a demonstrated role
in G1 phase, regulating the function of the
retinoblastoma susceptibility gene product (Rb). Previously, we have
shown that following treatment with low doses of UV radiation, cell
lines that express wild-type p16 and Cdk4 responded with a
G2 phase cell cycle delay. The UV-responsive lines
contained elevated levels of p16 post-treatment, and the accumulation
of p16 correlated with the G2 delay. Here we report that in
UV-irradiated HeLa and A2058 cells, p16 bound Cdk4 and Cdk6 complexes
with increased avidity and inhibited a cyclin D3-Cdk4 complex normally
activated in late S/early G2 phase. Activation of this
complex was correlated with the caffeine-induced release from the
UV-induced G2 delay and a decrease in the level of p16 bound to Cdk4. Finally, overexpression of a dominant-negative mutant of
Cdk4 blocked cells in G2 phase. These data indicate that
the cyclin D3-Cdk4 activity is necessary for cell cycle progression through G2 phase into mitosis and that the increased
binding of p16 blocks this activity and G2 phase
progression after UV exposure.
Loss of normal cell cycle control mechanisms is a common theme in
the development of most cancers, in particular the loss of mechanisms
involved in sensing and repairing DNA damage. An increasing number of
tumor suppressor genes have been found to be intimately involved in DNA
damage responses, including cell cycle responses. The best documented
of these are the tumor suppressor genes TP53 and Rb.
TP53 is mutated in a high proportion of tumors, and its gene
product (p53) is involved in cell cycle responses following DNA damage
(1). p53 knockout mice develop relatively normally, but are prone to a
range of tumors (2). Rb was identified as the retinoblastoma
susceptibility gene, and its gene product (Rb) has a pivotal role in
the late G1 phase restriction point (3). Knockout of one
allele of Rb in mice produced animals predisposed to
pituitary tumors, but they did not develop retinoblastoma, and the
homozygous knockout was embryonically lethal (4). These tumor
suppressors exert their cell cycle effects by ultimately modulating the
levels and activities of the regulatory elements underlying cell cycle
progression, the cyclin-dependent kinases.
The cyclin-dependent kinases are regulated in a complex
manner, through cyclin subunit association; a series of tightly
regulated phosphorylations and dephosphorylations (5); and the binding of inhibitory protein subunits, CKI proteins
(Cdk inhibitors) (6). p53 can
directly regulate the expression of one of these CKI proteins, p21 (1);
and in response to DNA damage, p53-induced p21 expression is
responsible for a G1 phase delay (7). Fibroblasts from p21
knockout mice fail to delay in G1 in response to similar damage (8). The mechanism by which Rb exerts its cell cycle inhibitory
effects is somewhat complex, although this is largely via its ability
to bind and inhibit the activity of a group of transcription factors
(E2F) that are essential for progression into S phase and the
expression of a number of positive and negative regulators of cell
cycle progression (3). Thus, loss of Rb function would be predicted to
result in a deregulation of entry into S phase. One of the cell cycle
regulators whose expression is controlled by Rb function is itself a
tumor suppressor, p16CDKN2A. p16 is another CKI that acts
specifically to inhibit cyclin D-Cdk4 and Cdk6, the
cyclin-dependent kinases responsible for phosphorylating Rb
during G1 (3, 6). CDKN2A itself is mutated, deleted, or hypermethylated in a high proportion of human tumors (9).
CDKN2A has also been identified as a melanoma susceptibility gene (10) and is deleted, mutated, or hypermethylated in a high proportion of melanoma cell lines (10).
Cell cycle responses to DNA damage induced by suberythemal doses of UV
radiation include both G1 and G2 phase delays.
G1 delays appear to be through a p53-dependent
mechanism, with increased expression of p21 resulting in inhibition of
G1 cyclin/cyclin-dependent kinases (11-13).
The G2 delay is p53-independent and is via a block in the
Cdc25-dependent activation of cyclin A-Cdk and cyclin
B-Cdc2 complexes (14, 15). Cells undergoing the UV-induced
G2 delay appear to be identical to normal early
G2 phase cells, and in many respects, the delay appears to
be a prolongation of that cell cycle state (15). The cells recover from
the UV-induced block after a delay of 24 h and recommence normal
proliferation (16). A major difference that has been noted between the
UV-induced G2-delayed cells and normally cycling cells at
the equivalent cell cycle stage was a large increase in the level of
p16 protein (17). Analysis of a number of mainly melanoma cell lines
either deleted for CDKN2A or that expressed either mutant
p16 or Cdk4 showed that only cells lines that expressed both wild-type
p16 and Cdk4 displayed a UV-induced G2 delay (16). Of the
melanoma cell lines that had lost their UV-induced G2 delay
response, two expressed p16 with mutations that abolished Cdk4 (but not
Cdk6) binding, and one line expressed the Cdk4 R24C mutation, which abrogated p16 binding (10, 16, 17). This suggested that the effects of
p16 on the G2 delay were being mediated through Cdk4, the
most likely mechanism being p16 binding and inhibition of a cyclin
D-Cdk4 activity. Here we show the existence of a cyclin D3-Cdk4
activity in late S/G2 phase cells that is inhibited in response to UV radiation through the increased association of p16 and
provide evidence that this Cdk4 activity is necessary for normal
G2 phase cell cycle progression.
Cell Culture and Metabolic Labeling--
The HeLa (human
cervical carcinoma) and human melanoma A2058 cell lines were cultured
in RPMI 1640 medium supplemented with 5 and 10% Serum Supreme
(BioWhittaker, Inc.), respectively. For synchrony experiments, the cell
lines were blocked overnight with 2 mM thymidine and then
released into fresh medium containing 24 µM thymidine and
deoxycytidine. S phase cell-enriched populations were obtained by
harvesting at 4 h after release, and late S/G2 phase
cell-enriched populations at 8 h after release. Mitotically enriched populations were collected when >30% of cells showed the
rounded mitotic phenotype. Asynchronous cultures were irradiated with
10 J m
Synchronized and UVC-irradiated cells were metabolically labeled with
0.3 mCi/ml 35S-Promix (Amersham Pharmacia Biotech) in
methionine- and cysteine-free medium for 2 h, chased in complete
medium for the indicated times, washed twice with phosphate-buffered
saline, and harvested. Synchronized late S/G2 phase cells
or UV-irradiated cells (24 h post-irradiation) were metabolically
labeled with 1 mCi/ml 32Pi in phosphate-free
medium for 3 h or, in the case of caffeine release, labeled for
2 h and then a further 1 h after caffeine addition. Cells
were washed twice with phosphate-buffered saline, harvested, and
processed as described below for immunoprecipitation.
Immunoprecipitation and Immunoblotting--
Cells were lysed in
NETN buffer (100 mM NaCl, 1 mM EDTA, 0.5%
Nonidet P-40, and 20 mM Tris, pH 8) supplemented with 150 mM NaCl, 5 µg/ml aprotinin, 5 µg/ml pepstatin, 5 µg/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 10 mM NaF, and 0.1 mM sodium orthovanadate. The
cleared supernatants were immunoprecipitated using anti-p16 (17),
anti-cyclin A (Pharmingen), anti-cyclin B1 (18), or anti-Cdk4 or
anti-Cdk6 (Santa Cruz Biotechnology) antibody with protein A-Sepharose.
Immunoprecipitates from metabolically 32P- and
35S-labeled samples were resolved on 15%
SDS-polyacrylamide gel and autoradiographed or quantitated by a
PhosphorImager (Molecular Dynamics, Inc.). Other immunoprecipitates
were immunoblotted for associated proteins using the indicated
antibody. Cyclin D3 was detected using two specific monoclonal
antibodies (Pharmingen G107-565 and Oncogene Research Products Ab 2).
Cdk4 immunoprecipitate Rb kinase assays were performed in a similar
manner to that described elsewhere (19). Briefly, cell pellets
(1-5 × 106 cells) were lysed in 50 mM
Hepes, pH 7.5, 1 mM EDTA, 2.5 mM EGTA, 1 mM dithiothreitol, 250 mM NaCl, 10% glycerol,
0.1% Tween 20, 1 mM sodium fluoride, 0.1 mM
sodium vanadate, and protease inhibitors as described above for 1 h on ice with occasional vortexing. The lysates were precleared with 30 µl of a 50% suspension of protein A-Sepharose for 30 min, and the
cleared supernatants were incubated with 1 µg of anti-Cdk4 or
anti-p16 antibody for 2-3 h and then precipitated by the addition of
20 µl of a 50% suspension of the protein A-Sepharose for an
additional 1 h. The precipitates were washed four times with the
lysis buffer and then twice with the kinase buffer, and the kinase
assay was performed with the addition of 30 µl of reaction mixture
containing 50 mM Hepes, pH 7.5, 10 mM
MgCl2, 50 mM ATP, 1 mM
dithiothreitol, and 1-3 µg of purified GST1-Rb-(773-923) and 10 µCi of [ Transient Transfections--
Expression constructs containing
hemagglutinin (HA)-tagged wild-type Cdk4 and Cdk6 and the
dominant-negative Cdk4 D158N and Cdk6 D163N mutants (10 µg/107 cells) (20) were transfected by electroporation
into HeLa cells. Cdk4 and Cdk6 were cotransfected with mouse CDC37 (10 µg/107 cells) (21). Cells were harvested 48 h
post-transfection, and overexpression of Cdk4 or Cdk6 was assessed by
staining with anti-Cdk4 or anti-Cdk6 antibody detected with a
fluorescein-labeled secondary antibody and DNA stained with propidium
iodide and then analyzed by two-parameter flow cytometry (18). Cells
expressing elevated levels of Cdk4 or Cdk6 were analyzed for their DNA
content for cell cycle status.
Gel Filtration Analysis--
Control unirradiated cells (either
asynchronous or synchronized late S/G2 phase) or
UV-irradiated G2-delayed cells were lysed in NETN buffer as
described above, and the cleared supernatant (2 mg of protein) was
applied to a Superose 12 column (Amersham Pharmacia Biotech)
equilibrated in 20 mM Tris, pH 8, 2 mM EDTA, 1 mM dithiothreitol, and 150 mM NaCl. The column
was developed by fast protein liquid chromatography (Amersham Pharmacia
Biotech) at a flow rate of 0.25 ml/min, and 0.5-ml fractions were
collected. Fractions were either immunoblotted for p16, Cdk4, Cdk6, or
cyclin D3 or immunoprecipitated with anti-p16 and anti-Cdk4 antibodies and then analyzed by immunoblotting.
Increased p16-Cdk4 and p16-Cdk6 Complex Stability following UV
Exposure--
We have previously reported elevated levels of p16 in
response to low dose UV radiation, which correlated with G2
phase cell cycle delay (16, 17). Here we have investigated the
consequences of p16 accumulation in two cell lines: HeLa cells, which
are human pappiloma virus-transformed epithelial cells derived from an
adenocarcinoma, and A2058 cells, derived from a malignant melanoma. The
following experiments were conducted in both cell lines with
essentially identical results, and consequently, only the data obtained
with HeLa cells are shown in most cases.
To identify proteins associated with p16 in HeLa and A2058 cells,
[35S]Met/Cys-labeled cells were lysed, and p16-associated
complexes were immunoprecipitated and analyzed by SDS-polyacrylamide
gel electrophoresis. Three bands were specifically precipitated with the antibody in both control and UV-irradiated HeLa cells (Fig. 2A), which were identified by
immunoblotting as p16, Cdk4, and Cdk6 (Fig. 3A). No other
35S-labeled bands were found to be specifically associated
with p16 in either control or UV-irradiated samples. p53 accumulation following DNA damage involves an increase in p53 protein stability (22), and 35S pulse-chase experiments were performed to
investigate whether the UV-induced accumulation of p16 utilized a
similar mechanism. There was little difference in the half-life of p16
in asynchronously growing and UV-irradiated G2-delayed
A2058 and HeLa cultures, although p16 had a shorter half-life in A2058
cells (6 h; n = 1) compared with HeLa cells (9.5 ± 0.5 h; n = 3) (Fig. 2, B and C). Unexpectedly, the half-life of Cdk4 and Cdk6 associated
with the p16 immunoprecipitates increased markedly (2-3-fold) in the UV-irradiated cells compared with unirradiated controls in both cell
lines. The half-life of the p16-Cdk4 complex increased from 5 ± 1 h in the control HeLa cells to 12 ± 1 h in the
UV-irradiated cells (n = 3). The increased half-life of
the p16-Cdk6 complex was similar, although the very low levels of Cdk6
in HeLa cells precluded accurate estimates. The increased half-life of
p16-Cdk4 and p16-Cdk6 in control cells compared with irradiated A2058
cells was similar, from 2 to 3 h to 10 to 14 h, respectively.
This was not due to any increase in the half-life of the Cdk4 and Cdk6 proteins themselves (Fig. 2C), but suggested a decrease in
the rate of exchange of newly synthesized p16-bound Cdk4 and Cdk6 with
the unlabeled pools of Cdk4 and Cdk6, reflecting an increase in the
stability of the p16-Cdk4 and p16-Cdk6 complexes following UV
radiation. Although asynchronously growing cultures were used as
controls in these experiments, pulse-chase data from synchronized G2 cell-enriched cultures gave essentially the same results
(data not shown).
UV Radiation Increases p16 Association with Cdk4 and Cyclin
D--
The association of p16 with Cdk4 and Cdk6 was further
investigated by immunoblotting. In HeLa cells, the levels of Cdk4 and Cdk6 were unchanged throughout the cell cycle or following UV radiation, whereas p16 levels increased >4-fold following UV radiation (Fig. 3A), as shown previously
(16, 17). The sole cyclin D partner for Cdk4 in HeLa cells, cyclin
D3,2 was also expressed at a
constant level in these samples. The very low level of cyclin D3
associated with Cdk4 was confirmed by immunoblotting with two separate
anti-cyclin D3 monoclonal antibodies (see "Materials and Methods").
When Cdk4 immunoprecipitates from these same cell lysates were
immunoblotted for cyclin D3, no change in the level of this protein was
apparent, whereas the level of p16 associated with Cdk4 increased in
the UV-irradiated sample, paralleling the increase in total p16 levels
(Fig. 3B). Immunoblotting of p16 immunoprecipitates from
similar lysates also showed constant levels of cyclin D3
associated with p16 in each sample and no change in the Cdk4 levels
associated with p16 in the UV-irradiated cells (Fig. 3C), in
contrast to the Cdk4-associated p16 levels (Fig. 3B).
To further investigate the p16-Cdk4-cyclin D3 complexes, UV-irradiated
and control HeLa cultures were lysed and fractionated on a Superose 12 gel filtration column, and the fractions were immunoblotted for p16,
Cdk4, Cdk6, or cyclin D3. p16 eluted as two distinct peaks; the level
of p16 eluting in the higher Mr peak increased
>4-fold in the UV-irradiated sample compared with the control, whereas
the increased level of p16 in the lower molecular weight peak was more
modest, ~2-fold (Fig. 4, A
and B). Cdk4 eluted as a broad peak from fractions 13 to 20, and Cdk6 eluted as a tighter peak around fraction 15, whereas the
majority of the cyclin D3 was associated with very high molecular
weight complexes, peaking in fraction 4, although very low levels were
detected through to fraction 14 (data not shown). The distribution of
Cdk4, Cdk6, and cyclin D3 did not change discernibly between control
(either asynchronous or synchronized late S/G2 phase
cultures) and UV-irradiated samples. The p16 and Cdk4 complexes in
these fractions were analyzed by sequential immunoprecipitation with
anti-p16 antibody under conditions that immunodepleted p16 and then
immunoprecipitated with anti-Cdk4 antibody to examine the Cdk4
complexes not containing p16. Both p16 and Cdk4 immunoprecipitates were
immunoblotted for p16, Cdk4, and cyclin D3. p16 immunoprecipitates of
the gel filtration fractions revealed that p16 was present in three
different complexes: with cyclin D3 and Cdk4 (fractions 13-15), a
complex with Cdk4 only (fractions 17-19) (Fig. 4, C and
D), and a monomer, as p16 immunoprecipitates from fractions
20-22 contained no Cdk4 or Cdk6 (data not shown). The level of cyclin
D3, Cdk4, and p16 detected in the p16 immunoprecipitate from fraction
13 was increased 2-fold in the UV-irradiated sample compared with the
late S/G2 phase control (Fig. 4, C and
D). Reciprocal Cdk4 immunoprecipitation and p16
immunoblotting of equivalent fractions in a separate experiment revealed the same increase of the p16-Cdk4-cyclin D3 complex in UV-irradiated samples (data not shown). Cdk4 immunoprecipitates from
the p16-immunodepleted fractions (demonstrated by the absence of
detectable p16 in the Cdk4 immunoprecipitates) (Fig. 4C)
revealed a higher level of cyclin D3 associated with Cdk4 (fractions 13 and 15) in the control compared with the irradiated samples. The relative intensity of the Cdk4 and cyclin D3 bands in the p16 and Cdk4
immunoprecipitates indicated that the majority of Cdk4 and cyclin
D3-Cdk4 was associated with p16, with only small pools of cyclin
D3-Cdk4 and Cdk4 (fraction 17, anti-Cdk4 immunoprecipitate) (Fig.
4C) not associated with p16, and that these free pools were reduced following UV exposure (Fig. 4C).
In an effort to identify novel p16-containing complexes in either
control or UV-irradiated cells, earlier eluting, higher molecular
weight fractions (fractions 1-4 and 5-8) were pooled, and the
presence of p16 was examined by immunoprecipitating with anti-p16
antibody from the pooled fractions and immunoblotting with the same
antibody. No p16 was detected in the earlier eluting fractions from
either control or irradiated samples.
A Cdk4 Activity in Late S/Early G2 Phase Is Inhibited
following UV Radiation--
The previous experiments showed that a
consequence of the increased p16 levels in the UV-irradiated cells was
an increase in the p16-Cdk4-cyclin D3 complex. This suggested that
cyclin D3-Cdk4 may be active in the late S/early G2 phase
of normally cycling cells and inhibited in the UV-irradiated
G2-delayed cells. The presence of such an activity was
assessed in HeLa cell cultures synchronized using a thymidine
block/release protocol. Cells were collected at times up to 14 h
post-release as they progressed from G1/S through mitosis
into the next G1 phase (Fig.
5). Cdk4 activity at each time point was
examined using a Cdk4 immunoprecipitate kinase assay with
GST-Rb-(733-928) as a substrate. A kinase activity associated with the
Cdk4 immunoprecipitates was detected as cells commenced entering
G2 phase (6 h), peaked at 8 h, and then decreased again as the cells existed mitosis (11 h). The peak of activity correlated with the shoulder of
p9CKSHs1-Sepharose-precipitable H1 kinase activity in
G2, prior to the major peak of H1 kinase activity in
mitosis (10 h). The immunoprecipitated kinase activity was shown to be
due to a Cdk4 complex, as nonreactive serum or immune peptide-blocked
anti-Cdk4 antibody immunoprecipitated only background Rb kinase
activity from late S/G2 phase HeLa cell lysates, and no
phosphorylated bands were detected in immunoprecipitates assayed
without addition of the Rb substrate (Fig.
6A). Furthermore, the
Cdk4-associated activity was not inhibited by 50 µM
olomoucine, a strong inhibitor of Cdk2 and Cdc2 kinases, but relatively
ineffective against Cdk4 (23), which resulted in 80% inhibition of
immunoprecipitated Cdk2 activity (Fig. 6, A and
B). When the Cdk4 activity in UV-irradiated G2-delayed samples was tested, it was found to be inhibited
in both HeLa and A2058 cells when compared with the respective cell cultures enriched for S, late S/G2, and M phase cells (Fig.
6C). In HeLa cells, Cdk4 activity was decreased by 60% in
the UV-irradiated cells compared with the G2 cell-enriched
cultures (Fig. 6D).
Activation and Phosphorylation of Cdk4 Are Correlated with Exit
from UV-induced G2 Delay--
We have previously shown
that the UV-induced G2 phase delay results in a block in
the Cdc25-dependent activation of the mitotic cyclin-Cdk
complexes in G2 and M (15). Here we have demonstrated that
the normal activation of cyclin D3-Cdk4 in early G2 phase was inhibited during the UV-induced G2 delay and that
cyclin D3-Cdk4 activation occurred in G2 prior to the
activation of cyclin B-Cdc2 at the G2/M transition (Fig.
7A). This suggested a possible
causal relationship between cyclin D3-Cdk4 activation and the
activation of the cyclin-Cdk complexes later in G2. This
relationship was examined in UV-irradiated HeLa cells induced to
progress through the G2 delay into mitosis with 5 mM caffeine (24), and the activities of Cdk4 and cyclin B1
kinases were measured. Addition of caffeine resulted in a rapid exit
from the G2 delay, and cells commenced accumulating in
G1 by 2 h following caffeine addition (Fig.
7B). Cdk4 activity was almost maximal within 1 h
following caffeine addition to the culture medium and then started to
decline by 3 h, whereas cyclin B1-Cdc2 activity was not maximal
until 3 h following caffeine addition (Fig. 7C). Thus,
the block of Cdk4 activation was relieved by the addition of caffeine,
was correlated with continued progression through G2, and
preceded cyclin B1-Cdc2 activation at the G2/M
transition.
We earlier demonstrated that the level of p16 associated with Cdk4
increased in UV-irradiated cells (Fig. 3B). The binding of
p16 to Cdk4 complexes after caffeine release was examined to see
whether the activation of Cdk4 correlated with decreased p16 association. By 1 h post-caffeine addition, the increased level of
p16 associated with Cdk4 complexes in the UV-irradiated cells had
returned to control levels, correlating with the activation of cyclin
D3-Cdk4 (Fig. 7, C and D). The levels of p16 did
not change discernibly during this time (data not shown) (16),
indicating that the reduced binding of p16 is not simply due to
reduction in the level of p16.
The phosphorylation status of Cdk4 was examined to determine whether
the activation of Cdk4 might involve a phosphorylation event. Cdk4
immunoprecipitates from metabolically
32Pi-labeled HeLa cells, either UV-irradiated
G2-delayed cells or at 1 h post-caffeine addition,
revealed a 1.8-fold increase in the phosphorylation of Cdk4 (Fig.
8A). Interestingly, p16
immunoprecipitates from equivalent samples did not contain any labeled
bands, although immunoblotting of 10% of these immunoprecipitates for
Cdk4 showed that all contained similar levels of Cdk4 protein (data not
shown). In a separate experiment, a 2-fold difference in the
phosphorylation state of Cdk4 was observed in Cdk4 immunoprecipitates
from UV-irradiated and synchronized late S/G2 cells (Fig.
8B). Again, no labeled bands were detected in anti-p16
immunoprecipitates. No phosphorylation of p16 was detected under any
condition.
Cdk4 Activity Is Required for Progression through G2
into Mitosis--
The data presented suggest a role for Cdk4 activity
in cell cycle progression through early G2 into mitosis. To
directly test whether Cdk4 is involved in cell cycle progression, the
dominant-negative Cdk4 D158N mutant was overexpressed in HeLa cells,
and the cell cycle status of the overexpressing population was assessed
by flow cytometry. Experiments using the equivalent mutants of Cdc2 and
Cdk2 have demonstrated the cell cycle transitions regulated by these
cyclin-dependent kinases (20). To obtain sufficiently high
levels of Cdk4 overexpression for the mutant to exert a
dominant-negative effect, the Cdk4 was cotransfected with mouse CDC37,
which increased the stability of the overexpressed Cdk4 protein (21).
Cotransfection of Cdk6 with CDC37 had little effect on Cdk6 expression,
as reported previously (21). Immunoblotting for Cdk4 and Cdk6 revealed
that wild-type and mutant versions of both proteins were strongly
expressed, with the ectopically expressed proteins being
distinguishable from the endogenous forms due their slightly retarded
electrophoretic mobility, a result of the HA tag (Fig.
9A). Cells overexpressing Cdk4
and Cdk6 were discriminated on the basis of increased Cdk4 or Cdk6
staining using flow cytometry, and the cell cycle distribution of the
overexpressing population was determined. The wild-type Cdk4-overexpressing population showed a small increase in the G2/M population compared with control vector-transfected
cells, but the mutant-overexpressing cells showed a 1.5-fold
accumulation of G2/M phase cells over the wild
type-overexpressing cells (Fig. 9B). Inspection by
immunofluorescent microscopy of transfected cells stained for ectopic
Cdk4 expression (HA tag) and endogenous cyclin B1 showed that a higher
proportion of cells strongly overexpressing the Cdk4 mutant contained
high levels of cytoplasmic cyclin B1, an indicator of G2
phase (25), compared with the untransfected population, indicating that
mutant Cdk4 overexpression resulted in accumulation of G2
phase cells. As a further control for the specificity of Cdk4 action,
wild-type and dominant-negative mutant forms of Cdk6 were also
transfected into HeLa cells. Cells overexpressing either form of Cdk6
produced a cell cycle profile similar to that produced by the wild-type
Cdk4-overexpressing cells. Thus, the increased accumulation of
G2 phase cells observed with mutant Cdk4 overexpression was
due to the specific inhibition of a Cdk4-dependent activity
and provides further evidence for a Cdk4-dependent step in
G2 phase cell cycle progression.
Cyclin D-Cdk4 complexes have been reported to exist throughout the
cell cycle in many cell types (26, 27), although the function of these
complexes through the cell cycle, apart from regulating the
Rb-dependent G1 checkpoint, is unknown. Here we have demonstrated that cyclin D3-Cdk4 complexes are present throughout the cell cycle and that this complex is activated in late S/early G2 phase in both HeLa and A2058 cells. The presence of a
Cdk4 activity in G2 phase was first indicated by Matsushime
et al. (27), who showed, in mouse fibroblasts overexpressing
Cdk4 and cyclin D, peaks of Cdk4 activity both in G1 and
G2 cell-enriched fractions. In the cell lines tested here,
little G1 phase Cdk4 activity was detected (the low level
of Cdk4 activity immunoprecipitated from lysate of asynchronous
cultures represents Cdk4 activity from >60% G1 phase
cells). The absence of G1 phase Cdk4 activity in
Rb-defective cell lines, a consequence of human pappilloma virus-E7
expression in HeLa cells and loss of Rb expression in A2058
cells,2 has been reported by others (28, 29).
The high levels of heterodimeric p16-Cdk4 and p16-Cdk6 complexes and
monomeric p16 detected in HeLa and A2058 cells are also a common
feature of Rb-defective cell lines (30, 31). In both HeLa and A2058
cells, only a small proportion of Cdk4 associated with cyclin D3, in
agreement with other studies that showed that high levels of p16
expression reduce cyclin D binding to Cdk4 (30, 31).
In response to UV exposure, p16 association with Cdk4 is elevated,
presumably a result of both increased p16 levels and the increased
stability of p16-Cdk4 and p16-Cdk6 complexes. A clear consequence of
the increased association of p16 with Cdk4 complexes is the inhibition
of cyclin D3-Cdk4 activity. This was correlated with decreased
phosphorylation of Cdk4 compared with either normal early
G2 phase cells or cells released from the UV-induced block with caffeine. The phosphorylation detected is likely to be at the
activating Thr-172 on Cdk4 catalyzed by CAK (32), which has been
demonstrated to be blocked by p16 in vitro (33). The absence
of phosphorylated Cdk4 in p16 immunoprecipitates fits well with the
proposal that the increased p16 association is inhibiting Thr-172
phosphorylation of Cdk4 and thereby blocking its activation.
The observed UV-induced increase in the stability of the p16-Cdk4 and
p16-Cdk6 complexes is unexpected. A recent mutational analysis of Cdk4
examining residues involved in p16 binding may provide a mechanism by
which this increased complex stability is acquired. This study
demonstrated that mutation of Thr-172 to the nonphosphorylatable Ala
residue increased p16 binding >2-fold (34). The crystal structure of
the p16-Cdk6 complex revealed the T-loop containing Thr-172 to lie
close to the first ankyrin repeat of p16 (35), and the presence of a
highly charged phosphoryl group at Thr-172 is likely to alter the
binding avidity of p16. Our findings that increased p16 binding
correlated with decreased Cdk4 phosphorylation in UV-irradiated cells
and that decreased p16 binding following caffeine-induced release from
the G2 block correlated with increased Cdk4 phosphorylation
support a mechanism where a decrease in Cdk4 Thr-172 phosphorylation
may increase p16 binding, which in turn will block the
rephosphorylation of Thr-172 by CAK. It is not clear how caffeine
changes the balance of phosphorylation and p16 binding so rapidly, and
this whole mechanism will require further study.
The decreased phosphorylation of Cdk4 during the G2 delay
following UV exposure, in particular the absence of any phosphorylation of p16-bound Cdk4, differentiates this Cdk4-dependent cell
cycle block from a Cdk4-dependent G1 phase
block also initiated by low dose UV radiation (36). It was demonstrated
that Cdk4 was phosphorylated at Tyr-17 in response to UV radiation of
cells in early G1 phase and that this blocked
G1 phase progression (36). However, this mechanism appeared
to be specific for the G1 block, and cells expressing
wild-type Cdk4 or a nonphosphorylatable Cdk4 Y17F mutant were equally
susceptible to DNA damage and displayed similar survival when
irradiated as they entered S phase, suggesting that later cell cycle
checkpoints did not utilize this mechanism.
A lot of attention has been given to the role of cyclin D-Cdk4
complexes in regulating the G1 phase Rb function, and there is currently little evidence of a role for these complexes later in the
cell cycle. Much of the work on cyclin D has focused on cyclin D1, and
although there is some degree of functional redundancy between the
D-type cyclins, it becoming evident that all D-type cyclins are not
equivalent. For example, overexpression of cyclin D1 (but not cyclin
D3) accelerated G1 progression in fibroblasts (27), and
overexpression of cyclins D2 and D3 (but not cyclin D1) inhibited
granulocyte differentiation (37). In estrogen-stimulated MCF-7 breast
cancer cells, cyclin D1 transcript and protein levels correlated with
G1 phase progression, whereas expression of cyclin D3
message and protein was delayed, with cyclin D3 protein reaching maximal levels as cells progressed into G2 phase (19).
Substrate specificity differences have also been found between the
cyclin D1-Cdk4 and D3-Cdk4 complexes in vivo and in
vitro (38). Thus, it is likely that different cyclin D-Cdk4
complexes may have distinct functions, dependent on the cell type in
which they are expressed and the timing of their expression and
activation during the cell cycle.
The existence of a G2 phase cyclin D-Cdk4 activity would
also imply a regulatory mechanism to control this activity. The
constitutive presence of the cyclin D3-Cdk4 complex throughout the cell
cycle precludes cyclin-Cdk complex formation; thus, activity must be regulated at the level of phosphorylation and/or CKI interaction, which
may act in concert to ensure very tight regulation. Agents or
conditions that increase CKI levels at a particular point in the cell
cycle presumably utilize these inhibitors to block cell cycle
progression beyond that point until this inhibition is relieved. There
are many reports demonstrating that overexpression of p16 inhibits
cyclin D-Cdk4-dependent phosphorylation of Rb and
G1 phase progression (29, 39, 40) and proliferation in an
Rb-dependent manner (29, 41, 42). Small increases in
endogenously expressed p16 protein were found as serum-stimulated cells
entered S phase (43) and at the S/G2 transition in
synchronized HeLa cells (17), although these were not correlated with
discernible cell cycle delays. Much larger increases in p16 levels have
been found in senescing cells, correlating with inhibition of the
G1 and S phase cyclin/cyclin-dependent kinases
(44), and as a delayed response to low doses of UV radiation,
correlating with a G2 delay (16, 17) and inhibition of
cyclin D3-Cdk4 (this report). The UV-induced elevated p16 levels appear
to be a specific response to UV-induced damage, as other genotoxic
agents that produce a G2 delay in HeLa and A2058 cells,
e.g. cisplatin and ionizing radiation, do not affect p16
protein levels.3
Interestingly, a recent report has provided evidence for a
p16-dependent checkpoint in G1 in response to a
wide variety of DNA-damaging agents in p53-deficient cell lines (45).
This checkpoint is dependent on Rb and Cdk4, although the levels of p16
appeared unchanged in this response, differentiating it from the UV
response investigated here, which appears to be independent of Rb function.
The data reported here provide evidence for a cyclin D-Cdk4 activity in
late S/G2 phase that appears to be required for normal cell
cycle progression through G2 phase into mitosis. This Cdk4 activity is inhibited in response to UV radiation, and inhibition of
this activity appears to be involved in the G2 phase delay observed. It will be of interest to see whether this role for p16 in a
G2 checkpoint is UV-specific or a more general cell cycle checkpoint control. It may also provide a mechanistic basis for the
strong genetic and epidemiological evidence linking UV radiation, p16CDKN2A function and melanoma.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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2 UVC in 2 ml of prewarmed phosphate-buffered
saline and then refed fresh medium. G2-delayed cells were
obtained by harvesting at 24 and 20 h post-irradiation for HeLa
and A2058 cells, respectively. The cell cycle status was confirmed by
flow cytometry of the DNA content. See Fig.
1 for typical cell cycle profiles of the
synchronized and UV-irradiated cultures.
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Fig. 1.
Fluorescence-activated cell sorter profiles
of HeLa cell cycle distribution of asynchronous (AS),
thymidine block release synchronized S (S) and late
S/G2 (G2), and UV-irradiated cultures
24 h post-exposure (UV).
-32P]ATP for 30 min. The reactions were
stopped by the addition of SDS sample buffer and boiled for 2 min, and
then the total reaction was separated on 10% SDS-polyacrylamide gel,
stained with Coomassie Blue, dried, and autoradiographed. Cyclin B1
kinase assays were performed as described previously (15). The activity
of the immunoprecipitated kinases was quantitated by phosphoimaging of the phosphorylated GST-Rb-(773-928) and histone H1 bands.
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Fig. 2.
UV radiation increases the stability of the
p16-Cdk4 and p16-Cdk6 complexes. A, lysates of either
control asynchronous or UV-irradiated G2-delayed HeLa cells
metabolically labeled with [35S]methionine and cysteine
were immunoprecipitated with either anti-p16 antibody or antibody
preincubated with the immunogen peptide, and the immunoprecipitates
were analyzed on 15% SDS-polyacrylamide gel. The positions of p16,
Cdk4, and Cdk6 are indicated. B and C, shown are
the relative levels of 35S-labeled p16, Cdk4, and Cdk6
associated with p16 immunoprecipitates during a pulse-chase experiment
of labeled A2058 and HeLa cultures. D, shown are the levels
of 35S-labeled Cdk4 and Cdk6 immunoprecipitated with their
respective antibodies from the same HeLa pulse-chase experiment as
C. The intensity of the labeled bands is in arbitrary units.
con, control.
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Fig. 3.
Level of p16 associated with Cdk4 increases
following UV exposure. Shown are the levels of cyclin D3, Cdk4,
Cdk6, and p16 in total lysates (A), associated with
anti-Cdk4 (B) or anti-p16 (C) immunoprecipitates,
and from synchronized or UV-irradiated HeLa cell cultures. The
specificity of the Cdk4 and cyclin D3 association with p16 was
demonstrated by preincubating anti-p16 antibody with the immunogen
peptide (UV + pep).
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Fig. 4.
Increased levels of p16 bound to cyclin
D3-Cdk4 complexes after UV exposure. Asynchronous control
( UVC) or UV-irradiated G2-delayed
(+UVC) HeLa cell lysates were fractionated on a Superose 12 gel filtration column, and the indicated column fractions were
immunoblotted for p16 (A). The level of p16 in the fractions
was quantitated by laser densitometry of the immunoblots
(B). In C, control synchronized late
S/G2 phase (G2) or UV-irradiated (UV)
HeLa cell lysates were fractionated as described for A.
Equivalent fractions were sequentially immunoprecipitated with anti-p16
and then anti-Cdk4 antibodies, and the immunoprecipitates
(IP) were immunoblotted for cyclin D3 (cyc D3),
Cdk4, and p16. The anti-p16 and anti-Cdk4 immunoprecipitates were
exposed together for the same period to demonstrate the relative levels
of Cdk4 and cyclin D3 complexed with p16 and not associated with p16.
The upper band in the UV-treated cyclin D3 blot is
cross-reactive IgG heavy chain. The shorter exposure of the immunoblots
shown in C shows the relative increase in cyclin D3 and p16
levels in anti-p16 immunoprecipitates from UV compared with control
samples (D).
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Fig. 5.
A Cdk4 activity is present in normally
cycling late S/G2 phase cells. HeLa cells were
synchronized in early S phase with a thymidine block and then released.
Cells were harvested at the indicated times after release and analyzed
for cell cycle status by flow cytometry and for Cdk4 activity by Cdk4
immunoprecipitate kinase assay using GST-Rb-(773-928)
(GT-Rb) as substrate or for Cdk2 and Cdc2 kinase activity
precipitated by p9CKSHs1-Sepharose using histone H1 as
substrate.
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Fig. 6.
G2 phase Cdk4 activity is
inhibited in UV-irradiated G2-delayed cells. A,
late S/G2 phase HeLa cell lysates were immunoprecipitated
with nonimmune serum (NI) or anti-Cdk4 or anti-Cdk2
antibody, and the immunoprecipitates were assayed either with or
without substrate and 50 µM olomoucine (Olo.)
as indicated. B, the level of Cdk4 and Cdk2
immunoprecipitate activity is expressed as a percentage of the
minus-olomoucine control. C, Cdk4 immunoprecipitate Rb
kinase activity from the indicated synchronized and UV-irradiated HeLa
and A2058 cells is shown. D, the Cdk4 Rb kinase activity in
UV-irradiated or control late S/G2 phase HeLa cells is
expressed as a percentage of the control from four separate
experiments. GT-Rb, GST-Rb-(773-928).
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Fig. 7.
Caffeine-induced release from the
G2 phase delay is correlated with increased Cdk4 activity
and decreased p16 binding. A, shown is the cyclin B1 and
Cdk4 immunoprecipitate kinase activity from asynchronous
(AS), UV-irradiated G2-delayed (UV),
late S/G2 (G2), and mitotically enriched
(M) HeLa cells. Phosphorylated GST-Rb-(773-928) is
indicated (arrowhead). B, UV-irradiated
G2-delayed HeLa cultures were treated with 2 mM
caffeine (time 0), the cells were harvested at the indicated times, and
their DNA content was analyzed by flow cytometry for cell cycle status.
C, lysates from equivalent samples were assayed for Cdk4 and
cyclin B1 immunoprecipitate kinase activity (C). The
activities are expressed as a percentage of their peak activity.
D, the level of p16 bound to Cdk4 from asynchronous,
UV-irradiated G2-delayed, and UV-irradiated cultures either
1 or 3 h post-caffeine (caff.) addition was assessed by
immunoblotting anti-Cdk4 immunoprecipitates for Cdk4 (as a loading
control) and p16.
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Fig. 8.
UV exposure decreases the phosphorylation of
Cdk4. A, UV-irradiated HeLa cells 22 h post-exposure
were labeled for 2 h with 32Pi in
phosphate-free medium; 5 mM caffeine (caff) was
added to half the plates and incubated an additional 1 h, and the
cells were then harvested. Lysates prepared from these samples were
initially incubated with nonimmune serum (NI) and then
either anti-Cdk4 or anti-p16 antibody. The immunoprecipitates were
washed and analyzed on 15% SDS-polyacrylamide gel. The position of
32P-labeled Cdk4 is indicated (arrowhead).
B, cultures of UV-irradiated cells 22 h post-exposure
and synchronized late S/G2 phase cells containing similar
numbers of cells were labeled for 3 h and processed as for
A. In this experiment, the UV sample was incubated with
anti-p16 antibody or with anti-p16 antibody preincubated with the p16
peptide to block immunoreactivity (see Fig. 2A). The
positions of molecular weight markers (×103) are shown,
and the 32P-labeled Cdk4 band is indicated
(arrowhead).
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Fig. 9.
Overexpression of a dominant-negative form of
Cdk4 produces a G2 phase delay. A, HeLa cells
were transiently transfected with either a control vector (SR
con) or vector containing the indicated HA-tagged wild-type
(wt) or dominant-negative mutant (D/N) Cdk4 or
Cdk6 cDNA. Cells were harvested and immunoblotted with anti-Cdk4
and anti-Cdk6 antibodies. The ectopically expressed proteins are
distinguished from the endogenous versions by their slower
electrophoretic mobility due the HA tag. B, cells
transfected as described for A were stained with either
anti-Cdk4 or anti-Cdk6 antibody detected with a fluorescein-labeled
secondary antibody and with propidium iodide for DNA. The Cdk4- and
Cdk6-overexpressing cells were gated as the cells with elevated
fluorescein signal over the empty vector control levels (Cdk
expression), and the DNA content of the gated overexpressing population
(population above the cutoff on dot plots on the left) is
shown in the histograms on the right. The vector control is
the total DNA histogram. The percentages of cells in each cell cycle
stage are mean ± S.D. from three separate experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. E. Harlow for the Cdk4 and Cdk6 expression plasmids; Dr. W. Harper for the CDC37 expression plasmid; G. Chojnowski for advise with fluorescence-activated cell sorter analysis; and Drs. S. Goldstone, N. Hayward, and G. Vairo for helpful discussions and encouragement during the course of this work.
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FOOTNOTES |
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* This work was supported in part by grants from the Queensland Cancer Fund and the National Health and Medical Research Council.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.
§ Recipient of an Australian Research Council fellowship. To whom correspondence should be addressed: Queensland Inst. of Medical Research, P. O. Royal Brisbane Hospital, Queensland 4029, Australia. Fax: 61-7-3362-0107; E-mail: brianG{at}qimr.edu.au.
2 X. Q. Wang and B. G. Gabrielli, unpublished observations.
3 S. Goldstone, J. Sinnamon, S. Pavey, and B. G. Gabrielli, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: GST, glutathione S-transferase; HA, hemagglutinin.
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