© 2012 Landes Bioscience.
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Cell Cycle 11:9, 1818-1826; May 1, 2012; © 2012 Landes Bioscience
Decreased translation of p21waf1 mRNA causes
attenuated p53 signaling in some
p53 wild-type tumors
1818 Cell Cycle Volume 11 Issue 9
*Correspondence to: Alan Eastman; Email: Alan.R.Eastman@Dartmouth.edu
Submitted: 02/17/12; Revised: 03/28/12; Accepted: 03/29/12
The DNA of a cell is constantly under attack by both external
insults, such as the sun’s radiation, and internal insults, such as
free radicals produced during normal metabolism. To ensure
integrity of the DNA, the cell utilizes DNA damage checkpoints
to arrest cell cycle progression and allow time for DNA repair.
When DNA double-strand breaks are detected, ATM kinase is
activated, which, in turn, activates Chk2 via phosphorylation of
threonine 68.1 Double-strand breaks are also processed to single-
stranded DNA that activates ATR, and, as a consequence, Chk1 is
phosphorylated at serine 345.2,3 Chk1 is then autophosphorylated
at serine 296 to become fully activated.4 Subsequently, activated
Chk1 and Chk2 inhibit the CDC25 family of phosphatases that
remove the inhibitory phosphorylation on the cyclin-dependent
kinase (CDK)/cyclin complexes.5 Thus, Chk1 and Chk2 acti-
vation leads to rapid cell cycle arrest. In addition, ATM, ATR,
Chk1 and Chk2 phosphorylate the p53 tumor suppressor at
serines 15 and 20, which disrupts the interaction between p53
and its negative regulator, MDM2.6 Once activated, p53 induces
transcription of the CDK inhibitor p21waf1 and thus provides a
second mechanism to arrest cell cycle progression.7
DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells
through induction of p21waf1 protein. Arrest permits cells to repair the damage and recover. the frequent loss of p53 in
tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild-type tumor cell lines,
such as HCt116 and U2oS, are also sensitive to inhibition of Chk1 due to attenuated p21waf1 induction upon DNA damage.
the purpose of this study is to determine the cause of this attenuated p21waf1 protein induction. We find that neither the
induction of p21waf1 mRNA nor protein half-life is sufficient to explain the low p21waf1 protein levels in HCt116 and U2oS
cells. the induced mRNA associates with polysomes, but little protein is made, suggesting that these two cell lines have
a reduced rate of p21waf1 mRNA translation. this represents a novel mechanism for disruption of the p53-p21waf1 pathway,
as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this
attenuated p21waf1 expression may render some p53 wild-type tumors sensitive to a combination of DNA damage plus
Li-Ju Chang and Alan eastman*
Department of pharmacology and toxicology; Dartmouth Medical School and Norris Cotton Cancer Center; Lebanon, NH USA
Key words: p53 response, p21waf1, cell cycle checkpoints, Chk1, UCN-01, MK-8776
Abbreviations: CDK, cyclin-dependent kinase; UCN-01, 7-hydroxystaurosporine; miRNA, microRNA; SN38,
As the p53-p21waf1 pathway requires the transcription and
accumulation of newly synthesized p21waf1 protein, it is slower
to induce arrest than the Chk1/2-CDC25 pathway.7 However,
the p53-p21waf1 pathway is crucial for maintenance of arrest, as
shown by our studies comparing isogenic cell lines.8 For example,
the topoisomerase I inhibitor SN38 induces S-phase arrest in the
p53 wild-type MCF10A cells as well as their p53- and p21waf1-
suppressed derivatives.8,9 Chk1 inhibition by 7-hydroxystau-
rosporine (UCN-01) had no impact on the p53 wild-type cells
but abrogated arrest in both the derivatives resulting in S and G2
phase progression. Based on these observations, it was expected
that all p53 wild-type tumors would be resistant to inhibition of
Chk1 by UCN-01, but we identified several that remained sensi-
tive. In HCT116 and MCF7 cells, Chk1 inhibition abrogated
SN38-induced arrest.9 We also demonstrated that this sensitivity
to checkpoint abrogation correlated with an attenuated induction
In this study, we examined the cause of the attenuated p21waf1
induction in HCT116 cells and in another p53 wild-type cell
line, U2OS. We find that this defect is not due to a failure
to induce p21waf1 mRNA or to a shorter protein half-life. The
induced mRNA associates with polysomes, but little protein is
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www.landesbioscience.com Cell Cycle 1819
p53 can prevent UCN-01-mediated abroga-
tion of S-phase arrest induced by SN38.8,10
We extended these experiments to p53 wild-
type tumors, and found that p53 could also
prevent UCN-01-mediated abrogation of
arrest in some, but not all, cell lines.9 Cell
lines that remained sensitive to checkpoint
abrogation included HCT116 and MCF7.
Here, we report that U2OS cells are also
sensitive to checkpoint abrogation.
As UCN-01 has been shown to have
many off-target effects, we reconfirmed
these findings with a more specific Chk1
inhibitor, MK-8776 (previously known as
SCH900776).11,12 SN38 at 10 ng/ml induces
S-phase arrest in MCF10A and U2OS
cells, but primarily a G2 arrest in HCT116
cell (Fig. 1). The limited S-phase arrest in
HCT116 cells has been attributed to a defect
in Mre11.13 On removal of SN38 after 24
h, MCF10A cells remained arrested in S
phase for at least an additional 24 h, whereas
U2OS slowly progressed to G2 and HCT116
remained in G2.
Addition of MK-8776 to SN38-arrested
cells did not abrogate arrest in MCF10A
cells (Fig. 1), while a similar experiment in
the p53 mutant MDA-MB-231 cells rap-
idly abrogated both S and G2 arrest.12 In the
U2OS cells, MK-8776 accelerated the rate
of progression through S phase and through
mitosis. After 24 h in MK-8776, a large pro-
portion of the U2OS cells are seen with G1
and sub-G1 DNA content. In the HCT116
cells, the majority of the cells remained in
G2/M upon incubation with MK-8776.
However, flow cytometry cannot discrimi-
nate between the G2/M populations. We
have previously shown that Chk1 inhibition
abrogates G2 arrest and induces mitosis in
HCT116 cells but they fail to undergo cyto-
kinesis.9 The result is tetraploid cells with
numerous micronuclei. This mitotic catas-
trophe was also observed with MK-8776.
These results demonstrate that some p53
wild-type cancer cells have limited capacity
to maintain cell cycle arrest when damaged,
and that the ability to arrest is dependent on
Attenuated p21waf1 induction by SN38
in HCT116 and U2OS. We previously noted a delayed p21waf1
protein induction in HCT116 cells compared with MCF10A
in SN38-treated cells. We thus hypothesized that an adequate
p21waf1 protein level is crucial for maintenance of cell cycle arrest,
and this was confirmed when we observed abrogation of S-phase
arrest by Chk1 inhibition in MCF10A/Δp21waf1 cells.9 To extend
made, suggesting these two tumor cell lines have a reduced rate
of p21waf1 mRNA translation.
Abrogation of cell cycle arrest by MK-8776 in HCT116 and
U2OS. Our previous studies using MCF10A cells showed that
Figure 1. Comparison of the efficacy of MK-8776 to abrogate SN38-induced S and G2 arrest in
p53 wild-type cell lines. Cell were incubated with 10 ng/ml SN38 for 24 h and then incubated in
either media with or without 1 μM MK-8776. Cells were harvested and assayed for DNA content
by flow cytometry.
© 2012 Landes Bioscience.
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was markedly elevated by 4 h and remained elevated through 24
h in all three cell lines (Fig. 3A–C). The results were expressed in
two different ways: (1) relative to the protein level at 0 h for each
cell line or (2) relative to the protein level at 0 h for MCF10A cells
(Fig. 3D and E). The latter expression provides a comparison of
the absolute level of the protein. Although the fold induction of
p53 was similar in all three cell lines, the lower basal levels of
p53 in U2OS resulted in lower absolute p53 levels than MCF10A
after 24 h.
Induction of p21waf1 protein was slightly slower than p53 in
MCF10A cells but was clearly detectable by 8–10 h. At compara-
ble exposures of the western blots, p21waf1 did not become detect-
able until 20–24 h in the other two cell lines. When expressed
as fold induction, MCF10A and HCT116 cells were fairly simi-
lar, but the absolute level of protein present was significantly less
in HCT116 cells. In U2OS cells, both the fold induction and
the absolute level of protein expressed was significantly less than
MCF10A cells. These results reiterate that the amount of p21waf1
protein does not reflect the level of p53 protein induced.
We concurrently assessed several DNA damage responses that
occur when cells are damaged. In all the cells, Chk1 was phos-
phorylated at serines 345 and 296 within 2 h, suggesting that
the delayed p21waf1 induction in HCT116 and U2OS is not due
to lack of drug uptake or DNA damage. Phosphorylation of p53
at serine 15 occurred with the same kinetics in MCF10A and
HCT116 cells, but only in the former did this correlate with the
kinetics of p21waf1 induction. U2OS cells showed a lower level of
p53 phosphorylation that may relate to the slightly lower level of
1820 Cell Cycle Volume 11 Issue 9
p53 induced in these cells. Overall, these results show that SN38
is able to damage the DNA, activate Chk1, induce phosphoryla-
tion and accumulation of p53 in all the cell lines, but they differ
markedly in their ability to express p21waf1.
We also analyzed the kinetics of protein expression follow-
ing removal of SN38. Upon release from SN38, MCF10A cells
appeared to partially recover, as reflected in the decrease in phos-
phorylation of Chk1 and p53, albeit p21waf1 remained high. In
HCT116 and U2OS cells, Chk1 and p53 remained phosphory-
lated, and in fact, serine 345-Chk1 phosphorylation continued to
rise in U2OS cells, suggesting that they were not recovering from
the insult. Most notable is the continued and dramatic increase
in p21waf1 in HCT116 and U2OS that occurs during the 24 h fol-
lowing drug removal.
p21waf1 mRNA is insufficient to explain the low p21waf1 pro-
tein levels in HCT116 and U2OS. We next determined whether
this research, we compared the absolute p53 and p21waf1 protein
levels between MCF10A, HCT116 and U2OS cells following
incubation with SN38 (Fig. 2A). The levels of protein presented
here and in subsequent figures were obtained from densitometry
of multiple exposures of western blots to avoid analysis of over-
exposed bands and from comparison to a standard curve gen-
erated for each antigen demonstrating that the values recorded
were in the linear range of detection. In addition, the same num-
ber of cells was loaded in each lane, and this resulted in a constant
amount of actin in each lane.
Overall, MCF10A and HCT116 cells showed fairly similar lev-
els of p53, while U2OS cells demonstrated only about 2-fold less
by 24 h of SN38 treatment. In contrast, there were marked dif-
ferences in the absolute levels of p21waf1. The p21waf1 expression in
HCT116 and U2OS was about 30% and 2%, respectively, com-
pared with MCF10A at 24 h of SN38. Interestingly, both the p53
and p21waf1 levels continued to increase in HCT116 and U2OS
cells after removal of SN38, whereas both proteins decreased in
MCF10A. To show that this attenuated p21waf1 induction is not
due to low p53 induction, we compared the ratio of p21waf1 to p53
protein level. The results clearly show that despite strong activa-
tion of p53, the HCT116 and U2OS cells exhibit a very attenu-
ated induction of p21waf1 at 24 h of SN38 (Fig. 2B). This p21waf1
to p53 ratio increased in all three cell lines within the next 24 h.
Comparison of p53 and p21waf1 protein kinetics induced by
SN38. To obtain further perspective on the relative induction of
p53 and p21waf1 between 0 h and 24 h, we performed a detailed
kinetic analysis during incubation with SN38. The p53 protein
Figure 2. SN38-induced p53 and p21waf1 protein levels. (A) Cells were
incubated with 10 ng/ml SN38 for 24 h and then released from SN38
for 24 h. the levels of p53 and p21waf1 protein were assessed by western
blotting. Numerical values compare expression level to the 24 h-treated
MCF10A cells. (B) the ratio of p21waf1 to p53 protein levels after 24 h of
SN38 and after an additional 24 h in fresh medium were compared with
that of MCF10A cells after 24 h of SN38.
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Figure 3. For figure legend, see page 1822.
© 2012 Landes Bioscience.
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(1) relative to the mRNA level at 0 h for each cell line or (2)
relative to the mRNA level at 0 h for MCF10A cells (Fig. 4).
The basal level of mRNA was only 2-fold lower in the HCT116
and U2OS cells compared with MCF10A. The fold induction by
SN38 was quite similar in all three cell lines. By 24 h, the abso-
lute level in HCT116 was the same as in MCF10A, while the level
in U2OS was about half. Hence, the difference is insufficient to
explain the very low protein level observed. Upon removal of
SN38, the p21waf1 mRNA began to decrease in MCF10A cells
but continued to increase in both HCT116 and U2OS cells. The
mRNA levels in these latter two cell lines seem to correlate with
the increase in protein between 24 and 48 h.
p21waf1 protein half-life is insufficient to explain the low
p21waf1 protein levels in HCT116 and U2OS. We next assessed
whether the difference in SN38-induced p21waf1 protein levels
could be explained by differences in p21waf1 protein half-life.
SN38-damaged cells were incubated with cycloheximide and the
decay of p21waf1 assessed by western blotting (Fig. 5). The p21waf1
protein half-life was about 30 min for all three cells lines and
therefore cannot explain the differences in p21waf1 protein levels.
Additionally, the p21waf1 protein half-life remained about 30 min
for all three cell lines 24 h after removal of drug, suggesting the
increase of p21waf1 protein in HCT116 and U2OS is not due to
changes in the protein half-life.
Inhibition of p21waf1 mRNA translation. Having been unable
to attribute the level of p21waf1 protein to transcriptional differ-
ences or protein half-life, we next investigated whether there
are differences in p21waf1 mRNA association with polysomes.
1822 Cell Cycle Volume 11 Issue 9
Polysomes were purified from SN38-damaged cells, and poly-
some-associated p21waf1 mRNA was assessed (Fig. 6A). The
majority of p21waf1 mRNA was associated with polysomes
(> 4 ribosomes/transcript) in all three cell lines, suggesting that
translation initiation had occurred. As the HCT116 and U2OS
cells synthesize little p21waf1 protein, we assume that these poly-
somes are arrested on the transcript.
As miRNAs have been shown to affect p21waf1 mRNA transla-
tional efficiency, we hypothesized that high levels of miRNAs in
HCT116 and U2OS are inhibiting p21waf1 mRNA translation.14
Recent studies identified a total of 44 miRNAs that inhibit p21waf1
expression and/or Ras-induced p21waf1-mediated growth arrest.15,16
We thus used microRNA microarray to analyze whether any of
these 44 miRNAs were highly expressed in HCT116 and U2OS
cells compared with MCF10A cells (Fig. 6B). The majority of
these miRNA were expressed at negligible levels. Three miRNAs
(miR-17-5p, miR-20a and miR-106a) were expressed at similar
levels in all three cell lines. However, miR-106b, miR-93 and
miR-130b were significantly elevated in the two tumor cell lines
compared with MCF10A cells. We confirmed this with real-time
PCR (Fig. 6C). These three miRNAs were not induced by SN38,
rather, they were constitutively high in both HCT116 and U2OS
compared with MCF10A cells. Additionally, we found miR125a
to be highly expressed in U2OS cells compared with the other
two cell lines (Fig. 6B). As miR-125a has also been shown to
repress p53 expression and subsequently p21waf1 levels, this cor-
relates with the low p53 induction observed in SN38-treated
U2OS cells.17 These results suggest that expression of these miR-
NAs may be responsible for the arrested translation observed in
HCT116 and U2OS cells.
the p21waf1 mRNA levels could explain the difference in SN38-
induced p21waf1 protein levels. The results were expressed either
Figure 3 (See previous page). Kinetics of p53 and p21waf1 protein expression following SN38 treatment. (A) MCF10A, (B) HCt116 and (C) U2oS cells
were incubated with 10 ng/ml SN38 from 0–24 h. the drug was removed, and cells incubated for an additional 24 h in fresh medium. Cells were
harvested at the indicated times and proteins detected by western blotting. (D) p53 and (e) p21waf1 protein levels were quantified by densitometry of
multiple exposures of western blots and from comparison to a standard curve generated for each antigen. the left panels show protein induction
compared with the untreated control of each cell line. the right panels show protein induction compared with the level in untreated MCF10A cells.
Figure 4. Kinetics of p21waf1 mRNA induction by SN38 treatment. Cells were incubated with 10 ng/ml SN38 from 0–24 h. the drug was removed and
cells were incubated for an additional 24 h in fresh medium. Cells were harvested at the indicated times and p21waf1 mRNA quantified by Rt-qpCR.
GApDH was used as an internal control. the left panel shows mRNA induction compared with the untreated control of each cell line. the right panel
shows mRNA induction compared with the untreated control of MCF10A. the error bars represent Se of at least three independent experiments.
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As the guardian of the genome, p53 suppresses cellular trans-
formation by inducing arrest, apoptosis, DNA repair and dif-
ferentiation in damaged cells.18 Thus, it is no surprise that the
p53 function is almost always compromised in tumor cells.18
This allows these tumor cells to survive DNA damage and
oncogene activation that would normally result in cell death.19
Approximately 50% of cancers have mutated p53, and some of
the remaining p53 wild-type cancers have subdued p53 levels or
activities.20 In this study, we show how dysregulation of the p53
pathway can still occur despite normal levels and activity of the
We found two well-characterized p53 wild-type cancer cell
lines, HCT116 and U2OS, which exhibit an attenuated p21waf1
induction upon treatment with SN38 when compared with the
non-tumorigenic MCF10A cells. This defect is not due to low
p53 induction or lack of p53 activity, as all the cell lines produce
similar levels of p21waf1 mRNA. As the p21waf1 protein half-life
in both cell lines is also comparable to MCF10A cell lines, we
looked for defects in p21waf1 mRNA translation. The major-
ity of p21waf1 mRNA was associated with polysomes in all three
cell lines, hence, we conclude that the differential expression of
p21waf1 protein is due to differences in translation of the mRNA,
a process that is regulated by microRNA.
MicroRNAs (miRNAs) regulate gene expression by affecting
translational efficiency and/or stability of the target mRNA.14 Of
the 44 miRNAs shown to either inhibit p21waf1 expression or Ras-
induced p21waf1-mediated growth arrest,15,16 we identified three
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(miR-106b, miR-93 and miR-130b) that were constitutively high
in HCT116 and U2OS compared with MCF10A cells. miR-106b
and miR-93 are both part of the miR-106b family and have pre-
viously been shown to directly downregulate p21waf1 expression
through binding to its 3' UTR.14,21-23 The other elevated miRNA,
miR-130b, has been predicted to bind to an alternate sequence
in the p21waf1 3' UTR. Overexpression of these miRNAs cause
increased cell proliferation and accelerated G1/S transition.15,22,23
Consequently, it is likely that the presence of these miRNA in
HCT116 and U2OS cells contributes to the attenuated induction
of p21waf1 protein.
Upon DNA damage, the rapidly activated Chk1-Cdc25 path-
way induces an immediate arrest, whereas the slow-operating p53-
p21waf1 pathway sustains the arrest. In this study, we observed that
Chk1 is downregulated once the p53-p21waf1 pathway becomes
fully activated in MCF10A cells. This may be accomplished
through p21waf1-mediated inhibition of Chk1 transcription. A
previous study also observed Chk1 downregulation in daunoru-
bicin-treated HCT116 cells,24 but we did not observe Chk1 sup-
pression in SN38-treated HCT116 cells. Even when the levels of
p21waf1 in HCT116 became twice that in MCF10A cells 24 h after
the removal of SN38, the Chk1 levels remained unchanged. In
U2OS cells, p21waf1 production is very low, and p21waf1 levels never
reach more than 20% of the levels observed in the other two cell
lines. As the impact of p53-activating agents on the cellular envi-
ronment can be different, it will be interesting to see if this defec-
tive p21waf1 protein production in HCT116 and U2OS also occurs
with other p53-activating agents that are either genotoxic, such as
doxorubin, or non-genotoxic, such as Nutlin-3.
Figure 5. p21waf1 protein half-life following SN38 treatment. Cells were incubated with 10 ng/ml SN38 for 24 h or for an additional 24 h in fresh medium.
Cells were then incubated with the protein synthesis inhibitor cycloheximide (10 μg/ml) for the indicated times. the half-lives are presented as the
means ± Se for three independent experiments.
© 2012 Landes Bioscience.
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1824 Cell Cycle Volume 11 Issue 9
certain p53 wild-type tumors are attenuated in DNA damage-
induced p21waf1 protein and thus sensitive to Chk1 inhibition.
Materials and Methods
active metabolite of the topoisomerase I inhibitor irinotecan,
was provided by Pfizer Global. MK-8776 (previously known as
SCH900776) was provided by Merck. Both drugs were dissolved
in DMSO at > 1,000 times the final concentration used in experi-
ments. Cycloheximide was obtained from Sigma-Aldrich.
Cell culture. MCF10A immortalized breast cell lines and
U2OS osteosarcoma cell lines were obtained from American
Type Culture Collection. HCT116 colorectal carcinoma cell lines
were obtained from Dr. Vogelstein (Johns Hopkins University).30
U2OS and MCF10A cells were maintained in DMEM/F12
SN38 (7-ethyl-10-hydroxycamptothecin), the
The tumor suppressive activity of p53 was first made clear
with the identification of p21waf1 as its mediator of growth sup-
pression.25 As p53 is often mutated in cancer, one might expect
to find inactivating mutations of its downstream target, p21waf1,
in wild-type p53 tumors.26 However, p21waf1 mutations are rare,
yet reduced p21waf1 expression has been associated with colorec-
tal, cervical, head and neck and small-cell lung cancers.27 Why
would cancer cells repress p21waf1 expression rather than delete its
function through mutation? This can perhaps be explained by
evidence showing that low basal levels of p21waf1 promote active
cyclin-CDK complex formation, and that cytoplasmic p21waf1
seems to exhibit antiapoptotic activites.14,27-29 But how would
cancer cells maintain p21waf1 protein levels low enough to avoid
arrest, especially when its mRNA is induced upon p53 activation?
In this study, we show that this can be accomplished through
suppression of p21waf1 mRNA translation. This helps explain why
Figure 6. Inhibition of p21waf1 mRNA translation. (A) Cells were incubated with 10 ng/ml SN38 for 24 h and then collected for polysome profiling. Sam-
ples were divided into four fractions. Fraction one includes the free mRNAs and monosomes, while fractions two, three and four include polysome of
increasing size. the bars represent the range of two independent experiments. (B) Cells were incubated with 10 ng/ml SN38 for 24 h and analyzed for
levels of 44 miRNAs shown to affect p21waf1 protein levels. (C) Cells were left untreated or incubated with 10 ng/ml SN38 for 24 h. Changes in miR-106b,
miR-93 and miR-130b levels were assessed by real-time pCR.
© 2012 Landes Bioscience.
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buffered saline and then lysed by addition of urea sample buffer
[4 M urea, 5% β-mercaptoethanol, 2% sodium dodecyl sulfate,
50 mM Tris (pH 6.8), 0.01% bromophenol blue and protease/
phosphatase inhibitor cocktail]. Samples were immediately
boiled for 5 min and stored at -80°C. Proteins were separated
by SDS-PAGE and transferred to polyvinylidene difluoride
membranes. Membranes were blocked with 5% non-fat milk
in Tris-buffered saline, 0.1% Tween 20, and then probed with
the appropriate primary antibody in 5% non-fat milk or 5%
BSA overnight at 4°C [p53 (DO-1) (Santa Cruz Biotechnology,
sc-126), p21waf1 (C-19) (Santa Cruz Biotechnology, sc-397),
Chk1 (G-4) (Santa Cruz Biotechnology, sc-8408), phos-
phoserine-15-p53 (Cell Signaling Technology, 9286), phos-
phoserine-345-Chk1 (Cell Signaling Technology, 2341),
phosphoserine-296-Chk1 (Cell Signaling Technology, 2349),
actin (Ab-1) (Calbiochem, CP01)]. Subsequently, membranes
were washed in Tris-buffered saline, 0.1% Tween 20, and
incubated with secondary antibody conjugated to horseradish
peroxidase (Bio-Rad). Proteins were visualized by enhanced
chemiluminescence (GE Healthcare).
Quantification of p21waf1 protein half-life. Cells were
incubated with the protein synthesis inhibitor cycloheximide
(10 μg/ ml) for the indicated times. p21waf1 protein levels were
detected by western blotting, and densitometry was performed
within the quantitative range to measure the intensity of the
bands. We assume that protein degradation follows first-order
decay kinetics.32 The quantified protein intensity data was ini-
tially log-transformed, and then a linear best-fit curve was used
www.landesbioscience.com Cell Cycle 1825
to determine the decay rate constant (k). From the decay rate
constant, the half-life (T1/2) was calculated.
Polysome fractionation. Cells were incubated with 0.1 mg/
ml cycloheximide for 3 min at 37°C and washed with ice-cold
phosphate-buffered saline containing 0.1 mg/ml cycloheximide.
Cells were lysed in 20 mM Tris, 2.5 mM MgCl2, 120 mM KCl
(pH 7.5), 0.5 mM dithiothreitol, 0.1 mg/ml cycloheximide,
0.5 mg/ml heparin, 0.5% NP-40 and 100 U/ml SUPERase-In
(Ambion, AM2696). The lysate was subsequently centrifuged at
3,000 rpm in a microcentrifuge for 5 min at 4°C. The superna-
tant was layered on a 10–50% w/v linear sucrose gradient and
centrifuged for 3 h at 4°C at 29,000 rpm in a Surespin 630 Rotor
(Sorvall). Gradients were fractionated using Gradient Station
(Biocomp) while continuously monitoring absorbance at 254
nm. RNA was extracted from individual fractions using RNeasy
Mini Kit (Qiagen).
Microarray. Total RNA was extracted using Tri Reagent
(Molecular Research Center Inc.,) according to the manufac-
turer’s instructions. 500 ng of total RNA was poly(A) tailed
and then directly ligated to a biotin-labeled dendrimer using the
FlashTag Biotin HSR kit (Genisphere). Labeled RNA was used
for hybridization to the GeneChip miRNA Array (Affymetrix)
for 16 h at 48°C and 60 rpm. Following hybridization, the
miRNA arrays were washed and stained with streptavidin-PE
(Affymetrix). Fluorescent images were obtained with 500GX
scanner (Illumina) and processed with GeneChip Command
Console software (Affymetrix).
Real-time PCR for mRNA. Total RNA was isolated using
RNeasy Mini Kit (Qiagen). cDNA was synthesized using iScript
cDNA synthesis kit (Bio-Rad). Real-time PCR was performed
with CFX96 real-time system (Bio-Rad) using iQSYBR Green
PCR Supermix (Bio-Rad). Amplification cycles were: 95°C for
10 min, then 30 cycles of 94°C for 30 sec, 56.7°C for 30 sec and
72°C for 1 min. The fold change of p21waf1 mRNA was deter-
mined by the equation of Pfaffl using GAPDH as the endogenous
control.33 The mRNA specific primer sequences are provided in
Real-time PCR for miRNA. Total RNA was isolated using
Tri Reagent (Molecular Research Center Inc.,) as above. cDNA
was synthesized from 400 ng of total RNA using miScript
Reverse Transcription Kit (Qiagen). Real-time PCR was per-
formed with CFX96 real-time system (Bio-Rad) using miScript
SYBR Green PCR Kit (Qiagen) according to the manufacturer’s
protocol. Amplification cycles were: 95°C for 15 min, then 45
cycles of 94°C for 15 sec, 55°C for 30 sec and 70°C for 30 sec.
The fold change of miRNA was determined by the equation of
Pfaffl using Z30 as the endogenous control.33 The miRNA spe-
cific primer sequences are provided in Table 1.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
This research was supported by NIH grant CA117874
(A. Eastman) and Cancer Center Support grant CA23108 to the
Norris Cotton Cancer Center.
supplemented with 10% fetal bovine serum plus antibiotic/anti-
mycotic. For MCF10A cells, the medium was also supplemented
with 8 μg/ml insulin, 20 ng/ml epidermal growth factor and
500 ng/ml hydrocortisone. HCT116 cells were maintained in
McCoy’s 5a medium supplemented with serum and antibiotics
Cell cycle analysis. Cell cycle analysis was performed as
described previously, whereby cells were harvested, fixed in
ethanol, incubated with ribonuclease and stained with propid-
ium iodide.31 DNA content was determined on a FACScan flow
cytometer (Becton Dickinson).
Western blot analysis. Cells were rinsed with phosphate-
Table 1. primers used for real-time pCR
Name Primer (5'-3')
p21waf1 (CDKN1A) Forward5'-AtG tCA GAA CCG GCt GGG GA-3'
Reverse5'-GCC Gtt ttC GAC CCt GAG AG-3'
5'-CtC AGA CAC CAt GGG GAA GGt
Reverse5'-AtG AtC ttG AGG CtG ttG tCA tA-3'
Reverse miScript SYBR Green PCR Kit (Qiagen)
miR-93 Forward5'-CAA AGt GCt Gtt CGt GCA GGt AG-3'
miR-106bForward 5'-tAA AGt GCt GAC AGt GCA GAt-3'
miR-130b Forward 5'-CAG tGC AAt GAt GAA AGG GCA t-3'
Z30Forward 5'-tGG Ctt tGA CCA GGG tAt GAt C-3'
© 2012 Landes Bioscience.
Do not distribute.
p21waf1 induction and cyclin B repression render-
ing them sensitive to Chk1 inhibitors that abrogate
DNA damage-induced S and G2 arrest. Mol Cancer
Ther 2008; 7:252-62; PMID:18281511; http://dx.doi.
10. Kohn EA, Ruth ND, Brown MK, Livingstone M,
Eastman A. Abrogation of the S phase DNA damage
checkpoint results in S phase progression or premature
mitosis depending on the concentration of 7-hydroxys-
taurosporine and the kinetics of Cdc25C activation. J
Biol Chem 2002; 277:26553-64; PMID:11953432;
11. Guzi TJ, Paruch K, Dwyer MP, Labroli M, Shanahan
F, Davis N, et al. Targeting the replication check-
point using SCH 900776, a potent and function-
ally selective CHK1 inhibitor identified via high con-
tent screening. Mol Cancer Ther 2011; 10:591-602;
1826 Cell Cycle Volume 11 Issue 9
1. Melchionna R, Chen XB, Blasina A, McGowan
CH. Threonine 68 is required for radiation-induced
phosphorylation and activation of Cds1. Nat Cell
Biol 2000; 2:762-5; PMID:11025670; http://dx.doi.
Jazayeri A, Falck J, Lukas C, Bartek J, Smith GC, Lukas
J, et al. ATM- and cell cycle-dependent regulation of
ATR in response to DNA double-strand breaks. Nat
Cell Biol 2006; 8:37-45; PMID:16327781; http://
Zhao H, Piwnica-Worms H. ATR-mediated check-
point pathways regulate phosphorylation and activa-
tion of human Chk1. Mol Cell Biol 2001; 21:4129-
39; PMID:11390642; http://dx.doi.org/10.1128/
Clarke CA, Clarke PR. DNA-dependent phosphoryla-
tion of Chk1 and Claspin in a human cell-free sys-
tem. Biochem J 2005; 388:705-12; PMID:15707391;
Niida H, Nakanishi M. DNA damage check-
points in mammals. Mutagenesis 2006; 21:3-9;
Kruse JP, Gu W. Modes of p53 regulation. Cell
2009; 137:609-22; PMID:19450511; http://dx.doi.
Kastan MB, Bartek J. Cell cycle checkpoints and
cancer. Nature 2004; 432:316-23; PMID:15549093;
Levesque AA, Kohn EA, Bresnick E, Eastman A.
Distinct roles for p53 transactivation and repres-
sion in preventing UCN-01-mediated abroga-
tion of DNA damage-induced arrest at S and G2
cell cycle checkpoints. Oncogene 2005; 24:3786-
96; PMID:15782134; http://dx.doi.org/10.1038/
Levesque AA, Fanous AA, Poh A, Eastman A. Defective
p53 signaling in p53 wild-type tumors attenuates
12. Montano R, Chung I, Garner KM, Parry D, Eastman
A. Preclinical development of the novel Chk1 inhibitor
SCH900776 in combination with DNA-damaging
agents and antimetabolites. Mol Cancer Ther
2012; 11:427-38; PMID:22203733; http://dx.doi.
13. Garner KM, Eastman A. Variations in Mre11/Rad50/
Nbs1 status and DNA damage-induced S-phase arrest
in the cell lines of the NCI60 part. BMC Cancer
2011; 11:206; PMID:21619594; http://dx.doi.
14. Jung YS, Qian Y, Chen X. Examination of the
expanding pathways for the regulation of p21 expres-
sion and activity. Cell Signal 2010; 22:1003-12;
15. Borgdorff V, Lleonart ME, Bishop CL, Fessart D,
Bergin AH, Overhoff MG, et al. Multiple microR-
NAs rescue from Ras-induced senescence by inhib-
iting p21(Waf1/Cip1). Oncogene 2010; 29:2262-
71; PMID:20101223; http://dx.doi.org/10.1038/
16. Wu S, Huang S, Ding J, Zhao Y, Liang L, Liu T, et
al. Multiple microRNAs modulate p21Cip1/Waf1 expres-
sion by directly targeting its 3' untranslated region.
Oncogene 2010; 29:2302-8; PMID:20190813; http://
17. Zhang Y, Gao JS, Tang X, Tucker LD, Quesenberry
P, Rigoutsos I, et al. MicroRNA 125a and its regula-
tion of the p53 tumor suppressor gene. FEBS Lett
2009; 583:3725-30; PMID:19818772; http://dx.doi.
18. Brosh R, Rotter V. When mutants gain new powers:
news from the mutant p53 field. Nat Rev Cancer 2009;
19. Massagué J. G1 cell cycle control and cancer. Nature
2004; 432:298-306; PMID:15549091; http://dx.doi.
20. Oren M. Decision making by p53: life, death and cancer.
Cell Death Differ 2003; 10:431-42; PMID:12719720;
21. Hu S, Dong TS, Dalal SR, Wu F, Bissonnette M, Kwon
JH, et al. The microbe-derived short chain fatty acid
butyrate targets miRNA-dependent p21 gene expres-
sion in human colon cancer. PLoS One 2011; 6:16221;
22. Kim YK, Yu J, Han TS, Park SY, Namkoong B,
Kim DH, et al. Functional links between clustered
microRNAs: suppression of cell cycle inhibitors by
microRNA clusters in gastric cancer. Nucleic Acids Res
2009; 37:1672-81; PMID:19153141; http://dx.doi.
23. Ivanovska I, Ball AS, Diaz RL, Magnus JF, Kibukawa
M, Schelter JM, et al. MicroRNAs in the miR-
106b family regulate p21/CDKN1A and promote
cell cycle progression. Mol Cell Biol 2008; 28:2167-
74; PMID:18212054; http://dx.doi.org/10.1128/
24. Gottifredi V, Karni-Schmidt O, Shieh SS, Prives
C. p53 downregulates CHK1 through p21 and the
retinoblastoma protein. Mol Cell Biol 2001; 21:1066-
76; PMID:11158294; http://dx.doi.org/10.1128/
25. el-Deiry WS, Tokino T, Velculescu VE, Levy DB,
Parsons R, Trent JM, et al. WAF1, a potential media-
tor of p53 tumor suppression. Cell 1993; 75:817-25;
26. McKenzie KE, Siva A, Maier S, Runnebaum IB,
Seshadri R, Sukumar S. Altered WAF1 genes do not
play a role in abnormal cell cycle regulation in breast
cancers lacking p53 mutations. Clin Cancer Res 1997;
27. Abbas T, Dutta A. p21 in cancer: intricate networks
and multiple activities. Nat Rev Cancer 2009; 9:400-
14; PMID:19440234; http://dx.doi.org/10.1038/
28. Erol A. Deciphering the intricate regulatory mecha-
nisms for the cellular choice between cell repair,
apoptosis or senescence in response to damaging sig-
nals. Cell Signal 2011; 23:1076-81; PMID:21144894;
29. Gartel AL. p21(WAF1/CIP1) and cancer: a shifting par-
adigm? Biofactors 2009; 35:161-4; PMID:19449443;
30. Waldman T, Kinzler KW, Vogelstein B. p21 is necessary
for the p53-mediated G1 arrest in human cancer cells.
Cancer Res 1995; 55:5187-90; PMID:7585571.
31. Demarcq C, Bunch RT, Creswell D, Eastman A. The
role of cell cycle progression in cisplatin-induced apop-
tosis in Chinese hamster ovary cells. Cell Growth Differ
1994; 5:983-93; PMID:7819136.
32. Belle A, Tanay A, Bitincka L, Shamir R, O’Shea
EK. Quantification of protein half-lives in the bud-
ding yeast proteome. Proc Natl Acad Sci USA 2006;
33. Pfaffl MW. A new mathematical model for relative
quantification in real-time RT-PCR. Nucleic Acids
Res 2001; 29:45; PMID:11328886; http://dx.doi.