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Oncogene (2000) 19, 4480 ± 4490
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UVB induced cell cycle checkpoints in an early stage human melanoma line,
WM35
T Petrocelli1 and J Slingerland*,1
1
Division of Cancer Biology Research, Toronto Sunnybrook Regional Cancer Centre, Sunnybrook and Women's College Health
Sciences Centre and University of Toronto, Toronto, Ontario, Canada
The activation of cell cycle checkpoints in response to
genotoxic stressors is essential for the maintenance of
genomic integrity. Although most prior studies of cell
cycle e€ects of UV irradiation have used UVC, this UV
range does not penetrate the earth's atmosphere. Thus,
we have investigated the mechanisms of ultraviolet B
(UVB) irradiation-induced cell cycle arrest in a
biologically relevant target cell type, the early stage
human melanoma cell line, WM35. Irradiation of WM35
cells with UVB resulted in arrests throughout the cell
cycle: at the G1/S transition, in S phase and in G2. G1
arrest was accompanied by increased association of p21
with cyclin E/cdk2 and cyclin A/cdk2, increased binding
of p27 to cyclin E/cdk2 and inhibition of these kinases.
A loss of Cdc25A expression was associated with an
increased inhibitory phosphotyrosine content of cyclin Eand cyclin A-associated cdk2 and may also contribute to
G1 arrest following UVB irradiation. The association of
Cdc25A with 14-3-3 was increased by UVB. Reduced
cyclin D1 protein and increased binding of p21 and p27
to cyclin D1/cdk4 complexes were also observed. The
loss of cyclin D1 could not be attributed to inhibition of
either MAPK or PI3K/PKB pathways, since both were
activated by UVB. Cdc25B levels fell and the remaining
protein showed an increased association with 14-3-3 in
response to UVB. Losses in cyclin B1 expression and an
increased binding of p21 to cyclin B1/cdk1 complexes
also contributed to inhibition of this kinase activity, and
G2/M arrest. Oncogene (2000) 19, 4480 ± 4490.
Keywords: UVB; cell cycle; checkpoint; DNA damage;
Cdc25A
Introduction
Much of what is known about the response of cells to
DNA damage has arisen from studies employing
ionizing radiation or ultraviolet C irradiation (UVC;
200 ± 290 nm). Far less is known about the e€ects of
UVA (320 ± 400 nm) and UVB (290 ± 320 nm), the
biologically relevant spectra that can penetrate the
earth's ozone and are implicated in skin carcinogenesis.
Maintaining genomic integrity during cellular proliferation is essential for the continued viability of the
organism. When cells bypass the normal restrictions on
*Correspondence: J Slingerland, Division of Biology Research,
Sunnybrook & Women's College Health Sciences Centre, Research
Building, S-218, 2075 Bayview Avenue, Toronto, Ontario M4N 3M5,
Canada
Received 27 July 1999; revised 10 July 2000; accepted 17 July 2000
entrance into S phase imposed by DNA damage, the
replication of damaged DNA can either result in cell
death or an accumulation of genetic changes leading
ultimately to cancer (Hartwell and kastan 1994). In
mammalian cells, progression through the cell cycle is
regulated by the ordered formation, activation, and
subsequent inactivation of a series of protein kinase
complexes, the cyclin dependent kinases or cdks
(reviewed in Morgan, 1995; Reed et al., 1994; Sherr,
1994). Cdks coordinate cell cycle checkpoints, a series
of biochemical pathways that ensure that the initiation
of cell cycle events occur only after successful
completion of others (Elledge, 1996; Hartwell, 1992;
Paulovich et al., 1997). Passage from G1 into S phase
requires the activation of cyclin D1-associated cdk4
and cdk6 (Baldin et al., 1993; Meyerson and Harlow,
1994) in addition to cyclin E/cdk2 (Dulic et al., 1992),
both of which contribute to phosphorylation of the
retinoblastoma protein, pRb (Dowdy et al., 1993;
Ewen et al., 1993; Kato et al., 1993). Cyclin A/cdk2
activation is essential for S phase progression (Fang
and Newport, 1991; Giordano et al., 1989) and both
cyclinA/cdc2 and cyclinB/cdc2 are required for the
transitions through G2/M (Girard et al., 1991; Pagano
et al., 1992).
For full activation, a cdk must bind its cyclin partner
and also undergo both activating phosphorylation and
dephosphorylation of inhibitory sites. Activation of
cdk2 requires phosphorylation of threonine 160 (thr
160) which is catalyzed by cdk activating kinase (CAK)
(Gu et al., 1992; Solomon and Kaldis, 1998).
Phosphorylation of thr 14 and/or tyrosine 15 (tyr15)
residues by human homologues of the wee1 and myt1
kinases inactivates cdk1 and homologous sites exist on
cdk2 and cdk4 (Gould and Nurse, 1989; Krek and
Nigg, 1991; McGowan and Russell, 1995; Parker et al.,
1995) and reviewed in (Lew and Kornbluth, 1996). The
Cdc25 family of phosphatases, which includes Cdc25A,
Cdc25B, and Cdc25C, remove the inhibitory phosphates on cdks. Cdc25A activates cyclin E-bound cdk2
and is essential for the G1-to-S phase transition, while
Cdc25B and Cdc25C act at the G2/M transition
(Draetta and Eckstein 1997).
The cdk inhibitors provide a further level of cyclin/
cdk regulation (Sherr and Roberts, 1995, 1999; Reed et
al., 1994). Two cdk inhibitor families have been
identi®ed: the KIP family which includes p21, p27,
and p57, and the INK4 family which includes p15, p16,
p18 and p19. While the latter are restricted in
speci®city, binding only to cdk4 or cdk6, the KIP
family bind a wider range of cyclin/cdks. The cdk
inhibitors inhibit cell cycle progression in response to
growth inhibitory signals including DNA damage.
UVB induced cell cycle checkpoints
T Petrocelli and J Slingerland
Most cells respond to DNA damage with delays in
cell cycle progression. This checkpoint allows repair of
damaged DNA prior to replication, thus ensuring the
®delity of genome transmission from one generation to
the next (Kaufmann et al., 1991; Lau and Pardee, 1994;
Li and Deshaies, 1993). Exposure to low doses of UV
light causes delays throughout the cell cycle, that are
both dose and cell type dependent (Barker et al., 1995;
Lu and Lane, 1993; Petrocelli et al., 1996; Poon et al.,
1996; Wang and Ellem, 1994). In mammalian cells
DNA damage by gamma, UVB and UVC irradiation,
causes an increase in p53, leading to induction of p21
gene expression, cyclin/cdk inhibition and G1 arrest
(Kastan et al., 1992; Liu and Pelling, 1995; Lu and
Lane, 1993; Petrocelli et al., 1996). UV irradiation has
been shown to cause an increase in binding of both p21
and p27 to cyclin E/cdk2 complexes (Petrocelli et al.,
1996; Poon et al., 1996). Furthermore, G1 arrest
following UVC may involve an increase in inhibitory
phosphorylation of cdk4 (Terada et al., 1995). An
increase in inhibitory tyrosine phosphorylation of cdc2,
resulting from loss of Cdc25C activity, has been
implicated in the G2/M cell cycle arrest in response
to DNA damage in both ®ssion yeast (Enoch and
Nurse, 1990; Rhind et al., 1997) and human cells
(Blasina et al., 1997; Jin et al., 1996). Recent work has
suggested a model whereby DNA damage activates
human Chk1 kinase which phosphorylates Cdc25C on
Ser216, leading to its association with 14-3-3 and
functional inactivation (Furnari et al., 1997; Peng et
al., 1997; Sanchez et al., 1997). In Xenopus and ®ssion
yeast, the binding of Cdc25 to 14-3-3 results in
cytoplasmic sequestration of Cdc25 away from its
target cyclin B/cdc2 (Kumagai and Dunphy, 1999;
Lopez-Girona et al., 1999; Yang et al., 1999). UV
induced G2 delays involve not only a block in the
Cdc25-dependent activation of cyclin B/cdc2 (Herzinger et al., 1995; Poon et al., 1996), but also that of
cyclin A/cdk2 and cyclin A/cdc2 (Gabrielli et al., 1997).
The central role of UVB in the induction of human
cutaneous malignancies and skin ageing has prompted
an examination of the speci®c cell cycle responses to
UVB exposure. To identify the cellular e€ects of this
genotoxic stressor, we have used a human melanoma
cell line derived from early stage disease, WM35.
Irradiation of WM35 cells induced arrests throughout
all phases of the cell cycle, in G1, S phase, and G2/M.
We demonstrate the involvement of p21 and p27 not
only in G1 arrest, but also in S and G2/M checkpoints
and show that UVB-mediated G1 arrest is associated
with a reduction of Cdc25A and its increased
association with 14-3-3.
Results
UVB irradiation induces checkpoints in G1, S phase and
G2/M
To examine the e€ect of UVB on asynchronously
growing WM35, cells were harvested at di€erent time
points following irradiation with 9 mJ/cm2 UVB. Flow
cytometry at di€erent intervals following irradiation
showed a prolonged G1 arrest (Figure 1). Inhibition of
G1-to-S phase progression was noted as early as 3 h
post-UVB (data not shown). The proportion of cells in
S phase fell from 33 to 5% at 12 h post-UVB, with a
nadir of 2 ± 3% S phase cells by 18 ± 24 h. Recovery of
G1-to-S phase progression was seen between 24 and
36 h. Cells that had a DNA content between 2N and
4N before irradiation failed to progress into G2,
indicating inhibition of replicative DNA synthesis, or
S phase arrest (Figure 1). A functional G2/M arrest
could be seen by the failure of cells in G2/M at the
time of irradiation to exit and enter into the next G1 at
subsequent time points (Figure 1).
To examine more speci®cally the G1-to-S phase
checkpoint, cells were synchronized by serum starvation and then released into the cell cycle by addition of
complete medium. Progression from G0 into S phase
was detected within 12 h in the control non-irradiated
cells. Cells irradiated in early G1, 4 h after release from
G0, showed no S phase entrance for up to 20 h postirradiation (Figure 2). Recovery of G1-to-S phase
progression was detected by 24 ± 36 h post irradiation
(not shown).
To determine the duration of the S phase arrest
following UVB irradiation, asynchronously growing
WM35 cells were pulsed with 10 mM BrdU for 2 h at
time T=0. BrdU labelling was followed immediately
by irradiation with a single dose of UVB, and cells
were then recovered at di€erent times thereafter for
¯ow cytometry (Figure 3, right column). Control, nonirradiated cells were also recovered at the same time
points after the initial BrdU pulse (Figure 3, left
column). Non-irradiated control WM35 cells that had
taken up BrdU exponentially during the pulse labelling
(T=0), proceeded through S phase and began entrance
into G2/M by 3 h (Figure 3). Within 9 h, most nonirradiated cells that were in S phase at time T=0 had
progressed into G2/M and some had already entered
G1. By 12 h, most of the BrdU-labelled cells had
moved into G1. Some re-entrance into S phase was
appreciable by 18 h. In contrast, cells that were BrdU
labelled and then irradiated at 0 h exhibited a
prolonged S phase arrest and no progression into the
G2 phase was observed for up to 12 h after UVB
irradiation (Figure 3).
The duration of G2 arrest post-UVB was assayed in
a manner similar to the S phase checkpoint above.
Following a 2 h BrdU pulse to label cells in S phase,
cells were chased into fresh media and irradiated (or
sham-irradiated for controls) 5 h later, at a time when
most of the BrdU-labelled cells had entered G2. The
progress of BrdU-labelled control non-irradiated cells
into the subsequent G1 phase can be seen on the left
column in Figure 4. In contrast, cells irradiated in G2
failed to exit G2/M and there was no progression of
these cells into G1 during the chase period for up to
13 h after-irradiation.
4481
Changes in expression of cell cycle regulators following
UVB irradiation
We and others have shown an increase in the level of
p53 protein followed by an increase in p21 expression
after UVB irradiation (Liu et al., 1994; Liu and Pelling,
1995; Petrocelli et al., 1996; Poon et al., 1996). Using a
biologically relevant melanoma model, the WM35 cell
line, we investigated the changes in cell cycle regulators
following UVB-irradiation of asynchronously growing
cells. Figure 1 shows the ¯ow cytometry results for
Oncogene
UVB induced cell cycle checkpoints
T Petrocelli and J Slingerland
4482
Figure 1 Cell cycle pro®le following UVB irradiation. Asynchronously growing WM35 cells were recovered either before or at the
indicated times after irradiation with 9 mJ/cm2 UVB. DNA content was assayed by BrdU pulse labelling (vertical axis) and
propidium iodide staining (horizontal axis) as described in Materials and methods. Time, in hours, after irradiation is indicated
data in Figures 5 ± 8. The p53 gene in this cell line is
wild-type (V Florenes, unpublished results). Asynchronously growing WM35 cells express low levels of p53
protein. p53 protein levels rose as early as 1 h
following UVB irradiation (data not shown), peaking
at 12 h, with a decline by 18 h (Figure 5). The increase
in p53 was followed by an increase in p21 protein. p21
protein increased by 6 h, with peak levels persisting
from 12 through 24 h post-irradiation. p27 protein
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levels also rose following UVB irradiation (Figure 5),
showing a pro®le similar to that of p21. The UVB
induced G1 arrest is re¯ected by dephosphorylation of
the retinoblastoma protein (pRb) (Figure 5). In
addition, the loss of total pRb proteins post UVB
may re¯ect activation of cell death pathways as
described (Pedley et al., 1996). Equal loading of
protein lysates was veri®ed by amino black staining
of immunoblotted proteins (not shown).
UVB induced cell cycle checkpoints
T Petrocelli and J Slingerland
4483
Figure 2 UVB mediated G1-to-S phase checkpoint. Cells were
synchronized in G0 as described in the text and then either
irradiated (right) or not irradiated (left), 4 h after release from
quiescence. Cells were pulse-labelled and recovered for FACS
analysis at the di€erent time points indicated after G0 release at
time T=0. The entrance to S phase can be seen in non-irradiated
cells at 12 h after release from G0 (left). Entrance to S phase was
signi®cantly delayed in the irradiated population (right)
There was no loss of cyclin E, cdk4, cdk2, and cdc2
proteins following UVB treatment. There was a
notable loss of cyclin D1 protein within 3 h of
irradiation, with a return to basal levels by 18 h
post-UVB. A loss of both cyclin A and cyclin B1
proteins was evident by 12 h following irradiation
(Figure 5).
Inhibition of signal transduction via PI3K/PKB can
lead to loss of cyclin D1. This can occur through the
resulting activation of GSK3-b, a kinase that can
Figure 3 UVB mediated S phase arrest in WM35 cells.
Asynchronously growing cells were incubated with 10 mM Brdu
for 2 h to label cells in S phase, and then chased in fresh media.
Cells were treated with (right) or without a single dose of UVB
(left) at time T=0, and harvested at di€erent time points
thereafter for FACS analysis. Cells that had been in S phase at
the time of BrdU labelling moved from S phase into G2 by 3 ± 6 h
(left) and could then be seen in G1 by 9 ± 12 h. Cells irradiated
while in S phase (right) showed no progression into G2 during the
chase period (up to 12 h post-irradiation)
phosphorylate cyclin D1 and target its ubiquitin
mediated degradation (Diehl et al., 1998). PI3K
activation is also reported to increase translation of
cyclin D1 (Muise-Helmericks et al., 1998). Activation
of the MAPK pathway has been shown to activate
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UVB induced cell cycle checkpoints
T Petrocelli and J Slingerland
4484
Figure 5 E€ects of UVB on cell cycle regulator proteins.
Asynchronously growing cells were irradiated with 9 mJ/cm2
UVB. Lysates were prepared from cells prior to (T=0 h) or at
di€erent time points following irradiation (T=3 ± 24 h). Equal
amounts of protein (30 mg) were resolved by SDS ± PAGE,
immunoblotted and probed with the indicated antibodies
Figure 4 UVB mediated G2/M checkpoint. Asynchronously
growing cells were incubated with 10 mM BrdU for 2 h to label
cells in S phase, and then chased in fresh media. Within 5 h, a
majority of the exponentially BrdU labelled cells had moved into
G2. At 5 h after BrdU labelling, cells in the right column were
UVB irradiated. Non-irradiated, BrdU labelled cells moved from
G2/M into the following G1 phase as early as 3 h later (T=8 h,
left column). Cells that were UVB irradiated while in G2 showed
no movement into G1 during the chase period (right column)
cyclin D1 transcription (Cheng et al., 1998). To
ascertain if inhibition of either the MAPK or PI3K
pathways may contribute to the observed UVB
mediated loss of cyclin D1 protein, we made use of
antibodies that detect either total levels of MAPK,
PKB or GSK3-b and antibodies speci®c to relevant
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phosphoforms of each of these proteins. PKB and
MAPK are both activated by phosphorylation (at
ser483 and thr202/tyr204 respectively), while GSK3-b
is inactivated by phosphorylation at ser9 (Alessi et al.,
1996, 1997; Hunter, 1995; Payne et al., 1991; Shaw et
al., 1997; Sutherland et al., 1993).
Total levels of MAPK, PKB and GSK3-b were
unchanged for up to 18 h following irradiation of
asynchronous WM35, while activation of both PKB
and MAPK was noted within 3 h post UVB (Figure 6).
The inhibitory phosphorylation of GSK3-b was
increased by UVB. Thus, the observed loss of cyclin
D1 protein could not be attributed to inhibition of
signal transduction via the PI3K/PKB or MAPK
pathways.
UVB induced cell cycle checkpoints
T Petrocelli and J Slingerland
UVB suppresses cyclin-associated kinase activities
To further study the timing and mechanisms of the
UVB mediated cell cycle arrests in WM35, cyclin/
cdk activities were assayed at various intervals
following UVB treatment of asynchronous cells
(Figure 7). Inhibition of both cyclin D1-dependent
kinase and cyclin E/cdk2 activities was detected
within 6 h of UVB irradiation and the activities of
both fell progressively to a nadir at 24 h. Inhibition
of cyclin A-associated kinase showed similar kinetics
to that of cyclin E/cdk2. Cyclin B1/cdc2 activity fell
by 6 h with no evidence of recovery within the 24-h
assay period.
4485
UVB effects on cyclin/cdk complex formation
The binding of KIPs, p21 and p27, to cyclin/cdk
complexes was assayed as a function of time postUVB. The amount of cdk4 bound to immunoprecipitated cyclin D1 paralleled the fall in the levels of
cellular cyclin D1 (Figure 8a). However, the levels of
both p21 and p27 in cyclin D1/cdk4 complexes were
increased at 18 and 24 h when compared to complexes
in non-irradiated cells (T=0 h). Cyclin E/cdk complex
formation was not inhibited post-UVB, but the
association of both p21 and p27 with cyclin E/cdk2
complexes was increased (Figure 8b). The association
of p21 with both cyclin A and cyclin B complexes also
increased following UVB irradiation (Figure 8c,d).
Figure 6 UVB increases PKB, MAPK, and GSK3-b phosphorylation. Asynchronously growing cells were irradiated with 9 mJ/
cm2 UVB. Lysates were prepared from cells prior to (T=0 h) or
at di€erent time points following irradiation (T=1 ± 18 h). Equal
amounts of protein (30 mg) were resolved by SDS ± PAGE,
immunoblotted and probed with the indicated antibodies. While
the levels of PKB, MAPK and GSK3-b proteins were unchanged
by UVB irradiation, their phosphorylation was increased within
1 h, with return toward baseline at later time points
Changes in Cdc25 complexes following UVB
In addition to increased cdk inhibitor binding, cdks
may be inhibited by phosphorylation. Loss of Cdc25B
and Cdc25C activity has been implicated in G2/M
arrest following di€erent forms of DNA damage
(Furnari et al., 1997; Poon et al., 1996; Sanchez et
Figure 7 Cyclin D1-cyclin E-, cyclin A-, and cyclin B1-associated kinase activities. Cyclin immunoprecipitates were recovered
before (T=0) and at intervals following UVB irradiation, and associated kinase activities were assayed using either pRb protein
fragment (cyclin D1) or histone H1 substrate (cyclins E, A and B). Incorporation of radioactivity into the substrate was quantitated
by PhosphorImager and expressed as a percentage maximum of that detected in asynchronous lysates at T=0
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UVB induced cell cycle checkpoints
T Petrocelli and J Slingerland
4486
Figure 8 Changes in cyclin/cdk complexes following UVB. Asynchronously growing WM35 cells were irradiated at T=0 and
lysates were prepared at di€erent time points thereafter. Cyclins D1, E, A and B1 were immunoprecipitates, resolved by SDS ±
PAGE and immunoblotted for associated cyclins, cdks and cdk inhibitors using speci®c antibodies
al., 1997). To determine whether e€ects on Cdc25A
may contribute to the G1/S phase checkpoint induced
by UVB irradiation, we assayed the levels of Cdc25A
and the phosphotyrosine content of G1-cyclin-bound
cdk2 before and after irradiation. As shown in Figure
9a, there was a modest reduction in the level of
Cdc25A following UVB irradiation of WM35 cells.
Moreover, as Cdc25A fell, its association with 14-3-3
increased signi®cantly. While cyclin E and cyclin Ebound cdk2 levels were constant post-irradiation
(Figure 8b), there was a signi®cant increase in the
phosphotyrosine content of cyclin E-associated cdk2
(Figure 9a). Thus, loss of Cdc25A protein is re¯ected
in an increased inhibitory phosphotyrosylation of
cyclin E-associated cdk2. When equivalent amounts
of cyclin A were immunoprecipitated from asynchronously growing WM35 and from cells at 12 and 24 h
post-irradiation, an increase in phosphotyrosine content of cyclin A-associated cdk2 was also observed
(data not shown). The antibodies used for Cdc25A
immunoprecipitation and blotting do not cross react
with Cdc25B and Cdc25C (P Worland, personal
communication). The levels of Cdc25B also fell
following UVB irradiation and the remaining phosphatase showed a progressive increase in association
with 14-3-3 (Figure 9b).
Discussion
Excessive exposure to the sun is implicated in the
etiology of skin cancers, including cutaneous melanoma (Epstein, 1983). Since UVC does not penetrate our
atmosphere and is therefore not relevant to skin
carcinogenesis in humans, we wished to examine the
cell cycle e€ects of UVB on biologically relevant target
cells. An understanding of UVB responses in early
stage melanoma would inform subsequent studies of
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the checkpoint loss during malignant melanoma
progression.
Our investigation was initiated with the WM35, a
cell line derived from a primary, radial growth phase
melanoma. Although itself not a normal cell line, it
retains wild-type p53 and cell cycle responses similar to
those already published for melanocytes, such as
increases in p53 and p21 following DNA damage
(Barker et al., 1995). This line is also responsive to
TGF-b (Florenes et al., 1996). As we detected
previously in normal rat keratinocytes (Petrocelli et
al., 1996), UVB irradiation of asynchronously growing
human melanoma cells resulted in inhibition of cyclin
E-, A-, and B-dependent kinase activities and cell cycle
arrests at G1, S phase and G2/M.
Checkpoints, or biological pathways at transition
points throughout the cell cycle, provide the cell with
sucient time to repair damaged DNA (Hartwell and
Weinert, 1989). Most cells activate a G1 checkpoint
following DNA damage, however the arrest mechanisms show some variation dependent on both the
stressor agent and cell type. For some cells, the p53dependent induction of p21 may be sucient for
inhibition of cdk2 activity (El-Deiry et al., 1993; Poon
et al., 1996). However, other mechanisms also contribute to G1 arrest following UV irradiation. An
increase in inhibitory thr14/tyr15 phosphorylation of
Cdk4 kinase may play a role in UVC-mediated G1
arrest in certain cell types (Terada et al., 1995). In
others, increased binding of both p21 and p27 to target
cdks has been demonstrated (Petrocelli et al., 1996;
Poon et al., 1996). Here we show that the UVB
mediated cell cycle arrests in WM35 melanoma cells
involve several mechanisms. These include loss of
cyclin D1, an increase in the cdk inhibitors p21 and
p27 and their increased association with G1, S and G2/
M phase targets. Increased inhibitory phosphorylation
of cyclin E-bound and cyclin A-bound cdk2 was
UVB induced cell cycle checkpoints
T Petrocelli and J Slingerland
Figure 9 UVB-mediated changes in Cdc25 phosphatases. (a)
Loss of Cdc25A and increased phosphotyrosylation of cyclin Eassociated cdk2 following UVB. Protein lysates were prepared
from asynchronously growing WM35 and from cells recovered 12
and 24 h after irradiation with 9 mJ/cm2 at T=0. Cell lysate
(800 mg) was immunoprecipitated with monoclonal anti-Cdc25A
antibody, and immunoprecipitates (Cdc25A IP) were blotted with
polyclonal antibodies against Cdc25A and against 14-3-3 proteins.
To assay changes in phosphotyrosine content of cyclin Eassociated cdk2 after UVB irradiation, cyclin E was immunoprecipitated (Cyclin E IP) from 400 mg cell lysate at the indicated
times, resolved by SDS ± PAGE, and the associated cdk2 was
immunoblotted with anti-phosphotyrosine antibody. (b) Loss of
Cdc25B and increased binding to 14-3-3. Cdc25B was immunoprecipitated from cells as in (a) above. Complexes were resolved
and blotted with antibodies to Cdc25B and to 14-3-3
associated with loss of Cdc25A phosphatase. Moreover, Cdc25B protein also fell and there was an
increased binding of Cdc25A and Cdc25B to 14-3-3
post UVB.
In WM35 cells, UVB caused a rapid and dramatic
loss of cyclin D1. Pagano et al. (1994) have shown that
following UVC radiation, loss in cyclin D1 protein is
associated with inhibition of replicative DNA synthesis
and initiation of DNA repair. They suggested that loss
of cyclin D1 may be required for the release of PCNA
from the cyclin D/cdk4/PCNA complex, liberating
PCNA to function in DNA repair (Pagano et al.,
1994).
MAPK activation by Mek1 has been shown to
induce cyclin D1 transcription (Cheng et al., 1998).
Cyclin D1 protein levels are regulated not only at the
level of transcription (Cheng et al., 1998; Muller et al.,
1994), but also post-translationally via ubiquitinmediated proteolysis (Diehl et al., 1997). Diehl et al.
(1998) recently demonstrated that phosphorylation of
cyclin D1 at threonine-286 by glycogen synthase
kinase3-b (GSK3-b) mediates nuclear to cytoplasmic
translocation and proteolysis of cyclin D1. To ascertain
whether the UVB-mediated loss of cyclin D1 in WM35
results from inhibition of PKB with subsequent
activation of GSK3-b, the phosphorylation of PKB
and GSK3-b were assayed post-UVB. Irradiation led
to activation of both PKB and MAPK pathways, and
the inhibitory phosphorylation of GSK3-b was increased in WM35. Thus, the dramatic fall in cyclin D1
protein observed following UVB irradiation of WM35
could not be accounted for by inhibition of the PI3K
pathway leading to accelerated cyclin D1 proteolysis
post UVB, nor was it due to loss of MAPK activity.
The mitogenic activation of PI3K/PKB and of MAPK
by ras and activation of these pathways by UVB
appear to have di€erent consequences with regard to
cyclin D1 levels. It is noteworthy that in other cell
types, prolonged MAPK activation is seen in association with G1 arrest (Alblas et al., 1998; Zimmermann
et al., 1999).
The present study con®rms and extends earlier
observations (Petrocelli et al., 1996; Poon et al.,
1996) that both the cdk inhibitors p21 and p27 have
a role in the UV induced cyclin-cdk inhibition. In
UVB-irradiated WM35, an increase in p21 followed
that of p53. Levels of cyclin E-, cyclin A- and cyclin
D1-associated p21 and p27 increased after irradiation,
consistent with both KIPs acting to inhibit these
respective kinase complexes. Following an initial rapid
loss post-UVB, cyclin D1 levels rose again at 12 ± 18 h.
In spite of the recovery of cyclin D1 protein expression,
cyclin D1-associated kinase inhibition persisted at 24 h
after UVB. The increased association of p21 and p27
to cyclin D1 complexes may not only facilitate
assembly in this context, but could also contribute to
the inhibition of cyclin D1-dependent kinase after
DNA damage by UVB. We saw no evidence for a
redistribution of p27 from cyclin D1/cdk4 complexes to
those of cyclin E/cdk2 as has been observed with UVC
irradiation (Poon et al., 1995). In addition, cyclin D1/
cdk6 complexes showed a similar increase in p27
association at later time points following UVB (data
not shown). The roles of p15 and p16 in UVBmediated G1 arrest could not be addressed in WM35,
because this line bears deletions of both p15 and p16
genes (Florenes et al., 1996).
Inhibition of G1 cyclin/cdk activity by UVB is not
dependent solely on the increased binding of p21 and
p27. Cdc25A has been shown to be essential for cdk2
activation and G1-to-S phase progression (Draetta and
Eckstein, 1997; Ho€mann et al., 1994; Jinno et al.,
1994). Following UVB irradiation of WM35 cells, there
was a loss of Cdc25A protein, and an increased
inhibitory phosphotyrosine content in cyclin E-bound
cdk2. Although levels of Cdc25A were modestly
reduced, the association of Cdc25A with 14-3-3
increased signi®cantly. The UVB-mediated binding of
Cdc25A phosphatase to 14-3-3 may contribute to
inactivation of the remaining phosphatase, as has been
shown for mitotic Cdc25C (Furnari et al., 1997; Peng
et al., 1997; Sanchez et al., 1997). Thus, both the loss
and inactivation of Cdc25A may contribute to the
inhibition of G1-to-S phase progression following UVB
irradiation.
Although DNA replication is central to the cell
division cycle, the mechanisms linking DNA damage to
S phase arrest have not been fully elucidated. Both
cyclin E- and cyclin A- associated kinase activities are
required for initiation and maintenance of DNA
replication (Pagano et al., 1992), while the cdk
4487
Oncogene
UVB induced cell cycle checkpoints
T Petrocelli and J Slingerland
4488
Oncogene
inhibitor, p21, is implicated in S phase arrest following
DNA damage (Chen et al., 1995, 1996; Flores-Rosas et
al., 1994; Ogryzko et al., 1997; Waga et al., 1994). Loss
and inhibition of Cdc25A may also be relevant to the S
phase arrest in irradiated WM35, since there was an
increase in the inhibitory phosphorylation of cyclin Aassociated cdk2 after UVB treatment. The increased
association of p21 with cyclin A may inhibit both cdk1
and cdk2 cyclin A-bound following UVB.
UVB irradiation of WM35 cells induced a prolonged
G2 arrest, re¯ected in an inhibition of cyclin B/cdk1
kinase activity. This loss in kinase activity is mediated,
in part by a loss in cyclin B protein. This loss of cyclin
B protein may re¯ect an arrest early in G2, prior to upregulation of cyclin B. UVB irradiation of WM35 cells
also increased binding of p21 to cyclin B/cdk1
complexes. Thus, both UVB (present study) and
UVC (Poon et al., 1996) induced G2 checkpoints
may involve p21 activity.
Activation of a G2 checkpoint following genotoxic
stress is highly conserved among eukaryotes, and
guards against mitotic entry in the presence of
unreplicated or damaged DNA (Hartwell and Weinert,
1989). Several mechanisms have been reported leading
to G2 arrest by DNA damaging agents. Inhibition of
Cdc25C phosphatase activity results in a G2 arrest
following treatment with nitrogen mustard (O'Connor
et al., 1994). G2 arrest following UVC irradiation of
normal human ®broblasts is associated with increased
thr14/tyr15 phosphorylation and inhibition of cdc2
(Poon et al., 1996). Moreover, UVB irradiationinduced G2 arrest in Hela and melanoma cells involves
a block in the Cdc25C dependent activation of cyclin
B/cdc2, cyclin A/cdk2 and cyclin A/cdc2 through
nuclear/cytoplasmic partitioning that segregates Cdc25
away from target cyclin/cdks (Herzinger et al., 1995;
Gabrielli et al., 1997). In WM35, the loss of Cdc25B is
also implicated in the UVB-mediated G2 checkpoint.
In addition, the remaining Cdc25B, as for Cdc25A,
showed an increased association with 14-3-3. DNA
damage induced activation of the Chk1 kinase
mediates ser216 phosphorylation of the mitotic
phosphatase, Cdc25C. Cdc25C phosphorylation leads
to increased association with 14-3-3 (Furnari et al.,
1997; Peng et al., 1997; Sanchez et al., 1997). In
Xenopus, phosphorylation of the homologous site,
ser287 on Cdc25, facilitates the binding of 14-3-3e
near the nuclear localization signal of Cdc25. This
inhibits nuclear import of Cdc25 and leads to its
accumulation in the cytoplasm (Kumagai and Dunphy,
1999; Yang et al., 1999). The UVB-mediated increases
in 14-3-3 binding to Cdc25A and Cdc25B observed in
WM35 may re¯ect similar mechanisms leading to
cytoplasmic sequestration of these phosphatases away
from target cyclin/cdks. Activation of pathways leading
to 14-3-3 association with Cdc25A and Cdc25B may
contribute importantly to UVB-mediated checkpoints
in WM35.
Environmental changes have allowed greater penetration of UVB through the earth's atmosphere,
leading to an increased incidence of skin cancers,
including melanomas. The incidence of melanoma is
rising at a rate second only to that of lung cancer in
women, with a lifetime risk of 1 in 75 in the U.S.
population. Lifetime risk for development of skin
cancer in the U.S. population is currently estimated
at 1 in 5 (Rigel et al., 1996). The elucidation of cell
cycle checkpoints following UVB mediated DNA
damage may provide important mechanistic insights
into the malignant progression of human tumours. The
present study reveals a multiplicity of mechanism of
UVB mediated cell cycle arrests, including the losses of
cyclin D1 and of Cdc25A, in addition to an increase in
binding and inhibition of cdks by the cdk inhibitors,
p21 and p27. Such a plurality of defence mechanisms
may provide an evolutionary advantage to ensure the
®delity of genome replication in long lived day-loving
species.
Materials and methods
Cell culture and UVB treatment
WM35, a human cell line derived from a radial growth
melanoma (gift of Dr M Herlyn, Philadelphia, PA, USA),
was grown in RPMI 1640 medium supplemented with 5%
foetal bovine serum (FBS, Gibco ± BRL), 0.1 mg/ml streptomycin, 100 U/ml penicillin (Gibco ± BRL) and 2 mM glutamine (Gibco ± BRL). Asynchronously growing cells were
irradiated with 9 mJ/cm2 UVB. The UVB light source
consisted of three General Electric UVB lights, which emit
between 280 ± 320 nm with peak emission at 310 nm. The
irradiance dose of UVB was monitored using an International Light IL1400A UVB radiometer/photometer. A dose
of 9 mJ/cm2 was selected, since this dose was sucient to
induce reversible cell cycle e€ects in a proportion of the
population (Petrocelli et al., 1996). Prior to UVB treatment,
culture medium was replaced with phosphate bu€ered saline
(PBS) and the lids removed from the culture dishes. Fresh
medium was replaced after irradiation. Sham irradiated 0 h
samples were treated identically, but not exposed to UVB.
Cells were harvested for cell cycle and protein analysis
immediately before (T=0 h) and at various time points after
irradiation (T=3, 6, 9, 12, 18 and 24 h).
Flow cytometric analysis
To assay the e€ects of UVB irradiation on cell cycle
parameters, asynchronously growing WM35 cells were
irradiated at time 0 with 9 mJ/cm2 UVB and harvested at
di€erent times thereafter. At the indicated times, cells were
pulse-labelled with 10 mM bromodeoxyuridine (BrdU) for 2 h
just prior to recovery for ¯ow cytometry and protein analysis.
BrdU incorporation was detected with anti-BrdU FITClabelled antibodies and DNA content assayed by propidium
iodide staining, as described (Petrocelli et al., 1996).
The e€ects of irradiation on cells in G1, S and G2 were
assayed as follows. To monitor the e€ects of UVB on cells
that were irradiated during the G1 phase, cells were
synchronized by serum starvation. Cells were plated at low
density and on the following day, transferred to serum
depleted medium (FBS 0.1%) for a further 72 h. Cells were
then restored to complete medium (5% FBS). At 4 h
following release from G0, when cells had entered G1, they
were either sham irradiated (control) or irradiated with 9 mJ/
cm2 UVB and progression through the cell cycle monitored
by BrdU pulse labelling at intervals thereafter (12, 18 and
24 h after readdition of high serum medium).
To assay the e€ects of UVB irradiation on cells that were
in S phase at the time of irradiation, asynchronously growing
cells were pulsed with 10 mM BrdU for 2 h and then treated
with or without a single dose of UVB at time 0 h. Cells were
harvested at di€erent time points thereafter for protein and
¯ow cytometric analysis. To follow a population of cells that
were irradiated while in G2, asynchronously growing cells
were pulsed with 10 mM BrdU for 2 h and then irradiated
UVB induced cell cycle checkpoints
T Petrocelli and J Slingerland
with UVB 5 h later. S phase cells that had taken up BrdU
exponentially during the pulse were mostly in G2 at the time
of UV irradiation 5 h later.
Antibodies
Monoclonal p53 antibody, mAb1801, was provided by S
Benchimol (OCI, Toronto, Ontario, Canada). Antibodies to
cdk2 and cdk4, cyclins A and B1, p21, GSK3-b, Cdc25C and
14-3-3b were obtained from Santa Cruz Biotechnology. The
14-3-3b antibody used cross reacts with several di€erent
isoforms of 14-3-3. PRb antibody was from Pharmingen and
p27 antibody from Transduction Labs. Cyclin E was detected
with mAbs E12 and E172 (Slingerland et al., 1994). Cyclin
D1 was detected with DCS-6 or DCS-11 (Neomarkers), while
monoclonal cyclin A antibody, E67 was provided by G
Gannon and T Hunt (ICRF, England, UK). Monoclonal and
polyclonal antibodies speci®c for Cdc25A and Cdc25B were
provided by Peter Worland (Mitotix Incorporated, Cambridge, MA, USA). Antibodies to PKB and MAPK, and
antibodies reactive with speci®c phosphoforms phospho-PKB
(ser473), phospho-p44/42 MAP kinase (thr202/tyr204) and
phospho-GSK3-b (ser9) were obtained from New England
Biolabs.
Immunoblotting
For Western blot analysis, cells were harvested and lysed at
the indicated times in ice cold lysis bu€er containing 0.1%
NP-40, 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM phenylmethlysulfonyl ¯uoride (PMSF) and 0.02 mg/ml each of
aprotinin, leupepsin and pepstatin. Lysates were sonicated
and clari®ed by centrifugation. Protein concentration was
quantitated by Bradford analysis. Thirty mg of protein per
lane were resolved by SDS-polyacrylamide gel electrophoresis
(SDS ± PAGE) and transferred onto PVDF membrane
(Millipore). Blots were reacted with appropriate primary
and secondary antibodies and proteins detected using
enhanced chemiluminescence.
For analysis of cyclin/cdk complexes, 100 ± 200 mg of
protein lysate was immunoprecipitated with speci®c cyclin
antibody and complexes resolved by SDS ± PAGE. Cyclinassociated proteins were detected by Western blotting with
antibodies to cdks, p21 and p27. Control lanes with antibody
but no lysate were run alongside all immunoprecipitates in
IP/Westerns. For detection of Cdc25A, 800 mg of protein was
immunoprecipitated with a monoclonal anti-Cdc25A and
immunoblots were reacted with a polyclonal anti-Cdc25A
and with antibody to 14-3-3. To assess the phosphotyrosine
content of cyclin-bound cdk2, cyclin E or cyclin A complexes
from 400 mg total cell lysate were immunoprecipitated, and
blots prepared as above. The phosphotyrosine content cyclinbound cdk2 was examined using phospho-speci®c cdc2
(tyr15) antibody (New England Biolabs) which cross reacts
with phosphotyrosylated cdk2. Cdc25B was immunoprecipitated with a monoclonal antibody and immunoblots reacted
with polyclonal antibodies to Cdc25B or to 14-3-3. Cdc25C
was also assayed by immunoprecipitation Western blotting.
4489
Cyclin dependent kinase assays
Cyclin E-, A- or B complexes were immunoprecipitated,
collected on protein A sepharose beads, washed and reacted
with g-[32P]ATP and histone H1 as described (Dulic et al.,
1992; Slingerland et al., 1994). Cyclin D-associated kinase
assays were performed using 200 mg protein lysate following
the method of LaBaer et al. (1997) using cyclin D1 antibody
DCS11 for immunoprecipitation and a carboxyl-terminal
fragment of pRb as substrate. Reaction products were
resolved by SDS ± PAGE and quantitation of radioactivity
incorporated in histone substrate was performed using
Molecular Dynamics PhosphorImager and ImageQuant software.
Phosphorylation status of PBK, MAPK and GSK3-b
Steady state levels of PBK, MAPK and GSK3-b were
detected using commercially available antibodies. To detect
PKB activation, blots were blocked with TBS containing 5%
bovine serum albumin (BSA) and 0.25% Tween 20 and then
reacted with antibody speci®c for PKB phosphorylated at
ser473. For detection of MAPK activation, we made use of
an antibody speci®c for MAPK phosphorylated at thr202
and tyr204. The GSK3-b phosphorylated at ser9 was detected
using a phosphorylation-speci®c antibody. GSK3-b is inactivated by phosphorylation at this site (Shaw et al., 1997;
Sutherland et al., 1993).
Acknowledgments
JM Slingerland is a clinician investigator supported by
Cancer Care Ontario, and by a career awards from the US
Army Department of Defense Breast Cancer Research
Program and from the Burroughs Wellcome Fund. We
thank Dr N Bhattacharya for excellent technical assistance
with tissue culture and irradiation of cells. This work was
supported by grants to JM Slingerland from the CBCRI/
National Cancer Institute of Canada.
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