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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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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.
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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.