From the Department of Medicine,
§ The Johns Hopkins Oncology Center, Baltimore, Maryland
21205,
Program in Cellular and Molecular Medicine, ** Cornell
University Graduate School of Medical Sciences,
New York, New York 10021, the ¶¶ Department of
Pediatrics and the Institute Of Genetic Medicine, The Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205, and
Gilead Sciences,
Foster City, California 94404
Received for publication, November 8, 2000
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ABSTRACT |
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Mammalian cellular responses to hypoxia include
adaptive metabolic changes and a G1 cell cycle
arrest. Although transcriptional regulation of metabolic genes by the
hypoxia-induced transcription factor (HIF-1) has been established, the
mechanism for the hypoxia-induced G1 arrest is not known.
By using genetically defined primary wild-type murine embryo
fibroblasts and those nullizygous for regulators of the
G1/S checkpoint, we observed that the retinoblastoma
protein is essential for the G1/S hypoxia-induced
checkpoint, whereas p53 and p21 are not required. In addition, we found
that the cyclin-dependent kinase inhibitor p27 is induced
by hypoxia, thereby inhibiting CDK2 activity and forestalling S phase
entry through retinoblastoma protein hypophosphorylation. Reduction or
absence of p27 abrogated the hypoxia-induced G1 checkpoint,
suggesting that it is a key regulator of G1/S transition in
hypoxic cells. Intriguingly, hypoxic induction of p27 appears to be
transcriptional and through an HIF-1-independent region of its proximal
promoter. This demonstration of the molecular mechanism of
hypoxia-induced G1/S regulation provides insight into a
fundamental response of mammalian cells to low oxygen tension.
Cellular hypoxia is an environmental stress with important
implications in developmental biology, normal physiology, and many pathological conditions, including cancer. Normal tissues display an
oxygen gradient across a distance of 400 µm from a blood supply; tumors often have disordered and diminished vascularization, and hypoxia occurs in tumor tissue that is >100-200 µm away from a functional blood supply (1-3). Cells may adapt to hypoxia in numerous
ways, including a transition from oxidative phosphorylation to
glycolysis and neovascularization. Many of these metabolic responses
are mediated by the transcription factor
HIF-1,1 a heterodimer of a
hypoxia-induced subunit HIF-1 Cells may also respond to hypoxia by diminishing their proliferative
rates. Both invasive and noninvasive studies of a variety of normal
tissues and tumors suggest that hypoxic cells may be viable but
nonproliferating (8-12). This low proliferative state may be related
to the phenomenon of tumor dormancy, described in nonvascularized
metastatic foci (13), and may also help explain why hypoxic tumors are
relatively chemoresistant and radioresistant (14). Although some
transformed cell lines undergo apoptosis in extreme hypoxia and an
acidic environment, nontransformed hypoxic cells remain viable but
arrest in G1 (15-17).
The best characterized molecular event necessary for the
G1/S phase transition is phosphorylation of the
retinoblastoma protein (RB) by specific cyclin-dependent
kinase (CDK)-cyclin complexes (18). CDK activity can be inhibited by
cyclin-dependent kinase inhibitors (CDKIs), such as p27 and
p21, which then promote RB hypophosphorylation. There is also evidence
that CDKIs may promote a G1 arrest that is RB-independent
(19, 20). Hypoxia-induced arrest is associated with hypophosphorylation
of RB (16, 21) and, as opposed to hypoxia-induced apoptosis, appears to
be independent of p53 induction (17, 22). A direct role for HIF-1 We therefore sought to characterize the molecular mechanisms
responsible for hypoxia-induced growth arrest, and the role of HIF-1 in
this response. Many previous studies exploring the effect of hypoxia on
the cell cycle have been limited by the use of transformed and/or
immortalized cell lines, which may have altered cell cycle regulators
and/or other mutations (23). In this ambiguous genetic background, and
without the ability to manipulate regulators of the G1/S
transition, conclusions regarding the significance of cell cycle
regulators in hypoxia-induced G1 arrest have not been definitive. We elected to first identify cell cycle regulatory elements
that are altered by hypoxia in immortalized fibroblasts, and then study
the effect of hypoxia on the cell cycle of wild-type murine embryo
fibroblasts (MEFs) and primary fibroblasts deficient in key regulators
of the G1/S checkpoint. We demonstrate that RB and p27 play
important roles in the hypoxia-induced G1 arrest of primary
fibroblasts. We then used immortalized fibroblasts for further
molecular manipulation to show that hypoxia transcriptionally induces
p27 and causes a G1 arrest in an HIF-1-independent manner. These observations suggest a molecular mechanism for hypoxia-induced cell cycle regulation.
Cell Culture and Hypoxic Induction--
Rat1a fibroblasts,
NIH-3T3 fibroblasts, Balb-3T3 fibroblasts, and mouse embryo fibroblasts
(MEFs) null for RB, p53, p21 (obtained from Dr. Tyler Jacks (24)), and
p27 (25) and their wild-type counterparts were cultured in Dulbecco's
modified Eagle's medium containing 3.7 g/liter bicarbonate, 1.6 g/liter glucose and supplemented with penicillin/streptomycin and 10%
fetal calf serum (Life Technologies, Inc.). All MEFs were used before
passage 14. Embryonal Stem (ES) cells were prepared and cultured in
Dulbecco's modified Eagle's medium with 4.6 g/liter glucose
supplemented with nonessential amino acids, insulin, monothioglycerol,
serum, and HEPES as described (7). AIN4 cells were cultured in Improved
MEM-Zinc Option supplemented with fetal calf serum, insulin, and
hydrocortisone (26).
For all experiments, 1 × 105 cells were plated in
10-cm dishes and incubated in 20% O2 at 37 °C for
24 h. The media were then changed and supplemented with 25 mM HEPES (pH 7.55). To render cells hypoxic, dishes were
placed in a modular incubator chamber (Billups-Rothenberg), flushed
with 95% N2, 5% CO2, and incubated at
37 °C. This resulted in ~0.1-0.5% O2 after several
hours. After 32 h, cells were released from hypoxia and quickly
scraped in ice-cold phosphate-buffered saline, and analyses were
performed as described below. For CoCl2 experiments, cells
were incubated with CoCl2 (200 µM) for
32 h prior to analysis.
Description of Plasmids and Transient Transfection--
Each
10-cm dish was plated with 1 × 105 cells and 24 h later transfected with Lipofectin (Life Technologies, Inc.); after a 24-h incubation in 20% O2, cells were rendered hypoxic or
incubated in 20% O2 for an additional 24 h prior to
analysis. The HIF-1 Manipulation of Cell Cycle Regulators--
Balb-3T3 cells were
plated at 2 × 105/10-cm plate and 24 h later
transfected with 30 nM of p27 antisense
TGGCTCTCXTGCGCC or missense
TGGCTCXCTTGCGCC oligonucleotides, where C = 5MeC and X = G-clamp (Gilead), in the
presence of 2.5 µg/ml GS3815 cytofectin (Glen Research). After 5 h buffered media were added, and cells were incubated in 20%
O2 or rendered hypoxic for an additional 32 h.
Recombinant adenoviruses containing full-length human p27 in the
antisense direction and, as control, GFP alone were prepared using the
AdEasy method (29) and titered so that >90% of infected cells
expressed GFP and no toxicity was noted. AIN4 cells, plated at
1 × 105/10-cm plate, were infected with virus for
6 h in the presence of 2% fetal calf serum, and 24 h later
media were exchanged and exposed rendered hypoxic or incubated in 20%
O2 for 48 h.
Cell Cycle Analysis--
After 32 h, cells were released
from hypoxia and quickly scraped in ice-cold phosphate-buffered saline,
washed, and suspended in a buffer containing sucrose and trisodium
citrate. Samples were then incubated for 10 min consecutively with
trypsin/Nonidet P-40/spermine tetrahydrochloride, trypsin
inhibitor/ribonuclease/and propidium iodide (PI) as described (30). PI
and forward light scattering were detected by using a Coulter EPICS 752 flow cytometer equipped with MDADS 11 software, version 1.0. Cell cycle
distribution profiles were determined with a curve fitting program
ELITE version 3.0 (Coulter).
To assess directly the rate of DNA synthesis, cells were exposed to
media containing BrdUrd (10 µM) for 30 min. Cells
were then trypsinized and removed from hypoxia. Nuclei were prepared and stained with a fluorescein isothiocyanate-labeled and BrdUrd antibody, and total DNA was stained with propidium iodide and analyzed
as described (26). Wild-type MEF controls from RB, p53, and p27
wild-type animals all showed similar degrees of hypoxia-induced growth
arrest and were averaged together.
Immunoblotting and Immunoprecipitation--
Cells were released
from 32 h of hypoxia, quickly washed with ice-cold
phosphate-buffered saline, and then resuspended in a solution
containing 1% SDS and boiled. Protein was quantitated by the BCA
method (Pierce), and equal amounts of total cellular protein were
resolved by SDS-polyacrylamide gel electrophoresis and subjected to
immunoblot analysis (26). RB antibody was obtained from PharMingen, and
cyclin A, D, and E were from Upstate Biotechnology, Inc.; for
immunoblots, p27, p53, and p21 were from Santa Cruz Biotechnology, and
HIF1 RNA Analysis--
After 28 h of hypoxic or nonhypoxic
conditions, medium was changed to contain 0.5 µg/ml actinomycin D
(Sigma), a concentration at which no toxicity was seen after 12 h
of incubation. Total RNA was harvested at 0, 30, 120, and 240 min after
actinomycin D addition under continued hypoxic or nonhypoxic conditions
using Trizol (Life Technologies, Inc.) and the supplier's protocol. 15 µg of RNA/lane was subjected to electrophoresis in 1%
agarose-formaldehyde gels, transferred to nylon membranes, and
hybridized with a p27 cDNA probe. Autoradiographic signals were
quantitated with a PhosphorImager and normalized to 18 S RNA.
Hypoxia Induces a G1 Cell Cycle Arrest in Immortalized
Fibroblasts and Is Associated with Hypoxia-mediated Changes in
G1 Regulators--
Since contact inhibition and serum
withdrawal are well documented to result in G1 cell cycle
arrest (31-34), all experiments were performed at low (<50%)
confluency in the presence of fresh, serum-containing, buffered media.
We initially studied a well characterized, easily manipulated,
immortalized fibroblast cell line to identify key cell cycle components
that could be further investigated. When Rat1a fibroblasts were exposed
to hypoxia for approximately two doubling times (32 h), propidium
iodide staining and flow cytometric analysis of cell cycle distribution
revealed a significant (p < 0.005) G1
arrest (Table I). To assess directly the
rate of DNA synthesis, fibroblasts were rendered hypoxic for 30 h, transferred to a large hypoxic atmospheric chamber
(PlasLabs), and BrdUrd labeling was performed as described (26).
Hypoxic cells incorporated significantly less BrdUrd (44 ± 0.1%
in normoxia versus 24 ± 0.3% in hypoxia) confirming a
decrease in S phase under hypoxic conditions.
We then sought to identify further components of the G1
checkpoint that respond to hypoxia and might contribute to cell cycle arrest. RB phosphorylation is promoted by CDK2 and CDK4 when they complex with cyclins E, A, and D. A G1 arrest occurs when
these CDK activities are inhibited by the CDKIs p21, p27, or p16. After two doubling times in either normoxia or hypoxia, Rat1a fibroblasts were collected, and the expression of several proteins important for
the G1-S phase transition was examined. As previously
reported in other immortalized cell lines (16, 21, 35-36), hypoxia
induced hypophosphorylation of RB in Rat1a cells, a hallmark of
G1 arrest in normal cells (Fig.
1A). This reduction in RB
phosphorylation was associated with a decrease in CDK2 activity (Fig.
1A). Despite this decrease in kinase activity, the amount of
immunoprecipitated CDK2 protein was unaffected by hypoxia (Fig.
1A), indicating that a modifier of CDK activity
(e.g. cyclin E, A, p21, and/or p27) is altered by hypoxia.
Although p53 may be induced in hypoxia by an
HIF-1 Wild-type MEFs Also Undergo a Hypoxia-induced G1
Arrest--
The decrease in CDK2 activity and hypophosphorylation of
RB associated with hypoxia that we and others observed in rat and other
immortalized cell lines suggest, but do not conclusively support, a
role for RB and p27 in hypoxia-induced G1 arrest.
Immortalized cells may have multiple genetic abnormalities. Alterations
seen in one regulatory component (e.g. p27 induction) may be
the result of hypoxia-induced growth arrest, not a cause of the
G1 arrest. In addition, both overexpression of p27 and
serum withdrawal may induce G1 arrest in an RB-independent
manner (19, 20, 24). To evaluate the significance of these
hypoxia-induced changes noted in immortalized cells, hypoxia-induced
arrest was examined in mouse embryo fibroblasts (MEFs). This approach
allows the identification of changes that occur in primary cells with
minimal genetic alterations and permits the further examination of the
effects of hypoxia on isogenic MEFs deficient in key regulators of the
G1 phase of the cell cycle.
Similar to the immortalized fibroblasts, wild-type MEFs underwent a
G1 arrest (Table I) and decreased BrdUrd uptake (Fig. 2A) when rendered hypoxic. The
extent of this hypoxia-induced arrest was most apparent when wild-type
MEFs were synchronized with 32 h of serum withdrawal. When
re-stimulated with serum, MEFs incubated in 20% O2 showed
a dramatic (>60%) percentage of S phase at 16 h, whereas
serum-stimulated hypoxic MEFs increased S phase only minimally (<12%)
throughout the 32-h experiment (Fig. 2B). These observations
indicate that hypoxia inhibits G1/S transition in normal
primary fibroblasts.
Several of the changes that we (Fig. 1A) and others (16, 21,
35, 36) noted in immortalized cells were not apparent in primary cell
lines. Specifically, we noted only mild decreases in cyclin E and
cyclin A in MEFs (Fig. 1C and data not shown). Also, in
contrast to Rat1a cells, we noted minimal kinase activities associated
with CDK4 and cyclin E in both hypoxic and normoxic MEFs (data not
shown). However, similar to the immortalized cells that underwent a
G1 arrest in hypoxia, we noted that hypoxia increased p27,
decreased CDK2 activity, and led to a hypophosphorylation of RB in
hypoxic MEFs (Fig. 1C and Fig. 3, B and
C). These data suggest that these changes may be key in
regulating hypoxia-induced growth arrest.
Isogenic p27 and RB-deficient MEFs Demonstrate the Importance of
These Proteins in Hypoxia-induced G1 Arrest--
To better
assess the significance of RB hypophosphorylation and p27 induction in
hypoxia, MEFs deficient in G1 cell cycle regulators were
subjected to 32 h of hypoxia. Previous studies have shown that
hypoxia-induced growth arrest occurs in transformed cells with mutant
or null p53 status (17, 22). Consistent with these reports, MEFs null
for p53 or p21 arrested in hypoxia to the same degree as wild-type
cells (Table I and Fig. 2A). Therefore, neither p53 nor p21
is required for hypoxia-induced G1 arrest. However, as
compared with these cells, the ability of RB-null MEFs to arrest in
G1 in response to hypoxia was markedly diminished (Table I
and Fig. 2A), indicating that RB participates in this arrest.
Although the decrease in cyclin E in wild-type MEFs was less dramatic
than that in immortalized fibroblasts (Fig. 1, A and C), either a decrease in cyclin E and/or an increase in p27
could contribute to diminished CDK2 activity leading to RB
hypophosphorylation in hypoxia. Previous studies have shown that p27
induction is necessary for the growth arrest observed in Balb-3T3
fibroblasts subjected to serum withdrawal (33). To determine the extent that p27 induction is responsible for hypoxia-induced growth arrest, p27 null MEFs were rendered hypoxic for two doubling times. There was
no significant change in cell cycle profile or incorporation of BrdUrd
(Table I and Fig. 2A) despite hypoxia, indicating that p27
is necessary for hypoxia-induced G1 arrest. The
contribution of p27 to hypoxia-induced G1 arrest was also
apparent when CDK2 activity and RB phosphorylation status were examined
in p27 null fibroblasts rendered hypoxic (Fig.
3, B and C). The
base-line RB phosphorylation appears enhanced in the p27 null
fibroblasts. Whereas hypoxia led to a 42% decrease in CDK2 activity as
well as RB hypophosphorylation in wild-type MEF cells, these changes were not evident in p27 null fibroblasts.
It could be argued that the increase of p27 in hypoxic wild-type MEFs
is a secondary effect of cell cycle arrest and not a mediator of
hypoxia-induced G1 arrest. However, p27 expression was also
induced in hypoxic yet cycling RB null MEFs (Fig. 1C), which
indicates that the induction of p27 is a direct effect of hypoxia
rather than a secondary feature of RB hypophosphorylation and/or
G1 cell cycle arrest. We also observed that cyclin E
decreased in the hypoxic RB null MEFs, although the absolute levels of
cyclin E in both hypoxic and normoxic RB null MEFs were greater than those in wild-type MEFs (Fig. 1C). Therefore, we cannot
exclude the possibility that the resistance of RB null cells to
hypoxia-induced arrest may be due, in part, to increased cyclin E,
which in turn could then titrate hypoxia-induced p27.
Transient Decreases in p27 Also Abrogate the Hypoxia-induced
G1 Arrest--
Because a total absence of p27 may result
in abnormalities of cyclin expression and CDK-cyclin complex formation
(39, 40), we assessed whether a transient decrease in p27 would also
diminish the hypoxia-induced G1 checkpoint. p27 antisense
and missense phosphorothioate oligonucleotides containing both the
"G-clamp" and 5-methylcytosine analogues (41) were transfected with
cytofectin under conditions that have been shown to result in >90%
efficiency of oligonucleotide delivery to the nucleus (42). Whereas
hypoxia led to a G1 arrest and p27 induction in
nontransfected Balb-3T3 cells (data not shown) and missense transfected
cells, the antisense-treated cells had markedly less p27 induction with
hypoxia (Fig. 3D) and failed to arrest in G1
(Table II). Antisense treatment also
diminished RB hypophosphorylation (data not shown), again indicating
that the hypoxic induction of p27 is an upstream event of RB
hypophosphorylation. To determine whether our finding of a p27-mediated
hypoxia-induced G1 arrest may be generalized to other cell
types, immortalized human mammary epithelial cells (A1N4) were infected
with a full-length antisense p27-expressing adenovirus. Whereas A1N4
cells infected with control adenovirus expressing GFP arrested in
hypoxia, AIN4 cells treated with the antisense p27 virus showed a
decrease in inducible p27 and cell cycle progression despite hypoxia
(Table II and Fig. 3D). These observations further support
the hypothesis that an increase in p27 is required for hypoxia-induced
cell cycle arrest.
Hypoxia-induced G1 Arrest Affects the CDK2-Cyclin A
Complex--
CDK2 is activated when complexed with either cyclin E or
cyclin A; both of these complexes are inhibited by p27 (39). Hypoxia led to an increase in the amount of p27 associated with CDK2 in wild-type MEFs (Fig. 3A) as has been been reported in
immortalized cell lines (21). We confirmed that radiolabeled in
vitro translated cyclin E and cyclin A associate with CDK2 present
in MEF lysates (data not shown). Despite the fact that our protocol
immunoprecipitated in vitro translated cyclin E, the CDK2
activity associated with immunoprecipitated cyclin E was minimal in
both wild-type and p27 null MEFs, and hypoxia decreased this minimal
cyclin E/CDK2 activity in both wild-type and p27 null cells (data not
shown). Additionally, immunodepletion of cyclin E had a negligible
effect on total CDK2 activity in these MEFs (data not shown). In
contrast, when CDK2- and cyclin A-associated kinase activities were
measured from the same lysates, >90% of total CDK2 activity was
recovered in cyclin A-associated complexes (Fig. 3B). Cyclin
A/CDK2 activity decreased in hypoxic wild-type MEFs but was unchanged
in p27 null cells. Although cyclin A is a target of E2F (43), is
directly inhibited by hypophosphorylated RB (44), and has also been
reported to be diminished by hypoxia in immortalized kidney cells (36), we did not observe cyclin A levels to change appreciably with hypoxia
in either wild-type or p27 null MEFs (data not shown). Therefore, our
data suggest that the predominant active CDK2 complex in MEFs is
CDK2-cyclin A and that increased p27 associated with this
complex in hypoxia is responsible for inhibiting CDK2 activity and
inhibiting exit from G1.
Hypoxia Induces p27 Transcription via an HIF-1-independent
Hypoxia-responsive Proximal Promoter Region--
Because of the
importance of p27 inhibition of CDK2-cyclin A complexes in
hypoxia-induced G1 arrest, we sought to characterize better
the mechanism of p27 induction in hypoxia. Although the regulation of
p27 during the cell cycle and tumor progression is thought to occur
through translational and post-translational mechanisms (45, 46), we
observed a unique hypoxic regulation of p27 at the RNA level. When
Rat1a fibroblasts were exposed to hypoxia for 32 h, p27 RNA levels
increased 3-fold (Fig. 4A).
When cells were treated with the RNA synthesis inhibitor actinomycin D
in either hypoxia or normoxia, the p27 mRNA half-life did not increase in hypoxia (220 min in hypoxia versus 260 min in
normoxia) (Fig. 4). These observations suggest that hypoxia induces p27 at the transcriptional level, which contrasts with the increased stability of some mRNAs, including vascular endothelial growth factor, in hypoxia (47).
The best described hypoxia-induced transcription factor is HIF-1. HIF-1
has also been reported to have a role in cellular proliferation and is
overexpressed in some tumors (6, 7, 48). We thus examined the role of
HIF-1 in hypoxia-induced growth arrest and, more specifically, on p27
induction. Rat1a fibroblasts were cotransfected with an HIF1
Recent studies on the proliferative capacity of HIF-1
Consistent with our finding that HIF-1 transactivation does not
contribute to the hypoxia-induced G1 arrest, induction of HIF-1 in normoxia did not increase p27 expression (Fig. 1B).
We thus sought to determine whether the p27 promoter contains a
hypoxia-responsive region. NIH-3T3 cells, which arrest in
G1 and induce p27 when hypoxic (data not shown), were
transiently transfected with various fragments of the p27 promoter
linked to a luciferase reporter and subsequently subjected to 24 h
of hypoxia. Hypoxia increased the transcriptional activity of a 1.1-kb
fragment of the p27 promoter almost 3-fold relative to normoxia (Fig.
4B). Consistent with our observation that p21 is not induced
by hypoxia, transient transfections with a p53-responsive p21
promoter-luciferase reporter construct did not show an increase in
luciferase activity in hypoxic cells (data not shown). This lack of
activity also argues against nonspecific activation of G1
CDKI promoters when cells are arrested in G1. Induction of
the p27 promoter was dependent on DNA sequences located 1133 to 602 bp
5' to the transcription start site. Although it is necessary for the
hypoxic response of the p27 promoter, this 531-bp region is not
sufficient, since it alone subcloned 5' to an SV40 promoter-luciferase
construct did not result in a hypoxia-induced increase in luciferase
activity above that of the SV40 promoter alone (data not shown).
Deletional analysis indicated that the 531-bp region, when joined with
more proximal promoter sequences, is sufficient for hypoxic induction
of luciferase activity (1.1 D249-602 in Fig.
4B). Cotransfection of the 1.1-kb p27 promoter and an
HIF-1 Cellular responses to hypoxia are fundamental adaptive changes
that are required for normal mammalian physiology and may be exploited
by cancer cells for a proliferative/survival advantage. Although the
understanding of hypoxia-induced cellular metabolic changes is rapidly
emerging, our understanding of the effects of oxygen deprivation on the
control of the cell cycle is still rudimentary. In this study, we have
observed that hypoxia induces an accumulation of cells in
G1. This was best demonstrated when cells were synchronized
prior to being rendered hypoxic. As we and others (24) have noted, even
after prolonged serum withdrawal ~25% of MEFs continue to
incorporate thymidine. The observation that cells in other stages of
the cell cycle are also present after prolonged incubation in hypoxia
suggests several possibilities. One is that some individual cells may
escape G1 arrest. Purging the external environment of
oxygen may not necessarily lead to equivalent amounts of intracellular
hypoxia, and cell cycle regulation may vary with the extent of hypoxia.
Indeed, truly anoxic cells show an immediate and dramatic decrease in
BrdUrd incorporation, possibly through a mechanism other than the one
we observed at more moderate (~0.1%) levels of
hypoxia.2 It is also possible
that other stages of the cell cycle are also delayed in hypoxia. We
observed a decrease in S phase in all cell lines that exhibited a
hypoxia-induced G1 arrest (Table I.); this hypoxia-induced
decrease in S phase was greatly diminished or absent in RB null cells,
in p27 null cells, or when p27 was transiently reduced with antisense
techniques (Table II and Fig. 3D). A hypoxia-induced
G2 delay has been reported in some transformed cell lines
(23), and indeed when hypoxic cells were manipulated to escape their
G1 arrest with antisense p27 oligonucleotides, a small
accumulation in G2 was noted (Table II). Our data, however, are most consistent with the activation of a hypoxia-induced
G1/S checkpoint.
Although several studies have demonstrated changes in cell cycle
regulatory proteins associated with hypoxia-induced growth arrest,
mechanistic insight was not forthcoming from these reports. Tumor cells
lines with altered G1 checkpoints have also been used in
several studies. For example, previous studies have revealed that
hypoxia-induced arrest neither occurs in osteosarcoma cell lines
lacking functional RB nor in cells infected with HPV-E7 or E1a, in
which RB is inactivated (17, 21). A G1 arrest, however,
does occur in transformed and immortalized cells with mutant or null
p53 status (17, 22). The use of tumor cell lines provides some insight
into cellular responses to hypoxia, but it is unclear if these cells
have other abnormalities in their cell cycle regulatory mechanisms
(51). Indeed we have noted subtle differences in the effects of hypoxia
on immortalized fibroblasts and early MEFs. The immortalization process
often leads to an inactivation of p53, RB, and/or p16/p19/ARF (52).
Additionally, immortalized rat fibroblasts have been found to have a
methylated p21 promoter that may not be responsive to p53
transactivation (53). We observed that immortalized fibroblasts have a
greater reduction of cyclin E and cyclin A in response to hypoxia, when compared with wild-type MEFs (Fig. 1, A and C,
and data not shown). Additionally, CDK2/cyclin E kinase activity was
higher in immortalized rat fibroblasts than in MEFs, where it was
negligible (data not shown). However, consistent changes in both
immortalized and primary fibroblasts rendered hypoxic included an
increase in p27, a decrease in CDK2 activity, and RB hypophosphorylation.
By using genetically defined primary fibroblasts, we demonstrate that
hypoxia-induced G1 arrest requires a functional RB but not
p53 or p21. Indeed, neither the protein levels nor the promoter of the
CDKI p21, which is transcriptionally regulated by p53, is induced under
our hypoxic conditions. MEFs null for p27 also failed to arrest in
hypoxia. The importance of p27 induction in hypoxia-induced arrest is
underscored by the observation that the hypoxia-induced G1
block can be overcome by decreasing p27 through antisense approaches.
These observations suggest that the major regulator of the
hypoxia-induced G1 arrest is p27.
p27 was first noted to bind to cyclin E-CDK complexes and promote the
dephosphorylation of RB (20, 54). However, overexpression of p27
induces G1 arrest in osteosarcoma cell lines with mutant RB
(54), and serum starvation of RB null MEFs also induces a G1 arrest (24). Together, these observations suggest that a p27 induced, RB-independent G1 arrest pathway exists.
Indeed, another CDKI, p16, has also been shown to induce both
RB-dependent and RB-independent G1 arrests in
glioma cells (19). Our data also revealed a small G1 arrest
in hypoxic RB null MEFs, although it was greatly diminished when
compared with wild-type MEFs (Table I). We also noticed diminished
hypoxia-induced RB dephosphorylation in p27 null MEFs and when p27 was
reduced with antisense oligonucleotides (Fig. 3D). The
absence of RB led to increases in cyclin E, which may also play a role
in titrating induced p27, decreasing CDK activity, and promoting entry
into S phase even in hypoxia. However, the observation that cyclin E
decreased minimally in wild-type MEFs, where a significant decrease in
CDK2 activity was noted, and the fact that CDK2 activity changed
minimally in hypoxic p27 null MEFs, argues for a predominant role of
p27 induction in hypoxia-induced G1 arrest. Thus, our data
strongly suggest that p27 induces a G1 arrest in hypoxia by
modulating RB, although we cannot exclude the additional presence of an
RB-independent mechanism or an additional contribution from the high
levels of cyclin E noted in p27 null cells.
We noted that the predominant cyclin associated with CDK2 in both
unsynchronized, cycling MEFs, as well as in hypoxic arrested wild-type
MEFs, was cyclin A and not cyclin E. Cyclin E/CDK2 collaborates with
cyclin D-dependent kinases to complete RB phosphorylation. Inhibition of cyclin E-associated CDK2 activity during G1
inhibits entry into S phase, whereas inhibition near the
G1/S transition does not delay S entry (55). Cyclin A is
necessary for G2 and S phase progression, is up-regulated
by E2F, and is repressed by hypophosphorylated RB (43, 44). However,
cyclin A-associated CDK2 activity is also vital for the
G1/S transition. Microinjection of anti-cyclin A antibodies
inhibits entry into S phase (56), and cyclin A can complex with CDK2 to
phosphorylate RB (57). p27 can bind to and inhibit CDK2/cyclin A
activity (57, 58), and increases in cyclin A activity found in late
G1 do not correlate with increased cyclin A levels but
rather decreased binding of p27 to the CDK2-cyclin A complex (57).
These observations, combined with our data, suggest that that in mid to
late G1 CDK2-cyclin E and CDK2-cyclin A complexes are
sequentially activated, and when cells are hypoxic, p27 inhibits cyclin
A/CDK2 activity and RB phosphorylation and prevents entry into S phase
(Fig. 5).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and a constitutive subunit HIF-1
(ARNT), which transactivates genes encoding several glycolytic
enzymes as well as the vascular endothelial growth factor gene
(4-7).
in
regulating the cell cycle in hypoxia has not been clearly demonstrated
(6, 7), and the events leading to the hypoxic hypophosphorylation of
RB, and indeed the very relevance of RB phosphorylation status in
hypoxia-induced G1 arrest, have not been well delineated.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
expression vector, pCEP4/HIF-1
,
was described previously (5). For the cotransfection experiments, Rat1a
fibroblasts were transfected with 10 µg of pCEP4/HIF-1
(or pCEP4) and 1 µg of green fluorescent protein (GFP)
expression plasmid (pEGFP-N1, CLONTECH). HIF-1
function was assessed by a construct containing a hypoxia-responsive
element from the human erythropoietin gene subcloned 5' to an
SV40 promoter luciferase reporter (pGL2, Promega) (5). The
murine p27 promoter and deletion constructs, subcloned into the
pGL2 basic luciferase vector (Promega), were the generous
gift of Dr. Sehng-Cai Lin (27) and the 249-602 deletion construct was
created by digesting the 1.1-kb promoter with EagI. The
p53-responsive p21 promoter/luciferase reporter construct was obtained
from Dr. Bert Vogelstein (28). Luciferase assays (Promega) were
performed, and the data were normalized for total protein.
was from Novus Biologicals. Coomassie Blue staining of total
cellular protein confirmed equal protein loading. For
immunoprecipitation (IP) experiments, cyclin A (H432) and CDK2 (M2)
antibodies were obtained from Santa Cruz Biotechnology, and cyclin E
antibody (Ab1) was obtained from Neomarkers. 100 µl of protein
A-Sepharose beads (Amersham Pharmacia Biotech) were loaded with 20-40
µg of antibodies in IP buffer (50 mM Tris-HCl (pH 7.4),
250 mM NaCl, 0.5 mM
Na3VO5, 20 mM
-glycerophosphate, 15 mM phosphatase substrate p-nitrophenyl
phosphate, protease inhibitor Complete tablets (Roche Molecular
Biochemicals), and 0.1% Nonidet P-40). Lysates from cells subjected to
hypoxic or nonhypoxic culture conditions for 32 h were lysed with
IP buffer, sonicated, and solubilized for 30 min at 4 °C, and
protein concentration was assayed with the BCA kit (Pierce). Washed
protein A beads were incubated with 300 µg of lysate for 2 h at
4 °C, and the beads were then washed three times with IP buffer.
Histone kinase assays were performed by incubating the beads in buffer
containing 50 mM Tris (pH 7.4), 1 mM
CaCl2, 5 mM MgCl2, 0.5 mM Na3VO5, 20 mM
-glycerophosphate, 15 mM phosphatase substrate
p-nitrophenyl phosphate, protease inhibitor Complete tablets
(Roche Molecular Biochemicals), 50 µM ATP, 0.2 µg of
purified histone H1 (Roche Molecular Biochemicals), and 10 µCi of
[
-32P]ATP at 30 °C for 30 min. The reaction was
stopped by the addition of 50 µl of 2× Laemmli buffer and heating
for 95 °C for 5 min. The products were separated by
SDS-polyacrylamide gel electrophoresis, and autoradiography was
performed. To assess the amount of CDK2 protein in the lysates, CDK2
was immunoprecipitated with a goat anti-CDK2 antibody, and the blot was
probed with a rabbit anti-CDK2 antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Effects of hypoxia on the cell cycle profiles of various cell lines
-dependent manner (37, 38), we observed only a minimal increase in p53 and actually observed a decrease in p21 under
our experimental conditions (Fig. 1A). In contrast, protein lysates of hypoxic cells revealed a decrease in cyclin E and a minimal
decrease in cyclin A (Fig. 1A). Also consistent with a decrease in CDK2 activity and RB phosphorylation, hypoxia led to a
significant increase in the CDKI p27 (Fig. 1B).
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Fig. 1.
Alterations of G1/S checkpoint
with hypoxia. A, protein immunoblots demonstrate the
phosphorylation status of RB and expression of cyclin E, p53,
and p21 in untreated and hypoxic cell lines. Coomassie Blue staining of
total cellular protein confirms equal protein loading. PP
represents the hyperphosphorylated forms of RB, and P
represents the hypophosphorylated forms of RB. The histone kinase
(HK) activities of immunoprecipitated CDK2 containing
complexes under normoxic or hypoxic conditions are also shown.
B, p27 protein levels with hypoxia or 200 µM
CoCl2 for 32 h. C, protein levels of p27
and cyclin E in MEFs after 32 h of hypoxia or normoxia.
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Fig. 2.
A, BrdUrd uptake in wild-type and
RB, p53, and p27 null MEFs. Cells were incubated with 10 µM BrdUrd for the last 30 min of a 32-h incubation in
either 20% O2 (top panels) or hypoxia
(bottom panels). Isolated nuclei were then stained with PI
and an anti-BrdUrd antibody, as described under "Materials and
Methods." Representative experiments are shown, along with the
average number of BrdUrd-positive cells ± S.E. from three
experiments performed in duplicate. B, effect of hypoxia on
cell cycle of synchronized MEFs. MEFs were synchronized in 0.1% serum
for 32 h. Media were then changed to include 10% serum. After
incubation in either normoxia or hypoxia, cells were harvested at 0, 8, 16, 24, and 32 h and stained with PI, and cell cycle was analyzed
as described. Qualitatively similar results were obtained from three
separate experiments, and a representative experiment is
displayed.
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Fig. 3.
A, p27 associates with CDK2 in hypoxia.
CDK2 was immunoprecipitated from MEF after 32 h of hypoxia or
normoxia, and the amount of p27 associated with this CDK2 was assessed
by immunoblotting with an anti-p27. 10% of the total protein used for
the CDK2 immunoprecipitation was run on a gel subsequently stained with
Coomassie Blue to ensure equal loading (input).
B, cyclin A and CDK2 histone kinase (HK) activity
in wild-type MEFs and p27 null MEFs. MEFs were normoxic or rendered for
32 h, and kinase activity associated with immunoprecipitated
cyclin A and CDK2 was assessed. The bottom panel displays
kinase activity of wild-type and p27 null MEFs in both normoxia and
after 32 h of hypoxia (dark bars). Hypoxic values
reflect mean percent ± S.E. of normoxic values, all averaged from
three separate experiments. Kinase activities of normoxic p27 null
lysates were normalized to normoxic wild-type levels. C,
phosphorylation status of RB in hypoxic MEFs. Cell lysates from
normoxic and hypoxic wild-type and p27 null MEFs were harvested. and RB
was immunoblotted. PP represents the hyperphosphorylated
forms of RB, and P represents the hypophosphorylated
forms of RB. Coomassie Blue staining of total cellular protein confirms
equal protein loading. D, p27 antisense oligonucleotides and
p27 antisense adenovirus decrease the hypoxia-induced accumulation of
p27 and hypoxia-induced G1 arrest. Balb-3T3 cells were
transfected with 30 nM p27 antisense or missense. A1N4
cells were infected with either full-length human p27 antisense virus
or, as control, GFP-expressing adenovirus. After a brief recovery
period, cells were exposed to 32 (Balb-3T3) or 48 h (A1N4) of
hypoxia and compared with cells incubated in normoxia. Coomassie Blue
staining of total cellular protein confirms equal protein
loading.
The effect p27 manipulation on hypoxia-induced growth arrest
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Fig. 4.
A, hypoxia induces p27 RNA expression
increases with hypoxia but does not prolong p27 RNA half-life. After
28 h of hypoxic or nonhypoxic conditions, media were changed to
contain 0.5 µg/ml actinomycin D (Sigma). Total RNA was harvested at
0, 30, 120, and 240 min after actinomycin D addition under continued
hypoxic or nonhypoxic conditions. The upper panel is a
representative experiment, with ethidium bromide-stained 18 S RNA
presented as a loading control. The lower panel shows the
average of three experiments, normalized to the amount of p27 RNA
present at time 0 in nonhypoxic cells, with error bars
reflecting S.D. Filled bars represent hypoxic conditions.
Autoradiographic signals were quantitated by PhosphorImager.
B, increased p27 promoter activity in hypoxic cells. NIH-3T3
fibroblasts were transiently transfected with fragments of the murine
p27 promoter linked to a luciferase reporter (1.5 µg) and
subsequently incubated in either 20% O2 or hypoxia as
described. Fold increase indicates the ratio of activity of the
constructs in hypoxia to their activity in normoxia. Numbers
represent the average ± S.E. of three to ten experiments
performed in duplicate.
expression vector and GFP expression plasmid in a 10:1 w/w ratio, and
48 h later cell cycle analysis was performed on GFP-positive
cells. In addition, Rat1a fibroblasts were treated for 32 h with
200 µM CoCl2, a known inducer of HIF-1
(5). For both conditions HIF-1
expression was assayed by protein immunoblot, and HIF-1 function was assessed by means of cotransfection experiments with an HIF-1
responsive luciferase reporter construct containing a hypoxia response element from the human erythropoietin gene (5). HIF-1
expression and HIF-1 activity were equivalent in
both conditions to those found in hypoxic cells (data not shown). Although transfection of Rat1a cells with control plasmid caused a
reproducible shift of cells distributed in the G1 fraction, high expression of functional HIF-1 did not further arrest cells in
G1 (p = 0.26, Table I). Similarly, the
CoCl2-induced expression of functional HIF-1 in normoxia
did not alter the cell cycle profile of control treated cells as
assessed by both propidium iodide staining (Table I) and by BrdUrd
uptake (41 ± 0.1% in CoCl2-treated cells
versus 44 ± 0.1% in control cells). These data
indicate that HIF-1 transactivation is not sufficient for
hypoxia-induced cell growth arrest.
-deficient ES
cells have been contradictory. Whereas one study suggested that ES
cells lacking HIF-1
do not have a lower S fraction in hypoxia than
in normoxia (6), another reported that HIF-1
null ES cells
proliferate more slowly in hypoxia as compared with normoxia (7). When
we subjected subconfluent ES cells (prepared and cultured as described
(7)) to 32 h of hypoxia, similar degrees of G1 arrest
were observed in wild-type cells (G1 29 ± 0.1% in normoxia versus 34 ± 0.1% in hypoxia) and in
HIF-1
-null ES cells (G1 26 ± 0.1% in normoxia
versus 31 ± 0.6% in hypoxia). Although aspects of
cell cycle regulation may be significantly different in ES cells as
compared with somatic cells (49, 50), these observations suggest that,
at least in ES cells, HIF-1
is not necessary for hypoxia-induced
growth arrest.
expression vector did not result in a significant increase in
luciferase activity, even in hypoxia where HIF-1 transactivation of the
erythropoietin promoter increased nearly 10-fold over base line (data
not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
A schematic of our proposed mechanism of cell
cycle arrest in hypoxic primary cells.
Although it might be argued that the hypoxia-induced increase in p27
protein levels is simply a secondary effect of G1 arrest, our observations that changes in both proteins also occur in RB null
cells, which continue to cycle in hypoxia, suggest that they are
primary effects of hypoxia. The regulation of p27 during the cell cycle
typically occurs at the protein level, regulated by translational
control and ubiquitination (45, 46). However, our experiments revealed
a hypoxia-induced elevation of the p27 transcript that has an unaltered
half-life in hypoxia. These results are consistent with a
transcriptional activation of p27 by hypoxia. The existence of a
physiological role for the transcriptional regulation of p27 is
supported by the recent finding that dioxins can bind to the Ah
receptor and increase transcription of p27 in hepatoma cells and
thymocytes (59). Although the Ah receptor is closely related to
HIF-1, we found that functional HIF-1 could not induce growth
arrest, activate the p27 promoter, or elevate p27 expression. Although
regulation of the cell cycle in somatic cells and ES cells may not be
directly comparable (49, 50), we found no abrogation of the
hypoxia-induced growth arrest in HIF-1
null ES cells. In addition,
p27 mRNA increases in hypoxic HIF-1
-null ES cells (6). In
aggregate, these studies suggest that p27 is induced by hypoxia at the
transcriptional level independent of HIF-1, and the hypoxia-induced
G1 arrest is also independent of HIF-1. Although we have
not formally excluded the other well characterized hypoxia-inducible
transcription factor, endothelial periodic acid-Schiff protein-1
(EPAS-1/HLF/HIF-2
) (60), this factor is also induced by
CoCl2 (61), a condition in which we noted no cell cycle arrest.
The p27 CDKI appears to play an important role in the response of cells to their environment. Induction of p27 expression is responsible for the growth arrest seen in serum-deprived fibroblasts (33). p27 protein is also induced by E-cadherin when transformed cells are grown in spheroid culture and is responsible for the growth arrest and chemotherapy resistance seen under these conditions (34, 42). Our data suggest that hypoxia is another example of environmental control of p27. In the potentially damaging environment of hypoxia, elevation of p27 inhibits DNA replication and may prevent the inappropriate proliferation of genetically damaged cells.
Two of the most common genetic abnormalities found in human tumors are
mutations or inactivation of the p53 and RB pathways (62). p53
expression in transformed hypoxic cells, possibly promoted by HIF-1
(37), leads to apoptosis and thus selects for cells with mutant p53
(15). Our data suggest that these cells will still be hypoproliferative
in the background of a functional RB. Further studies will be required
to determine whether other RB-related proteins such as p107 or p130 may
also participate in the cellular response to hypoxia. Hypoxic
proliferation of tumor cells could occur through inactivation of RB or,
as shown by our studies, a decrease in inducible p27. Although p27 is
not one of the many cell cycle regulators known to be commonly deleted in human cancers, its importance in neoplastic progression is underscored by the fact that even a heterozygous reduction of p27 leads
to spontaneous and radiation-induced tumors in mice (63). Furthermore,
p27 protein decreases during tumor development and progression in
breast, colon, prostate, and other cancers (64). It is intriguing that
some transformed cells have been reported to escape partially the
hypoxia-induced cell arrest (23); the role of RB and/or p27 in this
escape is not known. Further studies will be necessary to characterize
better the trans-acting factor(s) that regulate p27 gene expression in
hypoxia, to determine whether modulation of p27 expression accounts for
the ability of some neoplastic cells to escape this G1
arrest, and to determine whether modulation of p27 expression may alter
chemosensitivity or radiosensitivity of hypoxic tumors.
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ACKNOWLEDGEMENTS |
---|
We appreciate the comments from anonymous reviewers, which have significantly contributed to the quality of the paper. We thank J. Flook for fluorescence-activated cell sorter analysis and Andrew Koff and members of the Dang and Semenza labs for assistance, especially H. Shim, J. Prescott, E. Emison, and C. Sutter Hayes. We are especially thankful to Tyler Jacks for the MEFs; Sheng-Cai Lin for the p27 promoter; and Bert Vogelstein for the p21 promoter construct and plasmids necessary for the AdEasy generation.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant CA 51497.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Fellow of the Lymphoma Research Foundation of America.
§§ Current address: Sunesis Pharmaceuticals Redwood City, CA 94063.
To whom correspondence should be addressed: Ross
Research Bldg., Rm. 1025, 720 Rutland Ave., Baltimore, MD 21205. Tel.:
410-955-2773; Fax: 410-955-0185; E-mail: cvdang@jhmi.edu.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M010189200
2 S. L. Green and A. J. Giaccia, personal communications.
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ABBREVIATIONS |
---|
The abbreviations used are: HIF-1, hypoxia-inducible transcription factor-1; CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; RB, retinoblastoma protein; MEF, mouse embryo fibroblast; ES, embryonal stem cell; GFP, green fluorescent protein; BrdUrd, bromodeoxyuridine; PI, propidium iodide; IP, immunoprecipitate; kb, kilobase pair; bp, base pair.
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