63, 214 –222 (2001)
Copyright © 2001 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
p53-Dependent Induction of p21 Cip1/WAF1/Sdi1 Protects against
Oxygen-Induced Toxicity
Christopher E. Helt, Raymond C. Rancourt, Rhonda J. Staversky, and Michael A. O’Reilly 1
Departments of Pediatrics and Environmental Medicine, The University of Rochester, Rochester, New York 14642
Received April 17, 2001; accepted June 20, 2001
The beneficial effects of supplemental oxygen delivered to patients suffering from acute respiratory distress is offset by its
reduction to genotoxic reactive oxygen species (ROS) that inhibit
proliferation and kill pulmonary cells. Cells respond to oxygeninduced damage by expressing the tumor suppressor p53 and the
cyclin-dependent kinase inhibitor p21 Cip1/WAF1/Sdi1 (p21), which limits proliferation by blocking entry into S phase. Since preventing
DNA synthesis during genotoxic stress may enhance survival, the
current study examines whether hyperoxia induces p21 through a
p53-dependent pathway and whether p21 protects cells from the
toxic effects of oxygen. HCT116 colon carcinoma cells and clonal
lines lacking p53 or p21were used in this study because they allow
direct cytotoxic comparisons between isogenic cells, without complications arising from unknown genetic differences between nonhomologous cell lines. Hyperoxia (95% O 2, 5% CO 2) increased p53
abundance, phosphorylation of p53 on serine 15, and p21 mRNA
and protein in parental HCT116 cells that ceased proliferation. In
contrast, p21 was not detected in either p53- or p21-deficient
HCT116 cells, which exited the G1 compartment and were arrested in S and G2/M phases during hyperoxia. Trypan blue-dye
exclusion revealed that induction of p21 markedly enhanced survival during exposure and colony survival assays showed that p21
enhanced the ability to resume proliferation during recovery in
room air. The observation that p53-dependent induction of p21
prevents exit from G1 and promotes survival during hyperoxia is
consistent with the importance of limiting DNA replication during
genotoxic stress caused by oxygen exposure.
Key Words: cell cycle; DNA damage; oxidative stress; reactive
oxygen species (ROS).
It is well accepted that reactive oxygen species (ROS) are
continuously formed during normal aerobic respiration, due to
the partial reduction of oxygen. Even though molecular oxygen
is not genotoxic when exposed to naked DNA (Gilbert et al.,
1957), it does damage DNA when reduced to ROS (Conger
and Fairchild, 1952). ROS may also damage DNA indirectly
through oxidized lipid products (Sullivan et al., 1992). The
1
To whom correspondence should be addressed at the Department of
Pediatrics, Box 777, Children’s Hospital at Strong, The University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642. Fax: (716) 756-7780.
Email: [email protected].
damaging effects of ROS are counterbalanced by a variety of
antioxidant defense molecules that include enzymes such as
superoxide dismutase, catalase, and glutathione peroxidases or
low-molecular-weight vitamins (Michiels et al., 1994). These
molecules afford adequate protection against the low levels of
ROS produced during normal aerobic respiration. Damage
occurs when the oxidant burden overwhelms the cell’s capacity
to detoxify. Cells may repair damaged DNA, fixate the mutation in their genome, or die by apoptosis or necrosis. Because
apoptosis removes terminally injured cells from a tissue, it can
be argued that it is a beneficial method to deal with cells
injured by ROS. However, it is also advantageous for cells,
especially stem cells, to undergo repair so that the tissue and
organism is not compromised. Given that nearly 10,000 nucleotides per nucleus are oxidized each day during aerobic respiration (Nakamura et al., 1998), one must consider that DNA
repair is as important in the cellular response to oxygeninduced damage as is antioxidant enzymes or apoptosis.
The lung is particularly affected by oxidative stress, because
it functions to exchange oxygen with the environment and
blood. Higher oxygen tensions used to reduce tissue hypoxia,
as well as inhaled pollutants such as nitrogen dioxide and
ozone, injure and kill pulmonary cells. Since hyperoxia produces oxidant-free radicals (Zweier et al., 1989), many investigators use hydrogen peroxide as an in vitro model for hyperoxia-induced lung injury. Even though both can damage DNA,
it is unlikely that hydrogen peroxide mimics the toxic effects of
oxygen for several reasons. Studies in Chinese hamster ovary
cells revealed that hydrogen peroxide produces mutagenic single-strand DNA damage, whereas hyperoxia causes sister chromatid exchanges and other chromosome aberrations (Gille et
al., 1989). Iron chelators enhance survival of cells exposed to
hydrogen peroxide, but not when exposed to hyperoxia (Gille
et al., 1992). Furthermore, hydrogen peroxide causes acute
injury because it has a short half-life. In contrast, hyperoxiainduced stress may be continuously maintained until the cells
are returned to a normoxic environment. Because hyperoxia is
used clinically to reduce tissue hypoxia as well as provide a
useful model for cancer or aging processes (Gille and Joenje,
1992), it is important to understand how cells respond to its
toxic effects. Although numerous studies in both rodents
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CELL PROLIFERATION AND SURVIVAL DURING HYPEROXIA
(Barazzone et al., 1998; O⬘Reilly et al., 2000) and a variety of
cultured epithelial cell lines (Kazzaz et al., 1996; Rancourt et
al., 2001, Rancourt et al., 1999) have shown that oxygen, by
⬎90%, kills cells by necrosis and not apoptosis, little is known
about how cells prevent oxidant damage.
One of the earliest studies examining the cellular response to
hyperoxia revealed that it inhibited proliferation of HeLa cells
(Rueckert and Mueller, 1960). Clearly, preventing DNA replication during periods of genotoxic stress would be advantageous.
Unfortunately, the mechanism by which cells cease proliferation
during hyperoxia remained unclear until recently. Several studies
in adult mice and cultured cell lines have now shown that ⬎90%
oxygen levels increase expression of the tumor suppressor protein
p53 and its phosphorylation on serine 15 (Barazzone et al., 1998;
O⬘Reilly et al., 1998a; Rancourt et al., 2001; Shenberger and
Dixon, 1999). DNA damage activates a number of kinases that
phosphorylate p53 at several sites, including serine 15. Phosphorylation at this site prevents mdm2 from binding p53 and targeting
it for ubiquitin-mediated degradation (Shieh et al., 1997). Hyperoxia also increases expression of the cyclin-dependent kinase
inhibitor p21Cip1/WAF1/Sdi1 (p21; see O⬘Reilly et al., 1998b; Rancourt
et al., 2001; Shenberger and Dixon, 1999), which may be transcriptionally induced by p53 and various cytokines and steroids,
or posttranscriptionally stabilized by ROS (Bellido et al., 1998;
Datto et al., 1995; el-Deiry et al., 1993). p21 inhibits cell proliferation by binding and inactivating G1 and S cyclin-dependent
kinases (cdk; Luo et al., 1995). It also binds proliferating cell
nuclear antigen PCNA and blocks DNA polymerase activity. A
role for p21 in the growth-arresting activities of hyperoxia was
first described in SV40 transformed type-II epithelial cells. In that
study, hyperoxia increased p21, which inhibited cyclin E/cdk2
kinase activity (Corroyer et al., 1996). Active cyclin E/cdk2
kinase phosphorylates substrates that are required for DNA replication (Elledge, 1996). Additional studies using a variety of
p21-functional and deficient epithelial cell lines revealed that cells
expressing p21 accumulated in G1 while cells lacking p21 exited
G1 and accumulated in S phase (Rancourt et al., 2001). Analogous studies revealed that hyperoxia inhibited cell proliferation in
lungs of adult mice in vivo, but not in mice lacking p21 (O⬘Reilly
et al., 2001). Interestingly, the p21-deficient mice were markedly
sensitive to hyperoxia, as shown by rapid necrotic death of parenchymal cells and an ⬃50% reduction in mean survival time.
This observation was consistent with other studies showing that
p21 protects cells from the genotoxic effects of ionizing radiation,
ultraviolet light, cisplatin, methylmethane sulfonate, and nitrogen
mustard (Fan et al., 1997; McDonald et al., 1996; Sheikh et al.,
1997; Wang et al., 1997). Because the mammalian lung contains
nearly 40 distinct cell types, a simple cell-line model would be
useful for clarifying how p21 protects cells from oxygen-induced
toxicity.
The current study examines the toxic effects of hyperoxia on
HCT116 colon carcinoma cells that express wild-types p53 and
p21. An advantage of these cells over existing pulmonary cell
lines is that clonal variants lacking p53 or p21 were created by
215
homologous recombination (Bunz et al., 1998; Waldman et al.,
1995). This allows direct toxicologic comparisons between
genetically identical cells without additional complications involving unknown genetic differences that may exist when
studying nonhomologous cell lines. We now show that hyperoxia induces p53-dependent expression of p21, which prevents
cells from exiting G1 as well as enhancing their survival. Our
findings are consistent with the importance of limiting DNA
replication during oxidant genotoxic stress and suggest that
repair of oxidant DNA damage may be critical for survival.
Since hyperoxia damages cells indirectly through production of
toxic ROS, our findings with these colon cells may also have
clinical significance for patients suffering from ROS-mediated
inflammatory or ischemic bowel injuries.
MATERIALS AND METHODS
Cell culture. The parental HCT116 human colon carcinoma cell line and
isogenic lines lacking p53 or p21 were obtained from Dr. Bert Vogelstein
(Johns Hopkins Oncology Center and the Program in Human Genetics and
Molecular Biology, Baltimore, MD). The p21 locus was deleted in the parental
cells by homologous recombination resulting in the isogenic cell line HCT116
p21 (-/-; Waldman et al., 1995). The p53 locus was separately targeted in the
parental line, resulting in the isogenic line HCT116 p53 (-/-; Bunz et al., 1998).
All cells were incubated in room air plus 5% CO 2 at 37°C in McCoy’s
medium, containing 10% fetal bovine serum (HyClone Laboratories, Logan,
UT), 50 U/ml of penicillin, and 50 ␮g/ml of streptomycin (Life Technologies,
Rockville, MD). Cells were trypsinized (0.25% trypsin, Life Technologies),
counted with a hemacytometer (Sigma, St. Louis, MO) and plated at 5 ⫻ 10 5
cells per 100 mm culture dish. The cells were allowed to adhere to the plates
by incubating at 37°C overnight, at which time the medium was replaced and
the dishes were placed in a plexiglass box (Belco Glass, Vineland, NJ). The
box was flooded with 95% O 2/5% CO 2 for 10 min at a flow rate of 10 l per min
before sealing and incubating at 37°C.
Western-blot analysis. Cells were harvested by trypsinization, followed
by centrifugation at 300 ⫻ g for 6 min. The pellets were then washed with 1⫻
phosphate-buffered saline (PBS, Life Technologies) and resuspended in 50
mM Tris (pH 8.0), 120 mM NaCl, and 0.5% Nonidet P-40 supplemented with
2 ␮g/ml of aprotinin and 100 ␮g/ml phenylmethylsulfonyl fluoride. The lysate
was cleared by centrifugation at maximum speed in a microcentrifuge and
protein concentrations were determined by the Lowry Assay (DC Protein
Assay, Bio-Rad, Hercules, CA). The lysates were boiled for five min in 3⫻
Laemmli buffer (1⫻ Laemmli contains 50 mM Tris pH 6.8, 1% ␤-mercaptoethanol, 2% SDS, 0.1% bromophenol blue, and 10% glycerol). The extracted
protein was separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (PVDF, Millipore, Bedford
MA). The membranes were blocked for 1 h at room temperature in phosphate
or Tris-buffered saline (PBS or TBS) containing 5% nonfat dry milk plus
0.05% or 0.1% Tween-20 (PBST or TBST), respectively. TBST was used to
wash membranes intended for phosphorylation-specific antisera. The membranes were then incubated in primary antibody p53 (1:1000, Oncogene
Research Products, Cambridge, MA), p53 (ser-15; 1:1000, Cell Signaling
Technology, Beverly, MA), or p21 (1:500, PharMingen, San Diego, CA),
followed by 3 washes in PBS or TBS to remove nonspecific interactions.
Membranes were then incubated in secondary antibody (p53 or p21:goat
antimouse; Southern Biotechnology, Birmingham, AL or p53 (ser-15): goat
antirabbit; Jackson Labs, West Grove, PA) for one h at room temperature.
After washing the membranes with PBST or TBST, specific antibody interactions were visualized by chemiluminescence (Amersham, Arlington Heights,
IL). As a loading control, membranes were also blotted for ␤-actin (1:5000;
Sigma, St. Louis, MO).
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HELT ET AL.
Growth curve and cell viability assays. Cells were trypsinized, counted
with a hemacytometer, and plated in triplicate in 60 mm culture dishes before
exposing to room air or hyperoxia for 24, 48, 72, or 96 h. A hemacytometer
was used to count cells and viability was determined by their ability to exclude
0.04% trypan blue dye. Colony-survival assays were initiated by exposing cells
to room air or hyperoxia for 24 or 36 h. Cells were then trypsinized and
counted using a hemacytometer. Serial dilutions were replated at 100 cells per
60 mm culture dish and allowed to grow in room air at 37°C for 13 days, at
which time colonies were fixed in 15% methanol and stained with crystal violet
(2.5 g/l). The total number of colonies produced on each plate was then
recorded.
Flow cytometric cell cycle analysis. HCT116 cells exposed to room air or
hyperoxia were trypsinized, resuspended in their own media, and centrifuged
at 300 ⫻ g for 6 min. Pellets were resuspended in 75% ethanol and stored at
4°C for a minimum of 24 h. Fixed cells were then centrifuged at 300 ⫻ g for
6 min. The pellets were resuspended in PBS containing 1 mg/ml RNase
(Sigma, St. Louis, MO) and incubated at room temperature for 30 min, with
intermittent vortexing. Cells were centrifuged again at 300 ⫻ g. The pellets
were resuspended in PBS containing 20 ␮g/ml propidium iodide. Stained
samples were filtered through a 37 ␮m mesh prior to analysis on an Epics Elite
(Coulter Electronics) equipped with an argon laser excited at 488 nm. Relative
DNA content per cell was determined by measuring fluorescence of the treated
samples. Cell-phase percentages were determined by use of Multicycle by
Phoenix Flow Systems software (Phoenix Flow Systems, San Diego, CA).
RNA extraction and analysis. Cells grown on culture dishes were lysed by
adding 1 ml of 4 M guanidine isothiocyanate, 0.5% N-laurylsarcosine, 20 mM
sodium citrate, and 0.1 M ␤-mercaptoethanol followed by scraping the dish
surface. RNA was extracted from the resulting solution using acid phenol and
phase-lock gel columns (5 Prime-3 Prime, Boulder, Colorado) and resuspended in diethylpyrocarbonate-treated water. The amount of RNA in an
aqueous solution was determined by absorbance at 260 nm. RNase protection
assays were performed with human cell cycle-2 multi-probe template kit,
according to the manufacturer’s instructions (PharMingen, San Diego, CA).
Riboprobe synthesis reaction was incubated for 60 min at room temperature,
followed by an additional 30 min at 37°C in the presence of 2 units of
RNase-free DNase. Riboprobes were extracted with phenol/chloroform and
precipitated with ethanol and ammonium acetate. Probes were resuspended in
50 ␮l of hybridization buffer (400 mM NaCl, 40 mM PIPES, pH 6.7, 1 mM
EDTA, pH 8.0, and 80% formamide) and diluted to 3.37 ⫻ 10 5 cpm/␮l before
incubating with 5 ␮g denatured total RNA at 56°C for 16 h. Hybridized probes
were digested with RNase buffer for 45 min at 30°C. Samples were incubated
with proteinase K and yeast tRNA before extracting with phenol/chloroform
and precipitating with ethanol. Protected products were separated on a 6%
FIG. 1. Hyperoxia increases p53 and p21 proteins. Parental HCT116 and
cells lacking p53 or p21 were exposed to room air or hyperoxia for 24, 48, and
72 h. Cell homogenates were immunoblotted for p53, using the pan-specific
antibody DO-1, phosphoserine 15 p53 (ser-15), or p21. The blots contained 10
␮g of protein when probed for p53 and 75 ␮g of protein when blotted for p21.
The blots were reprobed for actin to ensure that comparable levels of protein
were in all lanes. Only one immunoreactive protein of the expected size was
detected with the antibodies. Blots are representative of a typical experiment
(n ⫽ 3– 4).
FIG. 2. Hyperoxia increases p53-dependent expression of p21. Parental
HCT116 and cells lacking p53 or p21 were exposed to room air or hyperoxia
for 24, 48, and 72 h. Total RNA was hybridized with radiolabeled cRNA
probes, digested with RNase, and the protected products separated by size.
Lane labeled P contains input probe before digestion.
acrylamide/8 M urea sequencing gel, dried, and visualized by exposure on
PhosphorImager screens.
Statistical analysis. Values are expressed as means ⫾ SD. Group means
were compared by ANOVA with Fisher’s procedure post hoc analysis, using
Excel software for the Macintosh with p ⬍ 0.05 considered as significant.
RESULTS
Hyperoxia Induces p53-Dependent Expression of p21
Western blot analysis was used to determine whether hyperoxia increased levels of p53 or p21. Low levels of p53 were
detected in the parental HCT116 cells exposed to room air and
increased after exposure to hyperoxia (Fig. 1). Increased p53
levels were associated with increased phosphorylation on
serine-15, which occurs when cells are exposed to DNAdamaging agents (Shieh et al., 1997). Hyperoxia also increased
p53 in p21 (-/-) cells and its phosphorylation on serine 15. As
expected, p21 was not detected in these cells at any time.
Interestingly, p21 was also not detected in p53 (-/-) cells
exposed to room air or hyperoxia. p53 or phosphoserine-15
p53 was not detected in the p53-deficient cells. These findings
reveal that hyperoxia increases p53, which is required for
induction of p21.
Since p53 regulates p21 induction by binding and increasing
transcription of the p21 promoter (el-Deiry et al., 1993), RNase
protection studies were performed in order to determine
whether hyperoxia also increased p21 mRNA expression. p21
mRNA was detected in parental HCT116 cells and increased
nearly 9-fold when exposed to hyperoxia when compared to
expression of ribosomal L32 or glyceraldehyde phosphate dehydrogenase (Fig. 2). In contrast, low levels of p21 mRNA
CELL PROLIFERATION AND SURVIVAL DURING HYPEROXIA
217
were detected in p53 (-/-) cells that minimally increased during
hyperoxia. As expected, hyperoxia did not increase p21 mRNA
in the p21 (-/-) cells. It remains unclear why a protected
product for p21 was faintly detected in the p21 (-/-) samples,
because the sequence of the input probe is proprietary information. Since only a portion of the p21 gene was replaced by
the gene-targeting event (Waldman et al., 1995), one potential
explanation is that the p21-deficient cells transcribe a mutant
p21 mRNA that contains sequence homology to the probe.
Nonetheless, hyperoxia significantly increases p21 mRNA and
protein in cells expressing p53, but not in the cells lacking p53
or p21. In contrast to the robust induction of p21 mRNA,
hyperoxia did not alter p53 mRNA in either the parental or
p21-deficient cells. Our observation that hyperoxia induced
p53 protein without altering the abundance of p53 mRNA is
consistent with activation of the DNA damage-dependent posttranscriptional phosphorylation of p53 leading to its increased
stability (Shieh et al., 1997).
The RNase protection assay also contained cRNA probes for
other cell-cycle regulators. p27 and p57 are structurally homologous to p21 and inhibit proliferation in G1 by inhibiting the
G1 kinases cdk4 and cdk2 (Toyoshima and Hunter, 1994). p15,
p16, p18, and p19 are members of the INK4 family that also
inhibit proliferation in G1 through their ability to bind cdk4,
thereby displacing cyclin D (Hall et al., 1995). p107 and p130
are related to the retinoblastoma gene product RB, which
regulates S-phase progression (Elledge, 1996). Interestingly,
the expression of all of these genes was not altered by hyperoxia, nor altered in the absence of p53 or p21. Thus, hyperoxia
specifically increases p21 mRNA through a p53-dependent
mechanism without affecting the mRNA levels of any of the
known G1 kinase inhibitors.
p21 Prevents Exit from G1 during Hyperoxia
The effect of hyperoxia on cell proliferation was determined
by counting cells with a hemacytometer (Fig. 3). Cultures of
parental HCT116 cells or those lacking p53 or p21 showed a
linear increase in cell number over time when exposed to room
air. In contrast, cell numbers did not increase in cultures
exposed to hyperoxia. The inhibitory effects of hyperoxia were
observed in the parental cells and in both the p53 and p21deficient lines after 48 h of exposure.
Several studies have now shown that the early inhibitory
effects of hyperoxia on cell number are mediated through
decreased proliferation (Corroyer et al., 1996; Rancourt et al.,
2001; Shenberger and Dixon, 1999). Since p21 inhibits DNA
replication by preventing cells from exiting G1 (Waldman et
al., 1995), we used flow cytometry to quantify the percentage
of cells in G1. Approximately 25% of parental cells exposed to
room air were in G1 phase of the cell cycle (Fig. 4A). This
slowly increased during hyperoxia until nearly 40% of the cells
were in G1 after 72 h. Hyperoxia also modestly increased the
percent of cells in S phase and decreased the percent of cells in
FIG. 3. Hyperoxia inhibits proliferation. Parental HCT116 (A) and cells
lacking p53 (B) or p21 (C) were exposed to room air or hyperoxia for 24, 48,
72, or 96 h. Cells were harvested and counted in triplicate each day. Values
represent mean ⫾ SD (*p ⬍ 0.01, n ⫽ 3). Error bars are not observed in all
points because of the small standard deviations obtained.
G2/M phase. p53-deficient cells, exposed to room air, showed
a comparable percent of cells in G1 as the parental cells, which
decreased markedly during hyperoxia until less than 5% of the
cells remained in G1 by 72 h (Fig. 4B). Hyperoxia did not
markedly alter the percent of cells in S phase, but did cause a
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HELT ET AL.
or p53-deficient lines, it also decreased during hyperoxia (Fig.
4C). The loss of cells in G1 phase of the cell cycle was
associated with a significant increase in percent of cells in S
phase and a decrease in G2/M phase. Thus, failure to induce
p21 in p53 or p21-deficient cells was associated with a decrease in the percent of cells in G1. The oxygen-exposed
p53-deficient cultures had more cells in G2/M phase while
p21-deficient cultures had more cells in S phase.
p21 Deficient Cells Exhibit Decreased Viability during
Hyperoxia
FIG. 4. p21 prevents cells from exiting G1. Parental HCT116 (A) and
cells lacking p53 (B) or p21 (C) were exposed to room air or hyperoxia for 24,
48, or 72 h. DNA content was assessed by incorporation of propidium iodide.
Values represent mean ⫾ SD (* for G1, † for S, and ¶ for G2 denote significant
changes relative to the room air-exposed samples p ⬍ 0.05, n ⫽ 3). Representative DNA histograms depicting DNA content of cells exposed to room air
(0) or 72 h of hyperoxia are displayed to the right of each graph.
significant increase in the fraction of cells arrested in G2/M
phase. Although the percent of p21-deficient cells in G1 exposed to room air was lower than that observed for the parental
Previous studies revealed that A549 and Mv1Lu pulmonary
adenocarcinoma cell lines died by necrosis when continuously
exposed to lethal levels of oxygen (Rancourt et al., 2001).
Since p21-induction limited DNA replication during exposure,
we hypothesized that the parental cells were more resistant to
hyperoxia than cells that lacked p21. Trypan blue dye exclusion was used to measure viability because cells dying by
necrosis do not exclude the dye. Greater than 95% of the
parental cells were capable of excluding the dye over the first
48 h of hyperoxia before they showed a modest decline in
viability (Fig. 5A). By 96 h, approximately 80% of the cells
still maintained sufficient membrane integrity to permit dye
exclusion. Similarly, p53 (-/-) cells were capable of excluding
the dye for the first several days; however, they showed a more
rapid decline in viability such that only 50% of the cells could
exclude dye by 96 h of exposure (Fig. 5B). In contrast, p21 (-/-)
cells showed an even more rapid decline in viability that was
first noticed by 48 h of exposure (Fig. 5C). Cell viability
continued to decline rapidly until only 30% of the cells remained viable after 96 h of exposure. Thus, hyperoxia elicited
a progressive decline in cell viability with p21-deficient cells
being the most sensitive.
Colony survival assays were performed in order to determine whether cells that survived exposure could resume proliferation. In this assay, cells are exposed to room air or
hyperoxia for 24 and 36 h. These times were chosen because
the dye exclusion assay revealed that greater than 85% of the
cells were still viable. We compared the viability of the parental cells to p21-deficient cells because they showed the greatest
difference in trypan blue dye exclusion during exposure. As
expected, there was no detectable difference in survival of
parental cells and p21-deficient cells exposed to room air (Fig.
6). Interestingly, the colony size of p21-deficient cells was
larger than the parental cells, suggesting that individual cells
proliferated faster than the parental cells, perhaps due to
shorter time in G1. p21-deficient cells displayed a marked
reduction in colony formation when exposed to hyperoxia for
24 or 36 h compared to the parental cells. Thus, p21 induction
enhances ability of cells to survive and resume proliferation
when recovered in room air.
CELL PROLIFERATION AND SURVIVAL DURING HYPEROXIA
219
enhances survival during exposure as well as during recovery.
Given that hyperoxia causes single- and double-strand DNA
breaks, our findings suggest that p21 may protect cells by
allowing additional time for repair to occur and/or prevent
replication of damaged DNA. Although these hypotheses remain to be experimentally tested, our findings clearly show that
p21 protects epithelial cells from oxidative stress caused by
exposure to hyperoxia.
One of the important observations in the current study was
that p53 was necessary for the induction of p21. Previous
studies exposing p53-deficient mice to 92% oxygen revealed
that p21 induction was dependent upon p53. This is consistent
with our recent finding that hyperoxia activates a p53-dependent transcriptional reporter in cultured cell lines (Rancourt et
al., 2001). On the other hand, we also showed that modest
FIG. 5. p21 enhances survival during exposure. Parental HCT116 (A) and
cells lacking p53 (B) or p21 (C) were exposed to room air or hyperoxia for 24,
48, 72, or 96 h. The percent of cells excluding trypan blue each day was used
to assess cell viability. Values represent mean ⫾ SD (*p ⬍ 0.03, n ⫽ 3). Error
bars are not observed in all points because of the small standard deviations
obtained.
DISCUSSION
Previous studies showed that oxygen levels greater than 90%
inhibited cell proliferation in G1 and S phases of the cell cycle
and caused chromosome abnormalities. It also increased expression of p53 and p21, where p21 prevented cells from
exiting G1. The current study extends these findings by showing that p53 is required for induction of p21 and that p21
FIG. 6. Survival and recovery in room air is enhanced by p21. (A)
Representative plates of colonies obtained from parental HCT116 or p21deficient cells exposed to room air or hyperoxia for 24 and 36 h, which were
recovered for 10 days under normoxic conditions. (B) Graphic representation
of the number of surviving colonies. Values represent mean ⫾ SD of colonies
formed normalized to the number of colonies formed during exposure to room
air. (*p ⬍ 0.05, n ⫽ 5). Inset graph depicts a magnified view of the values
obtained after 36 h of hyperoxia.
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HELT ET AL.
levels of p21 were still induced in p53-deficient mice who were
exposed to 100% FiO 2 in vivo, while maximal expression was
detected in p53-wild-type mice. The latter finding is consistent
with studies showing that hyperoxia increases p21 in SV40transformed pulmonary epithelial cells that presumably contain
nonfunctional p53, due to their expression of SV40 (Corroyer
et al., 1996). One explanation for these differences is that p21
is regulated by multiple signal transduction pathways. For
example, oxidative stress can increase the dithiol-reducing
protein thioredoxin, which potentiates redox factor (Ref)-1dependent regulation of p53 and p21 (Ueno et al., 1999).
p53-independent changes in p21 mRNA stability have been
reported in cells treated with diethylmaleate, a compound that
oxidizes cells by depleting them of reduced glutathione (Esposito et al., 1997). The cytokines transforming growth factor
(TGF)-␤ and interleukin (IL)-6 increase p53-independent transcription of p21 in keratinocytes and osteoblast cells, respectively (Bellido et al., 1998; Datto et al., 1995). Interestingly,
the expression of both cytokines increases in mouse lungs
exposed to hyperoxia (Johnston et al., 1998; O⬘Reilly et al.,
1997). Additional response elements have been identified in
the p21 promoter, which mediate regulation by phorbol esters,
steroids, cisplatin, epidermal growth factor, and UV radiation
(Gartel and Tyner, 1999). Although multiple pathways exist to
regulate p21, all of the available evidence suggests that p53
participates in the induction of p21 by hyperoxia, because it is
absolutely required in the HCT116 cell line model and is
required for maximal induction in vivo.
The current study confirmed previous observations that lethal levels of oxygen inhibit cell proliferation in both animal
models (Evans et al., 1969) and cultured cell lines (Rueckert
and Mueller, 1960). More recent findings revealed that hyperoxia inhibits proliferation in G1 phase of the cell cycle through
induction of p21 (Corroyer et al., 1996; Rancourt et al., 2001).
Cells that fail to induce p21 exit G1 where they arrest predominantly in S phase. As expected, the percent of p21-deficient
HCT116 cells in G1 phase of the cell cycle decreased when
they were exposed to hyperoxia compared to the parental cells
that expressed p21. This decrease in the G1 population correlated with an increase in the percentage of cells in S phase.
Although hyperoxia also decreased the percent of p53-deficient
HCT116 cells in G1, the percent of cells in S phase remained
constant while the G2/M population increased significantly. It
remains unclear why a greater percentage of p53-deficient cells
were able to successfully complete S phase, because the mechanism by which hyperoxia causes cells to accumulate in S
phase remains unknown. Nonetheless, failure to induce p21
during hyperoxia is associated with a significant percentage of
cells exiting the G1 compartment. Cells that vacate G1 enter S
phase, where they either accumulate or successfully progress
through and accumulate in G2.
Another important observation in the current study is that
p21 protects cells from the toxic effects of oxygen exposure.
This finding is consistent with our recent observation that p21
enhanced survival of adult mice exposed to 100% FiO 2
(O⬘Reilly et al., 2001). The mean survival of p21-deficient
mice was 72 h compared to 120 h for p21-wild-type mice.
Ultrastructural analyses of their lungs revealed rapid necrosis
of alveolar endothelial and of type I epithelial cells that lead to
pulmonary edema. The same cell types are injured and killed in
p21-wild-type mice after longer periods of exposure. These
observations suggest that p21 protects cells from hyperoxia by
delaying necrosis, rather than by changing the population of
cells that are sensitive to hyperoxia. The current study extends
these findings by showing that p21 also delayed death of
HCT116 cells. To date, there is no evidence that hyperoxia
kills cells in vitro by apoptosis (Kazzaz et al., 1996; Rancourt
et al., 1999) or in vivo as shown by ultrastructural findings
(O⬘Reilly et al., 2000, 2001). ROS may cause more damage to
p21-deficient cells because they are trapped in S phase where
their DNA is actively replicating and not protected by histones.
In contrast, the modest increase in survival of the p53-deficient
compared to p21-deficient cells may be attributed to the larger
percentage of p53-deficient cells that successfully complete S
phase and arrest in G2/M. Cells that arrest in G2 are also more
resistant to genotoxins, presumably because they can repair
damaged DNA before cytokinesis.
p21 may protect cells from oxygen-induced toxicity by
preventing them from replicating their damaged DNA before it
has been repaired. The observation that, cultures that express
p21 retain a greater percentage of cells in G1 compared to cells
that lack p21, supports this hypothesis. Alternatively, p21 may
participate in DNA repair activities through its ability to bind
PCNA that interacts with FEN-1 and DNA polymerase. p21
may function in nucleotide excision repair because p21-deficient HCT116 cells have a reduced capacity to repair DNA
damaged by UV or cisplatin (McDonald et al., 1996). Similar
results were found when cells were challenged with nitrogen
mustard, which also damages DNA (Fan et al., 1997). p21 also
enhanced protection and repair from UV-induced DNA damage in DLD1 colorectal carcinoma cells (Sheikh et al., 1997).
In contrast, p21-deficient mouse embryo fibroblasts exhibited
minimal repair capacity when damaged by UV (Smith et al.,
2000). Even though the biochemical evidence supports the
argument that p21 participates in DNA repair processes, this
issue remains to be clarified especially in the context of oxidative DNA damage.
Another important finding in this study was that hyperoxia
only affected the expression of p21 mRNA. As shown in
Figure 2, hyperoxia did not markedly alter mRNA levels of
p27 or p57, both of which share structural homology to p21.
Previous studies using rat type-II epithelial cells showed that
hyperoxia increased p27 mRNA but not p27 protein (Corroyer
et al., 1996). The current study also showed that hyperoxia did
not alter mRNA of the INK4 proteins p15, p16, p18, or p19.
The INK4 proteins prevent S-phase progression by binding
cdk4 and preventing association with its catalytic partner,
cyclin D. Finally, hyperoxia did not alter expression of Rb or
CELL PROLIFERATION AND SURVIVAL DURING HYPEROXIA
its related members p107 or p130. Interestingly, loss of p53 or
p21 did not effect the basal expression of the Kip or INK4
genes or when the cells were exposed to hyperoxia. Thus, the
effect of hyperoxia on mRNA levels of G1 kinase inhibitors is
specific for p21 and is dependent upon functional p53.
In summary, the current study demonstrates that hyperoxia induces p53-dependent expression of p21 that prevented cells from exiting G1 and enhanced their survival.
These findings are consistent with an in vivo mouse model
of oxygen-induced lung injury that showed p21 enhances
survival. Both models may now be exploited to understand
how hyperoxia activates the p53 suppressor pathway and
how its downstream targets, such as p21, modify the genotoxic effects of oxygen. A better understanding of how cells
respond to the toxic effects of hyperoxia can provide insight
into how to block the damaging effects of ROS associated
with supplemental oxygen exposure, reperfusion injury,
cancer, and the aging process.
ACKNOWLEDGMENTS
This work was funded by NIH grant HL58774. C.E.H. and R.C.R. were
supported by NIH training grant ES07026. We thank Dr. Peter Keng for
assisting with flow cytometric analyses and intellectual discussions.
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