Carcinogenesis vol.25 no.12 pp.2325--2335, 2004
doi:10.1093/carcin/bgh274
Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in
human cancer cell lines
Adrian Harold Box1--3 and Douglas James Demetrick1--5
1
2
3
Department of Pathology, Department of Oncology, Department of
Medical Biochemistry and 4The University of Calgary and Calgary
Laboratory Services, Calgary, Alberta, Canada, T2N 4N1
5
To whom correspondence should be addressed
Email: [email protected]
Flow cytometric analysis of fibroblasts, normal breast
epithelial cells and breast or other cancer cell lines identified variation in the abilities of cell lines to undergo cell
cycle arrest as a response to hypoxia. Human mammary
epithelial cells (HMEC), normal fibroblasts (Hs68 and
WI38), HeLa cervical carcinoma and HTB-30 breast carcinoma cells arrest in G1/S in response to severe hypoxia.
Hep3B hepatocellular carcinoma cells did not exhibit
orderly G1/S arrest in response to severe hypoxia. We
found a general decrease in p16INK4a (p16) mRNA levels,
with an associated decrease in p16 protein levels in both
normal cells and in cancer cells, regardless of their cell
cycle response to hypoxia. p27 protein levels did not correlate with the cell line’s ability to enter a hypoxic G1/S
arrest. Furthermore, cell lines that underwent G1/S arrest
showed decreased expression of hypoxia inducible factor 1
(HIF-1a) and at least one member of INK4 or Sdi cell cycle
kinase inhibitors families after 12--24 h of hypoxia.
Conversely, Hep3B, which did not exhibit orderly hypoxiaassociated G1/S arrest, also did not show decreased HIF1a, INK4 or Sdi protein levels in hypoxia. Furthermore,
Hep3B showed constitutive activating phosphorylation of
Akt and inhibitory phosphorylation of GSK3b, which was
the opposite pattern to that exhibited by the cell lines
showing the G1/S arrest phenotype. Inhibition of GSK3b
by lithium chloride treatment of HeLa cells converted the
HIF-1a, p16 and p27 loss to levels unchanged by hypoxic
exposure. Our results suggest that regulation of the cell
cycle during hypoxia in either normal or cancer cells is
not simply due to up-regulation of cell cycle kinase
inhibitors. Furthermore, decreased protein expression of
HIF-1a, p16 and p27 was associated with both a hypoxiainduced G1/S arrest phenotype and increased GSK3b
activity.
Introduction
Tumour hypoxia is an important mitigating factor in the treatment of many forms of cancer. As a tumour mass grows, it
experiences extended periods of low O2 levels due to the size
of the mass and the lack of an appropriate blood supply (1).
Abbreviations: CDK, cyclin-dependent kinase; CDKI, cell cycle kinase
inhibitors; HIF-1a, hypoxia inducible factor 1; HMEC, human mammary
epithelial cells; p15, p15INK4a; p16, p16INK4a; p18, p18INK4a; p19, p19INK4a;
p21, p21WAF1; p27, p27Kip1; p57, p57Kip2; pRb, retinoblastoma protein; RPA,
Ribonuclease Protection assay; TS, tumour suppressor.
Carcinogenesis vol.25 no.12 # Oxford University Press 2004; all rights reserved.
In order for a tumour to metastasize, it must survive this period
long enough for vascularization to occur (2). Even after vascularization, the new blood vessels are often too poorly formed
to supply adequate O2 perfusion, resulting in a continually
hypoxic tumour environment. Hypoxic tumours are more
aggressive and invasive than their normoxic counterparts and
exhibit a greater tolerance to many chemo- and radiotherapies,
probably due to the importance of the enhancing effects of O2
and/or blood perfusion in these treatments (3--5).
The most characterized molecular response to hypoxia is the
stabilization and activation of the transcription factor known as
hypoxia inducible factor 1 (HIF-1) (6,7). HIF-1 is a heterodimer composed of the HIF-1a subunit, which is a member of
the basic helix--loop--helix--PAS protein family, and the HIF1b subunit, which was later identified to be ARNT (8).
Although both subunits of HIF-1 are constitutively expressed,
HIF-1a is rapidly degraded in the presence of cellular O2 via
the ubiquitin-mediated proteosome pathway (9,10).
A shift from aerobic metabolism to glycolytic metabolism
during hypoxia, in a major part mediated by HIF-1, is accomplished by the up-regulation of various metabolic genes, such
as glucose transporters (11), aldolase, lactate dehydrogenase
(12) and pyruvate kinase (13). HIF-1 also up-regulates vascular endothelial growth factor (14,15), presumably in order to
induce the formation of new blood vessels, and erythropoietin
(7), which accelerates red blood cell maturation and extends
lifespan to increase blood O2 carrying capacity.
Another major consequence of cellular hypoxia is a proliferation arrest of cells (16,17). Cells subject to severe hypoxia
will arrest in G1 or early S phase. Cells in late S, G2 or M phase
will finish cell division and arrest in G1 (16), unless the
hypoxia is severe (18). Progression of cells through the cell
cycle is controlled by a series of periodically expressed proteins known as the cyclins, and their associated partners,
the cyclin-dependent kinases (CDK) (reviewed in ref. 19).
The major proteins associated with the G1/S boundary are the
cyclin D family (D1, D2 and D3), their respective CDKs,
CDK4 and CDK6, and cyclin E, with its respective CDK,
CDK2 (reviewed in ref. 20). The G1/S cyclin/CDKs function
to control the phosphorylation of the retinoblastoma protein
(pRb) (reviewed in ref. 21).
p16INK4a (p16) was identified and found to bind to the
catalytic component of the cyclin D/CDK4 complexes, thereby
preventing their ability to phosphorylate pRb (22). This protein
was also found to be inactivated in multiple tumour cell lines,
suggesting that it was a tumour suppressor (TS) gene (23--25).
p16 knockout mice were shown to spontaneously develop
tumours early on in life (26), providing further evidence that
it is a putative TS.
Three other members of the INK4 gene family were identified based upon their sequence similarity to p16. p15INK4a
(p15), p18INK4a (p18) and p19INK4a (p19) were all identified
as members of the INK4 gene family and shared with p16 an
ability to bind to CDK4/6 to cause a G1 arrest (27,28). p16 has
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A.H.Box and D.J.Demetrick
been implicated in the induction of cellular senescence, or
withdrawal from normal cell cycle (29) and for contact inhibition of confluent cells (30). p15 is induced by and mediates cell
cycle arrest in response to transforming growth factor beta
(TGF-b) (27). p18 and p19 expression varies throughout the
cell cycle, reaching maximum levels at the G1/S transition
(28), and it has been suggested that they function in cell
cycle arrest coupled to specific differentiation pathways
(reviewed in ref. 31). Furthermore, data from transgenic
mouse studies suggests that the INK4 gene family may have
non-overlapping functions (32). Another group of CDK inhibitors, the Sdi family is represented by the prototype p21WAF1
(p21) (33--35), p27Kip1 (p27) (36) and p57Kip2 (p57) (37).
These proteins have inhibitory effects on several CDKs, and
p27 has been shown to be a likely TS gene.
Studies examining the effects of hypoxia on cell cycle
components have yielded conflicting results. Initial experiments have shown decreases in CDK4 and CDK2 kinase
activities, and accumulation of pRb in its hypophosphorylated state (38--40). Analysis of CKIs in hypoxia has shown
an increase in p27 protein levels (41), which has been
suggested as the cause of the hypoxic G1/S arrest.
Recently, this idea has come under fire (18), with evidence
showing that p27 is not necessary for hypoxic arrest. Analysis of the INK4 gene family in hypoxia has been inconclusive, with p16 levels initially reported to be below
detection levels (40), although a study using monkey CV1P cells showed hypoxia-induced up-regulation of p16 and
association with cdk4 (42). Plainly, any of the INK4 or Sdi
cell cycle kinase inhibitors (CDKI) may be good candidates
to mediate G1 arrest in response to hypoxia. With both
CDK4 and CDK2 kinase activities reduced in hypoxia, it
may be that a coordinated up-regulation of both CDKI
families, INK4 and Cip/Kip, is necessary for hypoxiainduced cell cycle arrest. p15 and p27 have been documented to cooperate in TGF-b-induced cellular arrest (43),
supporting this idea of coordination. Also, there is evidence
that induction of p16 causes a rearrangement of p27 and
p21 from cyclinD/CDK4 to cyclinE/CDK2, effectively inhibiting both G1/S kinase complexes (44--47). Therefore,
hypoxic cell cycle arrest may not be solely attributed to
one G1/S kinase pathway, and may require the coordinated
up-regulation of multiple CDKIs. In this study, we explore
the effects of hypoxia on a number of different cell lines in
order to determine the potential inducers of the hypoxiaassociated G1/S arrest.
Materials and methods
Tissue culture
WI-38, Hs68 (human fibroblasts), Hep3B (hepatocellular carcinoma), HeLa
(cervical carcinoma) and HTB-30 (also known as breast carcinoma, SK-BR-3)
cell lines (ATCC) were seeded on 175 cm2 dishes at 105 cells/ml in 20 ml of
recommended growth media (Invitrogen). Primary human mammary epithelial
cells (HMECs) were seeded and grown according to supplier’s recommended
conditions (Clonetics). All cells were incubated at 5% CO2/37 C for 48 h to
50--60% confluency prior to exposure to hypoxic conditions. Hypoxia was
established by incubating cells in the GasPak anaerobic incubator (BD
Bioscience), which reduces O2 levels while maintaining CO2 levels at ~5%.
O2 levels were monitored by a methylene blue indicator strip, which bleaches
white at 0.1--0.5% O2 levels, following which timing of hypoxia was measured.
Control cells were incubated alongside hypoxic samples under a normal tissue
culture gas mixture (20% O2/5% CO2). For treatment with lithium chloride
(LiCl, Sigma), cells were prepared as above, with the addition of 50 mM (final)
LiCl to the culture media immediately preceding hypoxia.
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Cell cycle analysis
For ploidy analysis, cells were prepared as follows, with all steps carried out at
4 C unless noted. Media was removed from the plates and the cells were rinsed
with 10 ml of ice-cold phosphate-buffered saline (PBS). The cells were lifted
by incubation with 2.5 ml trypsin and neutralized by the addition of 5 ml cell
culture media with FBS (Invitrogen). 1 106 cells were counted, spun down at
800 g and re-suspended in 1 ml of FBS-free media containing 1 mM EDTA to
prevent clumping. An equal volume of 100% ethanol was added drop wise and
the cells were stored at 4 C for 24 h. After storage, the cells were pelleted at
800 g, aspirated and re-suspended in 1 mg/ml RNase A (Invitrogen) in PBS and
incubated for 30 min at room temperature. 0.5 ml of 50 mg/ml propidium
iodide (Sigma-Aldrich) was added after RNase digestion and incubated for an
additional 30 min in the dark. Cells were then analysed for DNA content using
a Becton-Dickinson FACScan to determine cell cycle phase.
RNA extraction and preparation
Total cellular RNA was isolated using TRIzol LS (Invitrogen) according to
manufacturer’s protocol, which was modified as follows: RNA was precipitated using 0.5 vol of isopropanol and 0.5 vol of 1.2 M NaCl/0.8 M sodium
citrate, to prevent proteoglycan contamination. RNA was re-suspended in
DEPC-treated H2O, quantified, and used immediately or precipitated and
then stored in 100% ethanol at 80 C for later use.
Northern blot analysis and ribonuclease protection assay
For northern blot analysis, 60 mg of total RNA was loaded onto a 0.8%
formaldehyde agarose gel and transferred overnight to nitrocellulose using
standard procedures (48). Radiolabelled probe was synthesized by nick translation with 32P-CTP from full-length p16 cDNA cloned into pBluescript.
Membranes were incubated overnight with 2 106 c.p.m. of radiolabelled
probe and exposed to film for 2--7 days.
Transcriptional analysis was performed using the Ribonuclease Protection
assay (RPA). Total cellular RNA (10 ug) was analysed using the Human cell
cycle regulator probe set (BD BioSciences) following the manufacturer’s
protocol. Gels were exposed to autoradiography film overnight on a Kodak
intensifying screen, or for up to 5 days for weaker signals.
Western blot analysis
For analysis of cell cycle proteins, hypoxic and normoxic tissue culture cells
were prepared as described above, placed on ice and rinsed with 10 ml of icecold PBS. The cells were lysed on the plate by the addition of 1 ml of NP-40
lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1.5 mM
MgCl2, 1 mM EGTA, 100 mM NaF, 1% NP-40, 10 uM sodium vanadate,
10 uM PMSF, 10 ug/ml aprotinin, 5 ug/ml leupeptin). Protein concentration
was determined using the Bichronate assay (Pierce). Seventy micrograms of
cell lysate was loaded onto an SDS--PAGE gel and transferred to nitrocellulose
(for detection of HIF-1) or 0.1 m PVDF (Millipore) (for detection of proteins
530 kDa) by blotting overnight at 30 V/60 mA. Membranes were blocked for
1 h at room temp in 10% (w/v) powdered non-fat milk/Tris-buffered saline
with 0.1% (v/v) Tween-20 (Sigma). Western blotting was carried out overnight
at 4 C using the following antibodies: HIF-1a (# 610959; 1:250), p27 (# 25020;
1:2000) (Transduction Labs), Pan actin (Ab-5; 1:2000), p16 (Ab-1; 1:250), p15
(Ab-6; 1:200), p18 (Ab-3; 1:250) (Neomarkers), p19 (M-167; 1:500), p21 (F-5;
1:1000) and PARP (F-2; 1:2000) (Santa Cruz).
Detection of protein phosphorylation by western blotting was performed
using the conditions listed above, with the following modifications: after
transfer of proteins to nitrocellulose, membranes were incubated in 5% (w/v)
bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% (v/v)
Tween-20 and 0.1% (v/v) NP-40. Western blotting was performed overnight at
4 C using the following antibodies: GSK3b (#610201; 1/2000) (BD Transduction Labs), Phospho-GSK3a/b Ser 21/9 (#9331S; 1/2000), Phospho-Akt Ser
473 (#9271S; 1/2000), Akt (#9272; 1/2000) (Cell Signaling Technologies).
Results
Differing cell types exhibit differing arrest profiles in
response to hypoxia
We determined the cell cycle phenotype of a variety of cell
lines responding to hypoxia. As shown in Figure 1, HTB-30,
HeLa and HMEC exhibited hypoxia-associated G1/S arrest
occurring at 6--12 h after initial exposure to hypoxic conditions. In comparison, Hep3B, a hepatocellular carcinoma cell
line, lacked an observable G1/S arrest (defined as an increase
in the G0/G1 peak with a decrease in the S peak). All cell lines,
however, showed a lack of proliferation at 24 h in comparison
Cell cycle regulators in hypoxic cell cycle arrest
Fig. 1. Cell cycle analysis of selected cell lines in hypoxia. Cells were incubated in a normoxic (20% O2; UT) or hypoxic (50.5% O2; T) atmosphere for
2 or 24 h, fixed in ethanol, stained with propidium iodide and analysed for DNA content to determine G1 and S phase cell populations. (A) Graphical
representation of flow cytometric analysis of the HTB-30 cell line shows an increase in G0/G1 and a decrease in S phase, indicating cell cycle arrest
in response to hypoxia. (B) The HeLa cell line also shows hypoxia-induced cell cycle arrest. (C) Hep3B cells do not exhibit a significant increase in G0/G1
or decrease in S phase, indicating a lack of cell cycle arrest, even after 24 h of hypoxia. (D) Flow cytometry histograms of primary HMEC cells
show a gradual decrease in S phase and M phase starting at ~6 to 12 h after initial exposure to low O2, with a parallel increase in percentage of cells
in G0/G1. (E) Primary WI38 fibroblasts exhibit a decrease in cells transiting through S phase.
with untreated controls (not shown), indicating that the severe
hypoxia did cause proliferation arrest (probably due to insufficient cellular ATP for cell division), but only Hep3B failed to
undergo block at the G1/S boundary.
p16 RNA and protein levels are reduced by hypoxia
As p16 was a potential mediator of G1/S arrest seen in
the normal response to hypoxia, we evaluated its expression
in the treated and untreated cell lines. While p16 expression
is usually associated with cell cycle arrest, RPA analysis of
the diploid fibroblast cell line WI-38 revealed a reduction in
p16 mRNA levels after 12 and 24 h exposure to hypoxic
growth conditions (Figure 2A). This reduction in p16
mRNA was confirmed by northern blot analysis (Figure 2B).
Hep3B cells did not show a reduction in p16 mRNA
levels (data not shown). As well, HeLa p16 transcript levels
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Fig. 2. p16 mRNA levels are reduced in diploid fibroblasts WI-38. p16 mRNA levels were analysed by RPA or northern blot in cell lines incubated in
a normoxic (20% O2; UT) or hypoxic (50.5% O2; T) atmosphere for the times indicated. Levels of L32 transcript were used as a loading control for
RPA and 28S rRNA was used in northern blot analysis. Identically treated whole cell lysates were prepared for immunoblot analysis. (A) WI-38 fibroblast cells,
which undergo cell cycle arrest, reveal a reduction in p16 mRNA levels starting at 12 h (12H T) after exposure to hypoxia, as measured by RPA.
(B) Northern blots of p16 mRNA levels confirm p16 mRNA reductions in WI-38 fibroblasts treated with hypoxia for 24 h (24H T). (C) p16 mRNA levels
correlate with a reduction in p16 protein levels in WI-38 fibroblasts. (D) HMEC and HTB-30 cell lines exhibit a similar reduction in p16 mRNA levels,
as measured by RPA, while L32 ribosomal protein mRNA loading standards are unchanged.
showed no reliable changes in response to hypoxia (data not
shown).
Both WI-38 and Hs68, another normal diploid fibroblast cell
line, showed a reduction in p16 protein levels after 24 h in a
low oxygen environment (Figure 2C), in agreement with the
RPA data, as did both HMEC and HTB-30 cells (Figure 2D).
In HMEC and HeLa cell lysates, p16 protein levels again were
reduced after 12--24 h in hypoxia (Figure 3C). Hep3B, which
did not appear to undergo G1/S arrest in hypoxia, did not show
any variation in p16 protein level.
Cip/Kip and INK4 proteins are reduced in hypoxia
We chose to evaluate the expression of Cip/Kip and the p16related proteins to determine if the relative levels of the
remaining CDKIs were modulated in response to hypoxia. As
shown in Figure 3, all cell lines used in this study exhibited
down-regulation of at least one of the G1/S CDKIs in response
2328
to hypoxia. HMEC, HTB-30 and HeLa showed loss of p16
protein, while Hep3B, the cell line most resistant to hypoxiainduced cell cycle arrest, showed no loss of p16 protein after
24 h of treatment. There was also loss of p19 protein expression, which occurred in Hep3B, the breast cancer cell line
HTB-30, and HeLa cells (p19 was undetectable in HMEC
cells). Hep3B and HMEC exhibited a loss of p21, again after
12--24-h exposure to hypoxia, while p21 levels were undetectable in both HTB-30 and HeLa cells. Interestingly, HeLa was
the only cell line to show a drastic reduction in all detectable
CDKIs. In all cells, there was at least one instance of loss of a
CDKI of both the INK4 or Cip/Kip families. The loss of CDKI
expression does not appear to be due to a general reduction in
protein translation or stability, since levels of actin are stable,
and usually other related but less abundant proteins, such as
p15 or p18, were not affected. Analysis of p57Kip2 and Cyclin
D1 also showed a decreased level of these proteins associated
Cell cycle regulators in hypoxic cell cycle arrest
Fig. 3. Western blot analysis of cell cycle regulators reveals loss of at least one CDK inhibitor. 75 ug/lane of protein extracts were loaded onto 8% (for HIF-1 and
actin) or 12% acrylamide SDS--PAGE gels and transferred to the appropriate membrane. Pan actin blots were performed for all cell lines to normalize proteinloading amounts. (A) HTB-30 cells lines exhibited a loss of p16 protein, similar to WI-38 normal fibroblasts (previous figure) and also exhibited loss of another
INK4 family member, p19. HTB-30 also showed an induction of p27 protein levels late in the hypoxic response. (B) Hep3B exhibited an induction of p27 late in
the hypoxic response, but also showed loss of p19 and p21, similar to the observed reduction in the HTB-30 cell line. Hep3B p16 levels, however, were invariant in
hypoxia. (C) HeLa cells showed reductions in all CDKIs measured, including p27, although they still underwent hypoxia-induced cell cycle arrest. (D) Primary
breast epithelial cells (HMEC) showed dramatic reductions in p21 levels and p16, starting ~12 h after initial exposure to hypoxia. Other CDKI levels (p15, p18,
p19) were below detection thresholds in this cell strain.
with hypoxia, but with no correlation to cell cycle arrest
phenotype (data not shown).
Although all cell lines showed a reduction of at least one
CDKI protein level, neither the number of proteins reduced,
nor the identity of the CDKI lost, correlated with the ability of
the cell line to enter a hypoxia-induced G1/S arrest, although
Hep3B, which is resistant to hypoxic cell cycle arrest was the
only cell line to not show a reduction in either p27 or p16
levels. Of course, the data only indicate average results for the
cell lines, where cells could show either a generalized change
in protein level, or more drastic changes in a subclone of cells,
with less change in another subclone.
p27 is induced by hypoxia, but induction does not correlate
with G1/S arrest
It has been reported previously that hypoxia-induced G1/S
arrest was due to the induction of p27 (41), but other research
into p270 s involvement has been contradictory. Measurement
of p27 mRNA levels in our cell lines by RPA indicated that
only HTB-30 experienced a hypoxia-induced increase in p27
mRNA while the other cell lines, HMEC, HeLa and Hep3B,
did not show hypoxic modulation of p27 transcripts (data not
shown). Protein levels of p27 were increased in both HTB-30
and Hep3B (Figure 3A and B), but p27 was unchanged in
HMEC (Figure 3D) and reduced in HeLa cells (Figure 3C).
The most important observation was that induction of p27
could not be correlated with the ability of the cell line to
enter a hypoxia-induced G1/S arrest. Both HTB-30 and
Hep3B showed increased amounts of p27 protein, but only
HTB-30 exhibited a G1/S arrest. In addition, HMECs, which
could arrest during exposure to hypoxic conditions, did not
show any change in p27 levels and HeLa cells actually experienced a reduction in p27 while still arresting at the G1/S
boundary. Western blotting for pan-actin protein levels confirmed that overall cellular protein levels were constant. Considering the above data, we have determined that p27 transcript
or protein levels are unlikely to determine the hypoxia-induced
G1/S arrest of our cell lines.
Lack of apoptotic induction in all cell lines
It has been noted that induction of apoptosis in neuronal cells is
preceded by the loss of CDKIs, specifically p16 and p21 (49).
Considering this evidence, we decided to determine if the loss
of the CDKIs we have observed in hypoxia was due to
activation of an apoptotic pathway and/or cell loss. Flow
cytometric analysis did not reveal significant numbers of
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A.H.Box and D.J.Demetrick
Fig. 4. Western blots indicate a lack of PARP cleavage during hypoxia time course. Cell lines were incubated in a normoxic (20% O2; UT) or hypoxic
(50.5% O2; T) atmosphere for the times indicated and whole cell lysates were prepared for immunoblot analysis. Pan actin protein levels were used as a
protein loading control. Analysis of PARP in HeLa, Hep3B, HTB-30 and HMEC cells showed that PARP was maintained at its full-length size of ~119 kDa,
indicating a lack of caspase activity and subsequent apoptosis. No faster migrating versions of PARP were detected.
cells with sub-G1 content, indicating that significant apoptosis
was unlikely to have occurred (e.g. Figure 1D). We also
decided to evaluate PARP cleavage as an indicator of apoptosis. PARP is cleaved from its full-length size of ~119 kDa, via
apoptotically activated caspases, to a truncated form of ~85
kDa. As shown in Figure 4, there is no detectable cleavage of
PARP at any time point during exposure to hypoxia, in any of
the cell lines. Therefore, flow cytometry and PARP cleavage
assays do not indicate that significant apoptosis is occurring in
any of the four cell lines, even after 24 h of severe hypoxia.
The loss of CDKI proteins, therefore, does not appear to be
associated with markers of apoptosis.
Akt/GSK3b signalling is correlated with the ability to enter
G1/S arrest in hypoxia
Recent studies have indicated that Akt signalling through
GSK3b is involved in decreasing HIF-1a expression during
hypoxia (50). Using two epithelial cell lines from our studies,
HeLa cells, which arrested in response to hypoxia, and Hep3B
cells, which did not exhibit a G1/S arrest in hypoxia, we
examined if the loss of HIF-1 and CDKI expression was
associated with Akt/GSK3b status. As shown in Figure 5,
HeLa cells show an initial increase in Akt phosphorylation
between 2 and 6 h in hypoxia, but at 12--24 h there is a drastic
decrease in both phospho-Akt and total Akt levels in hypoxia.
This down-regulation of Akt levels is correlated with the loss
of HIF-1 and CDKI expression and cell cycle arrest. In addition, as the level of phospho-Akt decreases, there is a reciprocal loss of the inhibitory phosphorylation of serine 9 on
GSK3b, indicating a decrease in Akt activity and a probable
increase in GSK3b activity. In contrast, Hep3B cells
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Fig. 5. Phospho-specific western blots reveal differential AKT/GSK3b
activation in cells with and without an intact hypoxia-induced G1/S arrest.
Whole cell lysates were prepared from cells incubated in normoxia (20% O2;
UT) or hypoxic (50.5% O2; T) atmosphere for 2, 6, 12 and 24 h. Western
blotting of Akt and GSK3b showed that in hypoxia-treated HeLa cells (A),
Akt exhibited activating phosphorylation, but phosphoprotein and total
protein levels were lost between 6 and 12 h post-hypoxia. Concomitantly,
inhibitory phosphorylation on GSK3b was lost. (B) Hep3B cells did not
exhibit modulation of Akt and GSK3b phosphorylation with hypoxia
treatment.
Cell cycle regulators in hypoxic cell cycle arrest
Fig. 6. Inhibition of GSK3b activity prevents degradation of HIF-1 and CDKI proteins in hypoxia. HeLa cells were pre-treated with 50 mM LiCl (final), added
directly to the tissue culture media, immediately prior to incubation in a normoxic (20% O2; UT) or hypoxic (50.5% O2; T) atmosphere for the times indicated
(2, 6, 12 and 24 h). Whole cell lysates were prepared and protein levels were determined by immunoblotting. Pan actin levels were used to control for total
protein loading. (A) Normoxic HeLa cells were treated in the presence or absence of 50 mM LiCl for 12 or 24 h. Cell extracts were electrophoresed, blotted
and detected with antibodies to HIF-1a, p16, p27 and actin. (B) Hypoxic (T) or normoxic (UT) HeLa cells were treated in the presence of 50 mM LiCl for
12 or 24 h. Cell extracts were electrophoresed, blotted and detected with antibodies to HIF-1a, p16, p27 and actin. Over time, no decrease in HIF-1a, p16
or p27 was noted in the hypoxic cells treated with LiCl, in sharp contrast to their expression when LiCl was not present (Figure 3).
maintained high levels of phospho-Akt, irrespective of
hypoxia, and maintained GSK3b inhibition, as indicated by
high levels of inhibitory serine 9 phosphorylation. Associated
with a constitutively phosphorylated Akt is maintenance of
high levels of HIF-1 expression and a lack of a hypoxia
induced G1/S arrest.
To evaluate the effect of GSK3b on the CDKI proteins
levels, HeLa and Hep3B were treated with the GSK3b inhibitor LiCl and the results are shown in Figure 6. In the presence
of LiCl, HeLa levels of HIF-1a and p16 do not change appreciably with the 12- and 24-h hypoxia-treatment time points, in
sharp contrast to their expression in the absence of LiCl (Figure 3), although p27 levels were slightly reduced with 50 mM
LiCl treatment in non-hypoxic cells, as compared with
untreated cells. Hep3B showed no effect of LiCl treatment
on the levels of these three proteins in hypoxia. Essentially,
LiCl treatment converted the hypoxia-associated HeLa expression of HIF-1a, p16 and p27 proteins to a similar pattern
shown by Hep3B, reversing the low levels noted in nonlithium-treated HeLa cells. This indicates that GSK3b activity
may be responsible for the observed hypoxic reduction of these
proteins in HeLa and, perhaps, the other cell lines analysed.
Treatment of HeLa cells with hypoxia in the presence or
absence of 50 mM LiCl did not prevent a reduction in cells
transiting the S phase and arresting in G1 (Figure 6C).
Although there was a decrease in G1 cell arrest (as measured
by percent cells in G1 associated with LiCl at the 24-h hypoxia
time point) compared with untreated cells, there was actually
an increase as compared with normoxic cells treated for 24 h
with LiCl. Interestingly, HeLa cells pre-treated with LiCl, but
incubated in normoxia, showed an increase in the number of
cells entering the S phase (~2-fold) over untreated normoxic
HeLa cells, perhaps implying an overall stimulatory effect on
the cell cycle from LiCl.
Discussion
Hypoxia has been long known to initiate a profound, reversible
cell cycle arrest in many different cell types and tumour cell
lines (16,51). Cells in late S, G2 and M phase will finish the
cycle and arrest in G1, unless the hypoxia is severe (18). Cells
in G1 and early S phase will remain there (16). Arresting in
early S phase is easy to rationalize as due to a dependency on
cell energy levels for the completion of DNA synthesis. Finishing the cell cycle and arresting at G1 is typical of cells with
down-regulation of pRb phosphorylation. With initial research
identifying reductions in CDK2 and CDK4 activity, along with
hypophosphorylation of the pRb, it was therefore believed that
the hypoxia-induced cell cycle arrest was probably mediated
by one of the specific inhibitors of the G1 kinases from the
INK4 or Cip/Kip families, as is usually the case (reviewed in
ref. 52). Sequential modification of Rb by two separate cyclin/
cdk complexes is necessary for cell cycle progression, and
coordinated up-regulation of p27 and p15 is essential for
TGFb-mediated cell cycle arrest (43,53). Accordingly, simultaneous measurement of expression of both the INK4 and Cip/
Kip gene families may be necessary in order to elucidate the
mechanism of hypoxia-induced G1/S arrest.
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Response to hypoxia is cell line dependent
While generally confirming the hypoxia-induced cell cycle
arrest phenomenon, the panel of cell lines we analysed exhibited variability in their ability to undergo arrest. Normal
diploid cell strains, such HMECs, exhibited a coordinated
arrest, with a gradual decrease in S phase and an increase in
G0/G1 phase. HTB-30, a breast carcinoma cell line, and HeLa
a cervical carcinoma cell line also exhibited similar cell cycle
findings and followed essentially the same inhibition kinetics
as normal cells, showing peak arrest at ~12 h after exposure to
low atmospheric O2. Conversely, Hep3B cells, a line that is
commonly used in hypoxia studies, did not undergo a coordinated, hypoxia-associated cell cycle arrest even after 24 h in
hypoxic culture.
While the levels of G1 increase/S phase decrease are not
pronounced, it is likely that our severe hypoxia conditions
precluded the normal completion of the cell cycle in many of
the cells, including Hep3B (18); however, the ability to
undergo regulated G1/S blockade was the important observation rather than the magnitude of blockade. We expect that
titration of hypoxia using more elaborate equipment may
identify a higher O2 level correlating with an increase in the
magnitude of the G1/S blockade by providing enough oxygen
for cells to complete the cell cycle to the G1/S arrest point.
One could certainly argue that there are many differences
between these cell lines. Both HTB-30 and Hep3B have
mutated p53 (54,55), while HeLa p53 is down-regulated by
HPV E6. Hep3B lacks functional Rb (55), while HeLa pRb
function is down-regulated by HPV E7. HTB-30 has amplified
the HER2 gene. There does not appear to be a clear correlation
with the cell cycle arrest phenotype of these cells and p53, pRb
or HER2 status.
p16 expression is reduced under hypoxic conditions
p160 s importance as a TS has been widely established in a
number of tumour types (23--25) but previous studies of the
effects of hypoxia on p16 have yielded inconclusive results
(40,42). We evaluated p16 levels under hypoxia in the normal
diploid cells, WI-38 and HMECs, both of which exhibit a
normal G1/S arrest under hypoxia (Figure 1D and E). Ribonuclease protection assay and northern blot analysis of WI-38
cells indicated a reduction in p16 mRNA, which was concordant with a decrease in p16 protein levels. The same phenomenon was observed in HMECs and HTB-30 cells at both the
mRNA and protein levels and at the protein level in HeLa
cells. Hep3B cells, which did not show a hypoxia-induced
arrest, did not have an appreciable reduction in p16 mRNA
or protein levels.
It is unclear why p16 transcript and/or protein levels are
reduced in hypoxia. A recent paper reported that cerebral
ischaemia causes a reduction in INK4 gene family expression
as a precursor to apoptosis (49). Although our flow cytometry
analysis did not show a population of cells with a sub-G1 DNA
content at 24 h, or cleavage of PARP, further studies, using
other methods of analysis, may reveal if there is a correlation
between initiation of apoptosis and the loss of p16 expression.
p16 is constitutively regulated in cycling cells (56,57) and is
thought to be a relatively long lived protein (48 h) (58)
although we cannot rule out that a decrease in transcript level
may be responsible for low levels of the protein. HeLa, which
did not appear to show a reproducible decrease in p16 transcript with hypoxia, did show reduced protein levels, implying
2332
there is probably a change in the p16 protein lifespan in
hypoxia.
p27 is induced by hypoxia, but does not appear to mediate
hypoxia-induced G1/S arrest
Recently, there have been a number of conflicting papers
concerning the importance of p27 in hypoxia. It has been
reported that p27 is induced in mouse embryonic fibroblasts
at both the mRNA and protein levels (41). The induction of
p27 was not HIF-1 dependent, as determined by promoter
analysis. However, MEFs derived from p27/p21 double
knockout mice are still able to initiate a G1/S arrest, indicating
that neither p27 nor p21 is essential for hypoxia-induced
arrest (18).
In our study, HTB-30 alone experienced an increase in p27
transcript levels (data not shown). p27 protein levels were
elevated in HTB-30 and Hep3B cells, but this up-regulation
did not correlate with induction of a G1/S arrest, as
Hep3B cells are resistant to hypoxia-induced arrest. HeLa
cells exhibit a reduction in p27 protein levels while retaining
the ability to induce a cell cycle arrest. HMECs, a primary,
non-immortalized cell, exhibit no change in either p27 mRNA
or protein levels while undergoing arrest. Considering this
data, we conclude that an increased level of p27, although
induced in some cell lines by hypoxia, is not likely to be the
main effector of the observed G1/S arrest.
Loss of CDKI expression in hypoxia does not correlate with
hypoxia-induced G1/S arrest or apoptosis
Much of the recent research into hypoxia-induced arrest has
focused solely upon one or two of the CDKI family members.
Our study, evaluating all CDKI gene products, showed that
there is a rapid (~12 h) loss of one or more CDKIs from both of
the Cip/Kip and INK4 gene families after exposure to low
(50.5%) oxygen. The most commonly lost CDKIs were p21
and p19, although the pattern of CDKI loss could not be
correlated to either the cell line tissue origin, or the cell’s
ability to undergo hypoxic cell cycle arrest. Actin blotting of
hypoxic cell lysates reveals that general protein levels are
stable in hypoxia. It has been suggested that CDKIs may
confer a survival advantage to ischaemic neurons, and that
loss of their expression precedes induction of apoptosis (49).
Lack of sub-G1 DNA content in cytometric measurements and
the presence of intact PARP even after 24 h of exposure to
extreme hypoxia indicate that these cells are not undergoing
typical apoptosis.
Recent research into hypoxia-induced signalling effects may
give clues as to the mechanism of the phenomena that we have
described. Hypoxia may be associated with up-regulation
of the PI3K/Akt signalling pathway (59--61), which results in
up-regulation of HIF-1 via a variety of mechanisms (50,62,63),
although other investigators have reported that PI3K/Akt
signalling does not seem to be involved in the hypoxic
response (64,65).
It was found recently that short-term hypoxia (5 h) was
associated with an increase in Akt activity and increased
protein levels of HIF-1 (50). However, long-term hypoxia
(up to 16 h) was associated with inhibition of Akt function,
and activation of GSK3b. GSK3b activation was associated
with a reduction in HIF-1 protein levels. GSK3b activity was
also associated with loss of p21 (66) and cyclin D1 (67), with
p21 being a direct target for GSK3b (66). Furthermore,
GSK3b expression is increased in an expression microarray
Cell cycle regulators in hypoxic cell cycle arrest
analysis of hypoxia (68). The effect of GSK3b on INK4 family
regulation has not yet been reported.
In our study, we usually observed an initial activation of Akt,
as measured by protein phosphorylation, followed by a loss of
Akt phosphorylation and inhibitory GSK3b phosphorylation
late (412 h) in the hypoxic response. Correlated with this was
a reduction in HIF-1 protein levels even in the presence of
hypoxic conditions and a reduction in CDKI expression. Interestingly, the loss of Akt/GSK3b phosphorylation and HIF-1
expression levels was correlated with the ability of the cell line
to enter a hypoxic G1/S arrest. HeLa cells, which were permissive to hypoxia-induced G1/S arrest, exhibited modulation of
the Akt/GSK3b signalling pathway and loss of HIF-1 and
CDKI expression. Hep3B, which appeared to have constitutively activated Akt and consequently continually inhibited
GSK3b, was resistant to hypoxia-induced G1/S arrest and
had stable levels of HIF-1 and CDKI proteins throughout the
hypoxia time course. When GSK3b activity in HeLa cells was
inhibited with LiCl, there was no reduction in HIF-1 or CDKI
protein levels resulting in a pattern similar to that of hypoxic
cell cycle arrest.
Although incubation with LiCl prevented the observed
reduction in HIF-1 and CDKI protein levels, LiCl did not
prevent hypoxic cell cycle arrest. It appears that LiCl may
have a stimulatory effect on the HeLa cell cycle in general
(compare LiCl-treated versus -untreated normoxic cells), thus
while there is a cell cycle arrest observed between normoxic
versus hypoxic HeLa cells treated with LiCl, this effect is
much less pronounced in hypoxic cells treated with LiCl than
in untreated hypoxic cells, at the 24-h time point. The complexity of these findings makes it difficult to interpret the real
effect of LiCl on the cell cycle in HeLa. This result is of no
great surprise, as LiCl is well known to have pleotropic effects
on a variety of cell pathways in addition to serving as an
inhibitor of GSK3b (69,70), and these effects could affect
the cell cycle in many unpredictable ways.
It is intriguing to postulate that the same mechanism by
which GSK3b activity is associated with HIF-1, cyclin D1
and p21 degradation may also mediate INK4 loss. In our cell
lines, which exhibited hypoxia-induced cell cycle arrest, the
reduction in CDKI expression was mirrored by a reduction in
HIF-1 protein levels. Interestingly, inhibition of PI3K, an
upstream activator of Akt, with the chemical inhibitor
LY294002 resulted in the down-regulation of p15, p16, p19
and p21 protein levels but an increase in p27 protein levels
(71). As well, we have noticed that a consensus GSK3b phosphorylation site (S/T)XXXP(S/T) is present in p16 (Ser12),
and another potential site (S/T)XXXS is found in both p15
(Ser14) and p19 (Ser76). A short motif (TPLE) identical to that
phosphorylated by GSK3b on p21 (66) is also present within
p19 (Thr141) and five residues upstream of it is an aspartic
acid residue that could serve as a surrogate for substrate priming by prior phosphorylation (72), exactly as seems to occur
with p21. Other potential GSK3b sites can be found in both
p27 (Thr187, Ser110, Thr142, Ser161) and p57 (Thr310,
Ser32, Ser84, Ser258). Interestingly, the Thr187 GSK3b consensus site of p27 is identical to the Thr310 site in p57. The
significance of any of these is unknown, but the potential for
the CDKI’s to be direct targets of GSK3b phosphorylation and
degradation is worthy of study.
After evaluating our data, we propose that the observed loss
of CDKIs in hypoxia is downstream and due to the inhibition
of PI3K/Akt signalling in these cell lines (Figure 7). The loss
Fig. 7. A proposed model outlining an Akt/GSK3b-dependent mechanism
for INK4 and Cip/Kip down-regulation late in the hypoxic response.
of HIF-1 expression in our cells, we believe, is also due to the
hypoxic inactivation of Akt as noted above and subsequent
up-regulation of GSK3b. How down-regulation of Akt is
mediated in hypoxia is unknown, but upstream regulators of
Akt, such as PTEN (73), may be involved. The association of
resistance to hypoxia-induced cell cycle arrest and resistance
to hypoxia-associated degradation of p21 and INK4 proteins
may be associated with abnormally increased PI3K/Akt activity. It is tempting to speculate that such hypoxia-resistance
phenotypes may be selected in cancer cells with dysregulated PTEN function, in a manner similar to that proposed for
p53 (74).
Studies into the molecular events surrounding hypoxiainduced cell cycle arrest have been conflicting. Many reports
have utilized tumorigenic cell lines in which the major cell
cycle pathways are altered due to inactivating mutations in key
cell cycle regulators, such as p53, pRb and p16, leading to
inconclusive results (40). In addition, many of these studies
use animal-derived cell lines, which are capable of undergoing
spontaneous immortalization. Our study focused on the similarities and differences between a normal diploid cell strain
(HMEC) and cancerous cell lines (HeLa, HTB-30, Hep3B) in
their cell cycle responses to hypoxia, as well as their expression of cell cycle kinase inhibitors. While we still have not yet
identified the precise mediators of the hypoxia-induced cell
2333
A.H.Box and D.J.Demetrick
cycle arrest phenotype, we have accumulated evidence for the
involvement of the PI3K/Akt/GSK3b pathway in regulating this
response. Elucidation of the precise mediators will involve study
of the expression of other cell cycle regulatory proteins such
as CDKs, cyclins or phosphatases and/or post-translational
modifications of the CDKIs or other cell cycle proteins.
Acknowledgements
We would also like to thank undergraduate students Jason Morin, Eli Akbari
and Carol Yuen for their valuable help with the western blots, and Philip
Berardi, Chris Nichols and Alan Box for helpful discussions and/or review of
the manuscript. The authors wish to acknowledge the operating support of the
Cancer Research Society for making this work possible. Salary support for
D.J.D. was provided by Calgary Laboratory Services. A.H.B. is supported by a
stipend from the Alberta Cancer Board Strategic Training Program in Translational Cancer Research.
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Received May 10, 2004; revised August 11, 2004; accepted August 23, 2004
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