Hdm2-and proteasome-dependent turnover limits p21 accumulation during S phase

Article (PDF Available)inCell cycle (Georgetown, Tex.) 10(16):2714-23 · August 2011with30 Reads
DOI: 10.4161/cc.10.16.16725 · Source: PubMed
Abstract
Double-strand DNA breaks detected in different phases of the cell cycle induce molecularly distinct checkpoints downstream of the ATM kinase. p53 is known to induce arrest of cells in G 1 and occasionally G 2 phase but not S phase following ionizing radiation, a time at which the MRN complex and cdc25-dependent mechanisms induce arrest. Our understanding of how cell cycle phase modulates pathway choice and the reasons certain pathways might be favored at different times is limited. In this report, we examined how cell cycle phase affects the activation of the p53 checkpoint and its ability to induce accumulation of the cdk2 inhibitor p21. Using flow cytometric tools and centrifugal elutriation, we found that the p53 response to ionizing radiation is largely intact in all phases of the cell cycle; however, the accumulation of p21 protein is limited to the G 1 and G 2 phase of the cell cycle because of the activity of a proteasome-dependent p21 turnover pathway in S-phase cells. We found that the turnover of p21 was independent of the SCF (skp2) E3 ligase but could be inhibited, at least in part, by reducing hdm2, although this depended on the cell type studied. Our results suggest that there are several redundant pathways active in S-phase cells that can prevent the accumulation of p21.
Cell Cycle 10:16, 2714-2723; August 15, 2011; © 2011 Landes Bioscience
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2714 Cell Cycle Volume 10 Issue 16
*Correspondence to: Andrew Ko; Email: a-ko@ski.mskcc.org
Submitted: 05/27/11; Revised: 06/08/11; Accepted: 06/09/11
DOI: 10.4161/cc.10.16.16725
Introduction
Ionizing radiation induces double-strand DNA breaks, which
activate distinct molecular pathways to induce cell cycle arrest
depending on whether the cell is in the G
1
, S or G
2
phase of
the cell cycle.
1
In G
1
cells, a p53-dependent transcriptional pro-
gram induces cell cycle arrest in part by activating expression of
p21, a cdk inhibitor that targets cdk2-containing complexes. In
response to genotoxic stress, ATM- and chk2-dependent phos-
phorylation of hdm2 inhibits its ability to regulate p53 in three
ways: reducing its E3-ubiquitin ligase for p53, preventing the
binding of hdm2 to p53 (which can also block the transactiva-
tion function of p53) and by inhibiting the ability of hdm2 to
promote nuclear export of p53.
2
Additionally, hdm2 can promote
the proteasome-dependent but ubiquitin-independent degrada-
tion of p21. Phosphorylation of hdm2 may affect this activity
as well. The absence of p21 weakens p53-dependent G
1
arrest
in a variety of different cell lines and primary cells, both mouse
and human.
3-8
In S-phase cells, checkpoints are triggered by mul-
tiple mechanisms involving both the inhibition of cdc25A, which
removes an inhibitory phosphorylation on cyclin-cdk complexes,
and the MRN complex.
9-12
In G
2
cells, inactivation of cdc25
prevents the activation of cyclin B-cdc2, and in some cell types,
p53-dependent accumulation of p21 can also play a role.
4
Although there is extensive data for the involvement of p21
in causing G
1
arrest following DNA damage, cells might have
evolved mechanisms that prevent p21 accumulation in S phase,
because p21 can affect DNA repair and the ability of a cell to
Double-strand DNA breaks detected in dierent phases of the cell cycle induce molecularly distinct checkpoints
downstream of the ATM kinase. p53 is known to induce arrest of cells in G
1
and occasionally G
2
phase but not S phase
following ionizing radiation, a time at which the MRN complex and cdc25-dependent mechanisms induce arrest. Our
understanding of how cell cycle phase modulates pathway choice and the reasons certain pathways might be favored
at dierent times is limited. In this report, we examined how cell cycle phase aects the activation of the p53 checkpoint
and its ability to induce accumulation of the cdk2 inhibitor p21. Using ow cytometric tools and centrifugal elutriation, we
found that the p53 response to ionizing radiation is largely intact in all phases of the cell cycle; however, the accumulation
of p21 protein is limited to the G
1
and G
2
phase of the cell cycle because of the activity of a proteasome-dependent p21
turnover pathway in S-phase cells. We found that the turnover of p21 was independent of the SCF
skp2
E3 ligase but could
be inhibited, at least in part, by reducing hdm2, although this depended on the cell type studied. Our results suggest that
there are several redundant pathways active in S-phase cells that can prevent the accumulation of p21.
Hdm2- and proteasome-dependent turnover
limits p21 accumulation during S phase
Daniel Ciznadija, Xin-Hua Zhu and Andrew Ko*
Program in Molecular Biology; Sloan-Kettering Institute; Memorial Sloan-Kettering Cancer Center; New York, NY USA
Key words: p21, hdm2, skp2, cell cycle phase-dependent, protein turnover
restart DNA synthesis.
13-15
Thus, we were interested in determin-
ing whether the accumulation of p21 was prevented in S-phase
cells responding to DNA damage. In this report, we show that
an hdm2-dependent mechanism reduces accumulation of p21 in
S-phase cells. We suggest that this might prevent p21 from inhib-
iting PCNA ubiquitination and recovery from DNA damage.
Results
Accumulation of p21 was reduced in S-phase cells exposed to
ionizing radiation. We set out to determine a collection of cells
in which we could investigate p53-dependent p21 accumula-
tion following exposure to ionizing radiation. In MCF7 cells,
Darzynkeiwicz had reported that p53 accumulated throughout
the cell cycle, but accumulation of p21 was restricted to cells in
G
1
and G
2
phase of the cell cycle following an 816 h treatment
with camptothecin.
16
However, it was unclear whether this phe-
nomena was limited to MCF7 cells, whether it was due to the
extended length of time that the cells were in the presence of
the drug, or whether the antibodies used in the laser scanning
analysis were capable of detecting p21 species or complexes that
formed in S-phase cells. To avoid these caveats, we revisted these
results and began our analysis by screening a diverse collection
of five transformed cell lines at 3 and 6 h following exposure to
different doses of ionizing radiation (ranging from 1 Gy to 20
Gy). In a B-lymphoblast cell line (TK6), a colorectal carcinoma
cell line (HCT116), a mammary epithelial adenocarcinoma cell
line (MCF7) and a lung carcinoma cell line (A549), both p53 and
www.landesbioscience.com Cell Cycle 2715
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irradiation is a property shared by transformed, immortalized
and normal cells alike.
To determine if this pattern was due to a direct downstream
effect of the DNA damage response, we took advantage of the fact
that nutlin-3 can increase p53 without affecting other functions of
hdm2 or directly activating the DNA damage response.
17
Nutlin-3
fits into the small hydrophobic p53 binding pocket of hdm2, elim-
inating hdm2-induced turnover of p53. Thus, we took HCT116
cells and treated them with 1 or 3 μM nutlin-3 for 6 h and looked
at the accumulation of hdm2, p53 and p21 protein. Both p53 and
p21 increased following treatment of non-irradiated HCT116
cells; however, the increase in p21 was again greater in G
1
- and
G
2
phase compared with S phase (Fig. 1F). Taken together, these
results suggest that irrespective of how p53 was activated, p21
protein does not accumulate in this collection of S-phase cells.
p53 accumulation and transcriptional activity is cell cycle
phase-independent. After p53 protein is synthesized, it must
accumulate in the nucleus, bind to the appropriate promoters
and activate transcription. Blocking nuclear accumulation of p53
may contribute to the reduced p21 accumulation seen in S-phase
cells. Genotoxic stress induces phosphorylation of p53 and hdm2
by ATM and chk2, ultimately leading to an increase in nuclear
p53 and the transcription of p53 target genes. Other post-
translational modifications of p53 can affect the profile of the
transcriptional program but are not required for p53-dependent
transcription.
17
Thus we began to address whether p53 was acti-
vated to a similar extent in S-phase and G
1
- and G
2
-phase cells.
We found that the ability of elutriated cells to sense DNA dam-
age, as measured by p53 and chk2 phosphorylation, was largely
unaffected by cell cycle phase (Fig. 2A). In addition, p53 entered
the nucleus of S-phase A549 cells following ionizing radiation
(Fig. 2B). Similar results were also seen with MCF7 cells (data
not shown). Furthermore, PUMA and p21 transcripts were
present at nearly equivalent levels in irradiated G
2
- and S-phase
TK6 cells enriched by cell sorting, directly demonstrating that
p53 was activating transcription in a cell cycle phase-dependent
manner (Fig. 2C). Finally, we established that p53 levels were
equivalent at all phases of the cell cycle following irradiation
of TK6 (Fig. 2D), RPE-hTERT (Fig. 2E) and BJ-hTERT cells
(data not shown). Together, these data suggest that the lack of
p21 accumulation in S-phase cells in any of these cell lines does
not result from the inability of DNA damage to activate a p53
response.
Accumulation of p21 in S-phase cells is regulated at the
level of proteasome-dependent turnover. Many proteasome-
dependent pathways, both ubiquitin-dependent and ubiquitin-
independent, have been shown to suppress p21 accumulation in
cycling cells. These pathways can be inactivated by signals that
eventually lead to growth arrest. To begin determining whether
S-phase cells have a greater capacity to degrade p21 protein, we
prepared S-phase extracts from cells synchronized with hydroxy-
urea (Fig. 3A, lower right part) or G
1
/G
2
-phase extracts from cells
synchronized with nocodazole (Fig. 3A, lower left part). These
extracts were supplemented with rabbit reticulocyte lysate, and
35
S-methionine-labeled, in vitro-translated p21 protein was sub-
sequently added. We observed that p21 protein was degraded
p21 protein accumulated within 6 h post-irradiation (Fig. 1A).
In contrast, neither p53 nor p21 accumulated in the glioma cell
line U87 in this time. Similar results were obtained at 3 h and
with 5 Gy or 20 Gy as well (data not shown). These cell lines, as
well as others that are discussed below, were subsequently used
interchangeably in the experiments that followed.
We next measured whether there were cell cycle phase-spe-
cific differences in p21 and p53 accumulation by three indepen-
dent assays: centrifugal elutriation followed by immunoblotting,
cell sorting followed by immunoblotting and dual-staining flow
cytometry. TK6 cells were fractionated into G
1
-, S- and G
2
-phase
enriched populations by centrifugal elutriation, irradiated at dif-
ferent doses ranging from 1 to 20 Gy and allowed to grow for an
additional 3 to 6 h prior to analyzing their progression in the cell
cycle and measuring the accumulation of p53 and p21 (Fig. 1B).
Representative flow cytometric profiles of the G
1
- and S-phase
cells before and after 2 or 20 Gy irradiation are shown in the
figure. Similar results were obtained at 1, 5 and 10 Gy as well
(data not shown). In all cases, irradiation delayed progression
to the next phase of the cell cycle, with greater delays occurring
with increasing dose. We observed an increase in the amount
of p53 in all cell cycle phases following IR, but p21 expression
increased maximally in G
1
- and G
2
-phase cells. Similar results
were seen at both 3 and 6 h and with 2 Gy, 5 Gy and 20 Gy
of ionizing radiation (data not shown). Because of the contami-
nating G
1
- and G
2
-phase cells in the S-phase population, we
could not definitively determine whether the induction of p21
was prevented in S-phase cells. Furthermore, centrifugal elutria-
tion could not be used to enrich adherent cells into different cell
cycle phase fractions. Thus, we used flow sorting to isolate G
1
-,
S- and G
2
-phase populations of HCT116 (Fig. 1C) and TK6
cells following Hoescht staining. Under these conditions, p53
accumulated in a cell cycle phase-independent manner, whereas
p21 accumulation was clearly limited to cells in G
1
and G
2
phase
(Fig. 1C). Additionally, when asynchronously growing TK6
cells were irradiated and doubly stained 3 or 6 h later with p21
antibodies and propidium iodide, the level of p21 increased in
both G
1
- and G
2
-phase cells but not in S-phase cells (Fig. 1D). A
similar pattern of p21 accumulation in IMR90 lung fibroblasts
transformed with H-Ras and adenoviral E1A, MCF7, A549
and HCT116 cells was also noted (data not shown). Again, this
occurred irrespective of time post-irradiation (3 or 6 h) or dose
of irradiation (from 1 to 20 Gy). In all instances, less p21 protein
accumulated in S phase compared with the amount seen in G
1
or G
2
phase.
We next asked whether the inability to accumulate p21 in
S-phase cells following irradiation was a property restricted to
the five transformed cell lines. The flow cytometric analysis was
repeated on two epithelial cell lines immortalized by the catalytic
subunit of human telomerase (RPE-hTERT and BJ-hTERT),
as well as a normal non-immortalized lung fibroblast strain,
IMR90. We found that, similarly to transformed cells, these
cell lines showed increases in both p53 and p21 protein expres-
sion in response to irradiation, but the accumulation of p21 only
increased in G
1
and G
2
-phase (Fig. 1E and data not shown).
This suggests that the lack of p21 accumulation in S-phase after
2716 Cell Cycle Volume 10 Issue 16
Figure 1. For gure legend, see page 2717.
www.landesbioscience.com Cell Cycle 2717
of hdm2 was significantly decreased at 24 h post-transfection, cor-
relating with an increase in p53 (Fig. 5A, left). By 48 h the cells
were too sick to be used for experiments. Strikingly, when p21
expression was examined in cells treated with siRNA to hdm2
for 24 h, there was a nearly two-fold increase in p21 levels in S
and G
2
phase (Fig. 5A, right). The level of p21 increased another
2-fold upon irradiation in S and G
2
phase (Fig. 5A, right). The
amount of p53 induced by either reducing hdm2 or irradiating
cells was equivalent, suggesting that the effect on p21 accumu-
lation was not due to some change in the level of p53 protein.
However, when we knocked down hdm2 in RPE-hTERT cells,
there was no increase in p21 in S-phase cells following irradia-
tion (Fig. 5B). This was despite a strong increase in p53 levels
across all cell cycle phases (data not shown). In addition, when
we knocked-down either DDB1, cul5 or REGγ in RPE-hTERT,
there was never a reproducibly strong induction of p21 in S phase
following irradiation in such cells (data not shown). Hence, the
mechanisms regulating p21 turnover in S phase are cell-type
dependent. Consequently, the hdm2-and proteasome-dependent
mechanism of p21 turnover in HCT116 may explain why the p53
growth arrest response, which is dependent on the accumulation
of p21, is restricted to G
1
cells and cells that fail to arrest have
reduced viability and are lost.
Discussion
Our knowledge of the pathways that regulate p21 is extensive.
A variety of mechanisms operate at the transcriptional, post-
transcriptional and post-translational levels in different cell types
and under different conditions to dictate the level of p21. In this
manuscript, we demonstrate that hdm2-dependent p21 protein
turnover can control p21 accumulation in S and G
2
phase and
prevent the elaboration of a p53-induced, p21-dependent cell
cycle arrest in response to DNA damage at that time.
Using selective synchrony methods based on either cell volume
(elutriation) or DNA content (cell sorting), we enriched popula-
tions of cells in each phase of the cell cycle and showed that p21
accumulation was curtailed in S phase in transformed, immor-
talized and normal cells following exposure to ionizing radiation.
more rapidly in extracts prepared from S-phase cells (t
1/2
= 45.4
± 15.8 min) compared with extracts prepared from G
1
- and
G
2
-phase cells (t
1/2
= 116.8 ± 36.9 min) (Fig. 3A).
We also asked if the proteasome was required to keep p21
levels low during S phase. A 6-h treatment of asynchronously
growing HCT116 cells with the proteasome inhibitor MG132
increased p53 and p21 mRNA levels to the amounts seen in irra-
diated cells, while LLM, a calpain inhibitor, had no effect (data
not shown). However, unlike in the irradiated cells, the amount
of p21 protein was equally high at all phases of the cell cycle in
the MG132 treated cells (Fig. 3B). Similar results were seen using
two other proteasome inhibitors, lactacystin and ALLN (data not
shown). Irradiation only had a modest effect on p21 accumula-
tion in the MG132-treated cells (Fig. 3B), and LLM treatment
did not affect the cell cycle phase-dependent accumulation of p21
following irradiation (Fig. 3B). Similar results were seen when
RPE-hTERT cells were treated with MG132 (data not shown).
Together, these results indicate that p21 protein is subject to pro-
teosome-dependent turnover during all phases of the cell cycle,
but apparently with greater efficiency in S-phase.
p21 turnover in S-phase cells is skp2-independent but
involves hdm2. SCF
skp2
and hdm2 can contribute to p21 turn-
over (Bornstein, 2003; Jin, 2008). To examine the role that each
of these two pathways played in S phase-specific turnover, we first
treated HCT116 cells with pools of siRNAs designed to reduce
skp2 and looked at p21 accumulation by dual-color flow cytome-
try. The amount of skp2 was significantly decreased at 48 h post-
transfection (Fig. 4A and left). Consistent with this we observed
an accumulation of p27 (Fig. 4A, left), the best-documented sub-
strate of the SCF
skp2
E3 ligase.
18,19
Dual-color flow cytometry for
DNA content and accumulated p21 showed that the increase in
p21 was most evident in G
2
phase, with little change observed in
S phase (Fig. 4A, right). Similar results were obtained in RPE-
hTERT cells after knockdown of skp2 (Fig. 4B). Hence, S-phase
turnover of p21 in response to ionizing radiation appears to be
independent of skp2; however, skp2 clearly played a role in reduc-
ing the accumulation of p21 in G
2
cells.
We next assessed the effect of reducing hdm2 on the level of
p21 expression in S-phase cells following irradiation. The amount
Figure 1 (See opposite page). Cell cycle phase-dependent accumulation of p21 in cells after induction of p53. (A) p53 and p21 protein accumulation
following ionizing radiation. The indicated asynchronously growing transformed cells were irradiated and extracts prepared 6 h later and p21 and p53
were detected by immunoblot. Cell types are indicated above each autoradiograph, (-) no irradiation, (+) 10 Gy. (B) TK6 lymphoblasts were grown and
separated into cell cycle phase enriched populations by centrifugal elutriation prior to irradiation. Top part, propidium iodide staining was used to
monitor the position of the cells in the cell cycle; Bottom part, the amount of p21 and p53 was detected by immunoblot. The dose or ionizing radiation
and the time after treatment prior to harvest and analysis is indicated. (C) Asynchronously growing HCT116 cells were irradiated, allowed to grow for
an additional 3 h and then stained for 30 min with Hoescht 33,342 and sorted into populations. S-phase synchrony and S-phase contamination in G
1
and G
2
populations was assessed by a 15 min pulse of BrdU (data not shown). Extracts were prepared for immunoblot as indicated in each part. Cyclin
B1 is a loading and synchronization control. This experiment was repeated four times with dierent doses or ionizing radiation in dierent cell lines,
all with similar results. A representative example is shown. (D) Asynchronously growing TK6 cells were treated as indicated and incubated for an ad-
ditional 3 h, after which they were xed and stained with either an antibody to p21 or a control isotype as indicated in the parts. Antibody staining is
on the y-axis and DNA content on the x-axis. The median level of p21 expression at ve points along the DNA prole was determined and plotted. This
graph shows the mean and standard deviation compiled from six independent experiments performed at each indicated dose. (E) Immortalized RPE-
hTERT epithelial cells were irradiated with 5 Gy and stained as described in (D). In these parts, the line indicates above which no cells were detected
by isotype staining. The y-axis in the graph indicates the percent p21-positive cells after subtracting the cells below the gate in the ow cytometric
plots. The graphed data was compiled from three independent experiments. (F) Nutlin-3 did not induce S-phase accumulation of p21. In the top part,
extracts were prepared from HCT116 cells treated as described above each lane and the amount of hdm2, p53 and p21 determined by immunoblot. In
the bottom part, cells were stained as described in (E) and the percentage of p21 positive cells in each cell cycle phase determined.
2718 Cell Cycle Volume 10 Issue 16
in MCF7 cells treated with camptothecin. However, our results
extend their finding by showing that p53 was in the nucleus and
transcriptionally active in S-phase cells, but a proteolytic barrier
prevented p21 protein accumulation.
The effect of cell cycle phase on p53 activation had been
studied by a number of groups. Gottifredi et al. reported that
Ionizing radiation induces double-strand DNA breaks, leading
to activation of chk2 and a number of downstream checkpoints
dependent on p53, cdc25A, BRCA1 or Nbs1.
1
Our finding that
p53-induced p21 accumulation was dependent on the phase of
the cell cycle is consistent with the prior observation of Deptala
and colleagues,
16,20
who examined p53 and p21 accumulation
Figure 2. Transcriptionally active p53 accumulates in all phases of the cell cycle following irradiation. (A) Centrifugal elutriation. As described in the
legend to Figure 1B, we looked at the expression of serine15-phosphorylated p53, chk2, threonine68-phosphorylated chk2 and hdm2 by immunoblot.
(B) Asynchronously growing A549 cells were plated onto coverslips and p53 immunouorescence carried out. The percentage of S-phase cells with
nuclear p53 was plotted as a function of time. This experiment was repeated with MCF7 cells. (C) Enriched populations of TK6 cells were obtained by
cell sorting as described in the legend to Figure 1C and the expression of p21 and PUMA mRNA determined by RNA gel blotting. Ethidium bromide
staining of 28S and 18S rRNA is a loading control. (D) TK6 cells were irradiated with 5 Gy and assessed for p53 expression in dierent cell cycle phases
by ow cytometry as desribed in the legend to Figure 1D. p53 expression before and after irradiation is also depicted graphically (lower part). This is
a representative image from one experiment. (E) Immortalized RPE-hTERT epithelial cells were irradiated with 5 Gy and assessed for p53 expression as
described in the legend to Figure1E.
www.landesbioscience.com Cell Cycle 2719
p21 protein turnover is regulated by a number of pathways,
with both ubiquitin-dependent and ubiquitin-independent
mechanisms contributing to its regulation in S-phase cells. These
include a WISp39-associated chaperone pathway,
23
an SCF
skp2
-
dependent pathway
24-26
and an hdm2-dependent pathway.
27,28
Overwhelming cellular, biochemical and genetic evidence
a transcriptional blockade prevented p53-induced- accumulation
of p21 but not other p53 targets in S-phase RKO cells.
21
These
investigators also reported that a proteolytic mechanism could
contribute in other cell types,
13,14
but the molecular nature of this
mechanism was not addressed. The translocation of p53 to the
nucleus may also be regulated in some cells.
22
Figure 3. Proteasome activity inhibits p21 turnover in S phase. (A) Asynchronously growing TK6 cells were treated with either nocodazole or
hydroxyurea and subsequently released to enter G
1
/G
2
and S phase, respectively. Representative ow proles for the synchronized populations are
shown (up per left). Extracts were prepared from these enriched populations and tested for the ability to degrade in vitro-translated p21. Degrada-
tion reactions were prepared as outlined in the Materials and Methods section and incubated at 30°C for the times indicated above each lane prior to
separation by SDS-PAGE and detection by autoradiography, a representative example of which is shown in the lower left. On the right, the top graphs
combine data from four independent experiments using dierent preparations of substrate and extract, and the bottom part is from a representative
experiment in which a very rapid time course was examined. (B) HCT116 cells were exposed to ionizing radiation and immediately treated for 6 h with
either MG132 or LLM. After treatment, cells were xed and stained with an antibody to p21, which was detected with a secondary antibody conju-
gated to FITC, prior to counterstaining of DNA with propidium iodide. Representative ow cytometric proles are shown with treatment conditions
indicated above the dot-plots. The graph is compiled from at least three independent experiments for each condition.
2720 Cell Cycle Volume 10 Issue 16
cycle phase; however, in S-phase cells, unlike G
1
and G
2
cells,
the p53-dependent accumulation of p21 was limited by hdm2.
This raises an interesting question as to what cell cycle phase-
dependent events are controlling the ability of hdm2 to promote
p21 turnover.
Are their additional factors missing in G
1
cells? Are there fac-
tors that are present in G
1
cells preventing turnover? To address
such questions in the future, we are attempting to develop an in
vitro hdm2-p21 turnover system that will allow the biochemi-
cal identification of such factors (or modifications) and validate
their effect in cellular systems. Additionally, given recent reports
that the mTOR signaling environment could affect the outcome
of p53 induction vis a vis reversible growth arrest or irrevers-
ible senescence,
35,36
it is reasonable to speculate that cell cycle
phase-specific differences in the signaling environment might
also impact mdm2-dependent turnover of p21. Consistent with
this, we note that LY294002 and PD98059 were able to inhibit
G
1
accumulation of p21 protein, although the accumulation of
mdm2 mRNA and protein were unaffected (Bhakta R and Koff
A, unpublished data). In contrast, neither of these compounds,
indicates that p27 is a bona fide substrate for SCF
skp2
in S and
G
2
phase,
28-34
and, although it is generally accepted that skp2
can regulate p21 in cycling cells as well, the evidence for this is
largely drawn from the observation that p21 levels rise in qui-
escent serum-deprived, skp2-deficient mouse embryo fibroblasts
induced to re-enter the cell cycle.
24
Our inability to define a simi-
lar S-phase role for skp2 may reflect cell type- or species-specific
differences or, because we can see a strong G
2
-phase role for skp2
and a more modest one for G
1
-phase, could reflect the purity of
the populations that were studied by Bornstein and colleagues.
For example, the degree of synchronization obtained in mouse
embryo fibroblasts by serum starvation/release protocols is not
sufficient to eliminate G
1
and G
2
cell contamination, when skp2
promotes p21 turnover.
Hdm2 can regulate p21 accumulation in two ways. Indirectly,
hdm2 ubiquitinates p53, targeting it for degradation; reducing
hdm2 can increase p53-dependent transcription of p21. More
directly, hdm2 can bind p21 and target it to the proteasome for
degradation.
27,28
We noted that neither p53-dependent transcrip-
tion of p21 nor the accumulation of hdm2 was affected by cell
Figure 4. Skp2 does not contribute to p21 turnover in S-phase cells. (A) HCT116 cells were transfected with either a scrambled siRNA or a SMARTpool
skp2 siRNA by electroporation. Cells were harvested 48 h later and processed. Left part, immunoblot; Right part, cell cycle phase dependent ac-
cumulation of p21 as measured by ow cytometry. The graph compiles the average and standard deviation of three independent experiments. Cells
received 5Gy of ionizing radiation. (B) Similar to (A); however, we used RPE-hTERT epithelial cells.
www.landesbioscience.com Cell Cycle 2721
Flow cytometry. In order to simultaneously measure protein
and DNA, cells were washed with phosphate buffered saline
(PBS) and fixed in 1% paraformaldehyde at 4°C for 15 min. Cells
were rinsed twice in PBS, permeabilized in 70% ethanol (added
dropwise) and rotated overnight at 4°C. Following fixation, the
cells were washed twice with 1% bovine serum albumin in PBS
(BSA-PBS) and blocked in 5% goat serum (Vector) for 15 min at
room temperature. After washing, primary antibody was added
(diluted in BSA-PBS, in a total volume of 500 μL) and cells were
incubated in the dark at room temperature for the indicated
times: α-p21
WAF1
(clone 2G12, Becton Dickinson) at 1:300, 3
h; α-p53 (clone DO-7, Becton Dickinson) at 1:300, 3 h. Parallel
isotype-matched antibodies were used as negative controls. Cells
were washed twice in BSA-PBS and FITC-conjugated goat anti-
mouse secondary antibody (Dako) added at a 1:60 ratio diluted
in BSA-PBS for 1 h at room temperature. Following antibody
staining, cells were rinsed twice with BSA-PBS, resuspended
in a solution containing 5 μg/mL propidium iodide and 100
μg/mL RNase and processed using a FACScan flow cytometer
nor rapamycin, induced S-phase accumulation of p21 in treated
cells. This suggests that the activity of PI3-kinase and MEK
pathways may be required to control hdm2-p21 turnover events.
Having such an S- and G
2
-phase dependent proteolytic mech-
anism is consistent with the findings that persistent blockade of
cdk activity in S phase can lead to apoptosis (Shapiro 2006).
Thus, the presence of the hdm2-dependent regulatory mecha-
nism to eliminate p21 may allow for the dominance of cdc25-
dependent mechanisms to inhibit kinases, a process which is
more easily reversible. Thus, unraveling the pathways that pre-
vent p21 turnover in S phase may allow us to consider therapeu-
tic strategies that would drive a cell toward apoptosis through
p21-mediated inhibition of cdk activity in S phase.
Material and Methods
Protease inhibitors. MG132, N-Acetyl-L-leucyl-L-leucyl-L-
methioninal (LLM) and N-Acetyl-L-leucyl-L-leucyl-L-norleucinal
(ALLN) were acquired from Sigma and used at 50 μM.
Figure 5. hdm2 contributes to p21 turnover in S-phase HCT116 cells but not RPE-hTERT. This is exactly as described in the legend to Figure 4, except
hdm2 was knocked down instead of skp2.
2722 Cell Cycle Volume 10 Issue 16
Laser scanning cytometry. Adherent cells were plated in
4-well chamber slides (Nalge Nunc) at a density of 85,000 cells/
well and allowed to attach overnight. Following irradiation, the
cells were fixed using 1% paraformaldehyde for 15 min at 4°C
and subsequently permeabilized in 80% ethanol at 4°C over-
night. Fixed slides were initially rinsed in BSA-PBS twice for
5 min and then blocked with 5% goat serum for 15 min. Either
α-p53-FITC-conjugated antibody or an IgG2b-FITC isotype
(1 mg/mL) diluted in BSA-PBS was carefully dropped onto the
slide and then covered with a layer of paralm to allow even dis-
tribution of the antibody along the slide. After a 2 h incubation at
room temperature in the dark, slides were thoroughly rinsed with
PBS and placed in a PI (5 μg/mL)/RNase (100 μg/mL) solu-
tion overnight at 4°C. The slides were then mounted with cover-
slips using PBS and analyzed under a laser-scanning microscope
(CompuCyte). Analysis included identification of 100 individual
S-phase cells.
siRNA transfection. SMARTpools of four siRNAs specific
for human skp2 or hdm2 were obtained from Dharmacon.
Approximately 1 x 10
6
HCT116 cells were resuspended in 81.8 μl
of nucleofector solution V, 18.2 μl supplement and 8 μl 20 μM
siRNA (2 μg) and electroporated using the Nucleofector device
as recommended by the manufacturer (Amaxa). Cells were har-
vested either 24 (hdm2) or 48 (skp2) hours after electroporation.
Acknowledgments
The authors thank Jacob Jacobberger (Case Western Reserve
University) and Frank Traganos (Brander Cancer Institute, New
York Medical College) for advice with the p21 dual-color flow
cytometry; Gloria Juan and Carlos Cordon-Cardo (MSKCC,
Department of Pathology) for assistance with the analysis of
p53 localization; and Vincent Sahi, Patrick Anderson and Cris
Bare (MSKCC, Flow Cytometry Core Facilities) for devel-
oping the conditions for cell sorting. We also thank Stephen
Jones (University of Massachussetts/Worcester), Carol Prives
(Columbia University), Vanesa Gottifredi (Fundacion Instituto
Leloir) and Pengbo Zhao (Weill College of Medicine, Cornell
University) for helpful discussions as our work progressed and
Roshni Mody-Bhakta and Carmen Carneiro, former members of
the laboratory who provided the initial insight and preliminary
data on the S phase-dependent regulation of p21. This work was
supported by grants to Andrew Koff from the NCI (CA89563)
and Golfers Against Cancer Foundation. Additional support was
provided by an Institutional Core Grant to Memorial Sloan-
Kettering Cancer Center (NCI). Daniel Ciznadija was sup-
ported by fellowships from the Brain Tumor Center (Memorial
Sloan-Kettering Cancer Center) and the Joel A. Gingras Jr.
Basic Research Fellowship from the American Brain Tumor
Association.
(Becton Dickinson). Data was analyzed on FlowJo version 6.0
software (Tree Star).
Western blot analysis. Protein lysates were prepared and
analyzed as we previously described in reference 28 and 37.
Lysates (80 μg) were resolved by sodium dodecyl sulfate (SDS)
PAGE and transferred onto PVDF membranes (Millipore,
Bedford, MA). Primary antibodies were added overnight at room
temperature, diluted in TNT (25 mM Tris base; 150 mM NaCl;
0.5% Tween-20). Following washing in TNT, the appropriate
secondary antibodies conjugated to horseradish peroxidase were
added (Jackson Laboratories), diluted 1:5,000 in TNT for 1 h
at room temperature. Membranes were washed, Supersignal Pico
ECL chemiluminescent reagent (Pierce) added and the mem-
branes exposed to film (Kodak, XAR). The following primary
antibodies from Santa Cruz were used: p21 (F5; 1:200), hdm2
(SMP14; 1:200), cyclin B1 (GNS1; 1:1,000), skp2 (H435;
1:200). The p53S15P, chk2T68P and chk2 were all obtained
from Cell Signaling Technologies and used at 1:500. The p53
antibody (clone DO-7) was obtained from Becton Dickinson
and used at 1:200.
Isolation of cell cycle-phase specific populations (cell sort-
ing). HCT116 and TK6 cells at an approximate density of
6 x 10
5
cells/mL were aliquoted and incubated with 3 μg/mL of
Hoechst 33,342 at 37°C for 30 min. The cells were then pelleted
and resuspended in 5 mL of media containing Hoechst 33,342
and placed on ice for sorting. Sorts were completed within 3 h.
RNA gel blotting. Total RNA was collected using the RNeasy
kit (Qiagen, Valencia, CA) and resuspended in diethyl pyrocar-
bonate (DEPC)-treated water. The samples were lyophilized
and resuspended in 20 mM morpholine-propanesulfonic acid,
5 mM sodium acetate and 1 mM EDTA, 6.5% formaldehyde
and 25% formamide heated to 55°C for 15 min and then chilled
on ice. RNA was resolved on a 0.6%-agarose formaldehyde gel
containing 20 ng/mL ethidium bromide (EtBr) at 100 V for 3 h.
The RNA was blotted to Hybond-N
+
membrane (Amersham
Biosciences) and RNA crosslinked to the membrane with UV
light for 12 sec (Stratalinker; Stratagene).
cDNAs containing the entire open reading frame of p21 and
PUMA were used to generate probes using the Prime-It Random
Primer Labeling kit (Stratagene, La Jolla, CA). QuickHyb
hybridization solution (Stratagene, La Jolla, CA) was used to pre-
hybridize the membrane for 30 min at 68°. A mixture of 1 x 10
7
CPM of probe and 100 μg of sonicated salmon sperm DNA were
boiled for 5 min and added to the hybridization buffer for 2 h.
The membrane was then washed with 2x SSC containing 0.1%
SDS twice for 15 min at room temperature and once for 35 min
with 0.1x SSC containing 0.1% SDS at 68°C. For detection of
the specific RNA, the membrane was exposed to XMR film
(Eastman Kodak, Chedex, France) overnight at -80°C.
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  • [Show abstract] [Hide abstract] ABSTRACT: Comment on: Ciznadija D, et al. Cell Cycle 2011; 10:2714-23.
    Article · Oct 2011
  • [Show abstract] [Hide abstract] ABSTRACT: Comment on: Ciznadija D, et al. Cell Cycle 2011; 10:2714-23.
    Article · Oct 2011
  • [Show abstract] [Hide abstract] ABSTRACT: The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) is a regulatory factor of the cell cycle. Its transcriptional activation and its protein stability are tightly controlled by several distinct mechanisms. S100A11 is a member of the S100 family of Ca(2+) -binding proteins involved in several biological processes including cell cycle progression and signal transduction. Here, we show that down-regulation of S100A11 results in the reduction of p21 protein in human HaCaT keratinocytes. It seems that a ubiquitin-independent proteasomal degradation process is involved in p21 degradation in S100A11 down-regulated cells. The application of a proteasome inhibitor stabilized p21 protein in these cells. Analyses of distinct signal transduction pathways revealed a disturbed phosphatidylinositol-3-kinase/Akt pathway after S100A11 knock-down. We determined that the glycogen synthase kinase-3, which is negatively regulated by PI3K/Akt, was activated in cells possessing knocked-down S100A11 and seems to be involved in p21 protein destabilization. The application of a specific inhibitor of GSK3 resulted in an increase of the p21 protein level in S100A11 down-regulated HaCaT cells. GSK3 is able to phosphorylate p21 at T57 that induces p21 proteasomal turnover. Mutation of the GSK3 site threonine-57 into alanine (T57A) stabilizes p21 in HaCaT cells lacking S100A11. Beside decreased p21 protein, down-regulation of S100A11 triggered the induction of apoptosis in HaCaT cells. These observations suggest that S100A11 is involved in maintenance of p21 protein stability and seems to function as an inhibitor of apoptosis in human HaCaT keratinocyte cells. Thus, the data shed light on a novel pathway regulating p21 protein stability. This article is protected by copyright. All rights reserved.
    Article · Jun 2013
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