Signaling and apoptosis differences betweensevere hypoxia and desferoxamine treatment ofhuman epithelial cells

Adrian Harold Box, Carol Yuen, Dragana Ponjevic, Gordon H. Fick, andDouglas James Demetrick

Abstract: The mechanisms by which cells undergo proliferation arrest or cell death in response to hypoxia are still notcompletely understood. Originally, we showed that HeLa and Hep3B carcinoma cells undergo different proliferation re-sponses in hypoxia. We now show that these 2 cell lines also have different cell death responses to severe hypoxia, withHeLa showing both cell cycle arrest and apoptosis (as early as 12 h after hypoxia treatment), and Hep3B showing resist-ance to both. Hypoxia-induced apoptosis in Hela was associated with decreases of both phospho-S473- and -T308-AKTand loss of AKT function, whereas Hep3B cells were resistant to hypoxia-induced apoptosis and did not lose phospho-AKT or AKT function. We then decided to test if our observations were confirmed using a hypoxia mimic, desferoxamine.Desferoxamine treatment yielded cell cycle arrest in HeLa and moderate arrest in Hep3B but, surprisingly, did not inducenotable apoptosis of either cell line with up to 24 h of treatment. Hypoxia-treated normal human mammary epithelial cellsalso showed hypoxia-induced apoptosis. Interestingly, in these cell lines, there was a complete correlation between loss ofphospho-AKT and (or) total AKT, and susceptibility to hypoxia-induced apoptosis. Our data suggests a model in whichregulated loss of active AKT at a precise time point in hypoxia may be associated with apoptosis in susceptible cells.

Key words: AKT, apoptosis, cell cycle, desferoxamine, hypoxia.

Resume : Les mecanismes par lesquels les cellules s’engagent vers l’arret de la proliferation ou la mort cellulaire en re-ponse a l’hypoxie ne sont pas encore completement compris. A l’origine, nous avons demontre que les cellules de carci-nome HeLa et Hep3B repondent de facon differente a l’hypoxie en ce qui a trait a la proliferation. Nous demontronsmaintenant que ces deux lignees cellulaires repondent aussi differemment quant a la mort cellulaire suite a une hypoxie se-vere, les HeLa repondant par un arret du cycle cellulaire et par l’apoptose (aussi tot que 12 h apres un traitement hypo-xique) alors que les Hep3B etaient resistantes aux deux phenomenes. L’apoptose induite par l’hypoxie chez les HeLa etaitassociee a une diminution des formes phospho-S473- et -T308-AKT et a une perte de fonction de l’AKT, alors que chezles cellules Hep3B resistantes a l’apoptose induite par l’hypoxie, les formes phosphorylees de l’AKT n’etaient pas dimi-nuees et la fonction de l’AKT n’etait pas perdue. Nous avons alors decide de verifier si nos observations se confirmaientavec un agent mimant l’apoptose, la desferoxamine. Le traitement a la desferoxamine causait un arret du cycle cellulairedes HeLa et un arret modere des Hep3B, mais, de facon surprenante, il n’induisait l’apoptose chez aucune de ces deux li-gnees apres un traitement allant jusqu’a 24 h. Des cellules epitheliales mammaires humaines normales soumises a l’hy-poxie entraient egalement en apoptose. Fait interessant, chez ces lignees cellulaires, il existait une correlation completeentre la perte des formes phosphorylees de l’AKT et (ou) de l’AKT totale et la susceptibilite a l’apoptose induite par l’hy-poxie. Nos resultats suggerent l’existence d’un modele dans lequel une perte regulee d’AKT active a des moments precisde l’hypoxie peut etre associee a l’apoptose dans les cellules sensibles.

Mots-cles : AKT, apoptose, cycle cellulaire, desferoxamine, hypoxie.

[Traduit par la Redaction]

Introduction

Hypoxia plays a critical role in the development, progres-sion, and treatment of a large number of cancers. In the ini-

tial stages of tumour formation, small tumours can quicklyoutstrip the ability of the existing vasculature to provide suf-ficient O2 and nutrients because of the differing metabolicactivities of tumour cells. As the tumour mass grows, the

Received 24 January 2008. Revision received 7 July 2008. Accepted 10 July 2008. Published on the NRC Research Press Web site atbcb.nrc.ca on 7 October 2008.

A.H. Box, C. Yuen, and D. Ponjevic. Department of Pathology, Department of Oncology, and Department of Medical Biochemistry,University of Calgary, 3330 Hospital Dr. NW, Calgary, AB T2N 4N1, Canada.G.H. Fick. Community Health Sciences, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB T2N 4N1, Canada.D.J. Demetrick.1 Department of Pathology, Department of Oncology, Department of Medical Biochemistry, and Calgary LaboratoryServices, University of Calgary, 3330 Hospital Dr. NW, Calgary, AB T2N 4N1, Canada.

1Corresponding author (e-mail: demetric@ucalgary.ca).

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centrally located cells are not adequately perfused and entera state of chronic hypoxia. The presence of hypoxic growthconditions can have significant impact on the effectivenessof standard cancer treatments. Hypoxic tumours are resistantto radio- and chemo-therapies, since these treatments relyupon the generation of oxygen free radicals to induce lethalDNA damage (Gatenby et al. 1988; Schwickert et al. 1995;Tomida and Tsuruo 1999).

Exposure to hypoxia induces the stabilization of hypoxiainducible factor 1 (HIF-1) (Semenza and Wang 1992; Wangand Semenza, 1993a). HIF-1 is a heterodimeric transcriptionfactor composed of a DNA binding basic helix-loop-helix–Per-ARNT-Sim (bHLH–PAS) containing protein, calledHIF-1a, and a commonly shared aryl hydrocarbon nucleartranslocator (ARNT), referred to as HIF-1b (Wang et al.1995). Although both HIF-1a and -1b are constitutively ex-pressed, HIF-1a is rapidly degraded in the presence of O2 bythe ubquitin-mediated proteosome pathway (Huang et al.1998; Salceda and Caro 1997). HIF-1a is recognized andpolyubiquitinated by the Von Hippel-Lindau (VHL) protein,an E3 ubiquitin ligase (Lisztwan et al. 1999; Maxwell et al.1999; Tanimoto et al. 2000). A critical protein modificationof HIF-1 for recognition by pVHL is proline-directedhydroxylation (Ivan et al. 2001; Jaakkola et al. 2001;Masson et al. 2001). HIF-1a prolyl hydroxylase (HIF-PH)uses molecular O2 to hydroxylate HIF-1a on proline 564 inthe oxygen-dependent degradation (ODD) domain. The post-translational modification of Pro-564 is required for VHLbinding to HIF-1a to initiate degradation.

Aside from hypoxia, several chemical mimics of hypoxiasuch as cobalt chloride, desferoxamine (DFX) (Fandrey et al.1997), and N-mercaptopropionylglycine (NMPG) (Salcedaand Caro 1997) are commonly used to study the hypoxiaphenotype, most typically in the context of heart and strokeresearch. These surrogates are valuable experimentally, ascellular hypoxia can be difficult to measure and similar at-mospheric oxygen concentrations may yield solution concen-trations that differ from lab to lab depending on theexperimental methods. Iron chelators such as DFX chelateiron away from HIF-1a prolyl hydroxylase. Treatment ofcells with DFX will cause stabilization of HIF-1a and in-crease expression of hypoxia-associated genes, such glyco-lytic enzymes, erythropoetin, and angiogenic proteins(Gleadle et al. 1995; Minchenko et al. 2003; Oexle et al.1999; Wang and Semenza, 1993b). In addition, DFX hasbeen reported to induce G1 cell cycle arrest, similar to hy-poxia (Brodie et al. 1993; Renton and Jeitner 1996). Thus,iron chelator hypoxia mimics share strong upregulation ofHIF-1 in common and are frequently used to study HIF-1-mediated signaling (Le and Richardson 2002). While onecan hardly assume specificity between a general iron-chelating compound and HIF-1 upregulation, the predomi-nant effect of compounds such as DFX, at their commonlyused dosages, is upregulation of the HIF signaling path-way.

Exposure of cells to hypoxia results in a cell cycle arrestat the G1/S boundary (hypoxia-induced cell cycle arrest)(Box and Demetrick 2004). Cells subject to severe hypoxiawill arrest in G1 or early S phase. Cells in late S, G2, or Mphase will finish cell division and arrest in G1 (Amellemand Pettersen 1991) unless the hypoxia is severe (Green et

al. 2001). Surprisingly, the major mechanisms regulatingthe cell cycle response to hypoxia are still unclear. We havecharacterized some of the signaling and cell cycle regulatoryresponses to hypoxia in 2 well-known epithelial cell linemodels for hypoxia research (Box and Demetrick 2004).One of these, HeLa, is permissive to hypoxic arrest. Theother, Hep3B, is resistant to hypoxic arrest. To facilitate fur-ther study of hypoxia-induced cell cycle arrest, the ability touse a hypoxia mimic such as DFX would provide technicaladvantages in performing hypoxia experiments, providedthat the cell cycle response to DFX was a good model forthat of hypoxia. We chose, therefore, to evaluate the effectof DFX on proliferation and cell cycle signaling to deter-mine the fidelity of DFX at inducing cell cycle arrest.

Methods and materials

Tissue cultureHeLa (cervical carcinoma) and Hep3B (liver adenocarci-

noma) were seeded at 1 � 105 to 2 � 105 cells/mL in rec-ommended growth media (Invitrogen). Primary humanmammary epithelial cells (HMECs) were seeded and grownaccording to the supplier’s recommended conditions(Clonetics). Cells were incubated in 5% CO2 at 37 8C for48 h to 50%–60% confluency prior to exposure to hypoxicconditions. Hypoxia was established by incubating cells inthe GasPak anaerobic incubator (BD Biosciences), which re-duces O2 levels while maintaining CO2 levels at approxi-mately 5%. O2 levels were monitored by a methylene blueindicator strip, which bleaches white at 0.1%–0.5% O2, fol-lowing which the timing of hypoxia was measured. For titra-tion of O2 levels, cells were placed in a Billups-Rothenberghypoxia chamber. The chamber was then flushed with theappropriate O2 gas at 20 L/min for 5 min, balanced with5% CO2 – 95% N2. O2 levels in the chamber were monitoredusing a polaragraphic O2 sensor (Billups-Rothenberg Inc.).Untreated controls were incubated alongside hypoxicsamples under a normal tissue culture gas mixture(20% O2 – 5% CO2). Media containing detached cells wascentrifuged at 800g and added back to the cell populationfor all procedures. Desferoxamine treated cells were culturedas above and incubated in a 260 mmol/L final concentrationof DFX for the indicated time points. Untreated cells wereincubated in identical conditions, with the addition of anequal (200 mL) volume of PBS as an untreated control. Me-dia and ice cold PBS washes from treated and untreatedcells were centrifuged and either pooled with trypsinizedcells for counts, or extracted and the extracts pooled forblots, to ensure that all cells (adherent and nonadherent)were evaluated.

Cell cycle analysisFor ploidy analysis, cells were prepared as previously re-

ported (Box and Demetrick 2004). Cells were then analyzedfor DNA content using a FACScan (Becton, Dickinson, andCompany) to determine cell cycle phase.

Determination of cell viabilityCells were cultured in 6-well tissue culture dishes and

treated with hypoxia or DFX as previously mentioned. Cellswere collected by addition of 1 mL of trypsin, then incu-

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bated at 37 8C for 5 min and neutralized with 3 mL of theappropriate culture media, supplemented with 10% fetal bo-vine serum (FBS). Cells were stained with an equal volumeof 0.4% trypan blue stain (Invitrogen) for 2 min and countedusing an improved Neubauer–Levy hemacytometer (HausserScientific). Cell viability was calculated as follows:% viable = (unstained cell count / total cell count)100.

Annexin-V staining for detection of apoptosisCells used for annexin-V staining were established and in-

cubated in hypoxia or with 260 mmol/L DFX. For H2O2treatments, cells were incubated with the indicated concen-trations for 4 h prior to annexin-V staining. Media was re-moved and the cells washed with ice-cold PBS and thentrypsinized. Cells were washed in cold PBS and resuspendedin annexin binding buffer (10 mmol/L HEPES, 140 mmol/LNaCl, and 2.5mmol/L CaCl2; pH 7.4) at a density of 2 �106 cells/mL. One hundred microlitres of the cell suspensionwere transferred into a fresh tube and 5 mL of annexin-V-FITC conjugate (Biosource) was added. Propidium iodidewas added to a final concentration of 1 mg/mL as a deadcell indicator and the cells were incubated at room temper-ature for 15 min. After incubation, an additional 400 mL ofannexin binding buffer was added and the cells were anal-yzed immediately using a FACScan instrument.

Western blot analysisCell lysis, determination of protein concentration, West-

ern blotting, and immunodetection were performed as de-scribed previously, with the exception that 40 mg per laneof total lysate was loaded onto SDS–PAGE gels (Box andDemetrick 2004). For immunodetection, the following anti-bodies were used: HIF-1a (610959; 1:250) and p27 (25020;1:2000) (BD Biosciences Transduction Labs); Pan actin(Ab-5; 1:2000) and p16 (Ab-1; 1:250) (Neomarkers) andGSK3b (610201; 1/2000) (BD Biosciences TransductionLabs) Phospho-GSK3b Ser 9 (9336S; 1/2000), Phospho-Akt Ser 473 (9271S; 1/2000), Phospho-Akt Thr 308(9275S; 1/1000), and total Akt (9272; 1/2000) (Cell Signal-ing Technologies).

Statistical analysisStatistical evaluations were performed using the statistical

analysis tools (1-factor ANOVA) of Microsoft Excel (ver-sion 2002 SP3), Prism (version 4.1), or STATA.

Results

Hypoxia and DFX treatment results in similar cell cyclearrest profiles

Confirming our previously published results (Box andDemetrick 2004), exposure of HeLa cells to severe hypoxia(0%) resulted in a significant decrease of cells transitingthrough S phase (p = 0.0425) and a reciprocal increase incells in G1 (p = 0.0374) (Table 1), which we defined ashypoxia-induced cell cycle arrest. Titration of oxygen levelsshowed maximal G1 arrest with 0% oxygen, with an associ-ated minimal S phase population. We chose to use ~0% be-cause of the maximal G1 effect and thus performedsubsequent treatments with an anaerobic chamber.

Titration of DFX levels was performed to determine the

appropriate concentration that mimicked 0% O2. As shownin Table 1, treatment of HeLa cells with 0% O2 or260 mmol/L DFX resulted in similar arrest profiles, withcomparable increases and decreases in the G1 and S phasesof the cell cycle, thus, we chose to use 260 mmol/L of DFXas an appropriate concentration for our subsequent DFXtreatments.

In our previous work, Hep3B appeared to be resistant tohypoxic cell cycle arrest (Box and Demetrick 2004). Con-firming these observations (shown in Table 2), Hep3B cellsdid not show any change in the percentage of cells in G1after 24 h of growth in hypoxic conditions (p = 0.8197).Treatment of Hep3B cells with 260 mmol/L DFX, however,did result in a small, but measurable, increase in cells arrest-ing at the G1/S boundary (p = 0.0031) (Table 2). The de-crease in Hep3B proliferation, though, (S phase fraction)was approximately half of the observed average decrease inDFX-treated HeLa cells, suggesting that the DFX effect onHep3B is relatively weak.

Hypoxia, but not DFX, induces robust, early apoptosis inHeLa

Simple vital dye exclusion indicated that HeLa cells ex-hibited cell death in response to >12 h of severe hypoxia(Fig. 1A). Annexin-V staining, usually interpreted to indicateapoptosis, of hypoxia-treated HeLa cells showed a steadilyincreasing percentage of apoptotic cells, beginning after12 h of exposure to hypoxic conditions (Fig. 1B). After 24 hof hypoxia, ~50% of the HeLa cells exhibited annexin-Vstaining. We defined this phenotype as hypoxia-inducedapoptosis. In contrast, exposure of cells to 260 mmol/L DFXhad no appreciable effect on the level of apoptosis, evenafter 24 h exposure. Additionally, titration of DFX levels upto 2 mmol/L did not result in any measurable induction ofapoptosis after 24 h exposure (Fig. 1C).

Hep3B cells exhibit resistance to apoptosis from eitherhypoxia or DFX treatment

Hep3B cells did not show decreased cell viability asmeasured by trypan blue exclusion (Fig. 1A), nor didHep3B cells exhibit significant apoptosis by annexin-Vstaining (Fig. 1D) with hypoxia (p = 0.1697) or 260 mmol/LDFX (p = 0.0621). Although Hep3B cells had higher basallevels of apoptosis than HeLa cells, the percentage ofannexin-V positive cells did not vary greatly during thecourse of the experiment.

DFX is not an inhibitor of either hypoxia- or H2O2-induced apoptosis

Since exposure to DFX for 24 h did not result in apopto-sis in HeLa cells, we wished to determine if DFX treatmentwas simply an inhibitor of the apoptotic process by virtue ofits iron-chelating function. As shown in Fig. 2A, incubationof HeLa cells with 260 mmol/L of DFX exposure did not re-sult in any significant reduction in apoptosis at either 12 h(p = 0.6471) or 24 h (p = 0.2948) of treatment. Further-more, incubation of HeLa cells with 260 mmol/L DFX in ad-dition to incubation with 2 or 4 mmol/L of H2O2 also didnot result in a significant reduction in H2O2-mediated apop-tosis (p = 0.8669 or p = 0.2804, respectively) (Fig. 2B).Thus, DFX certainly does not appear to be a general inhibi-

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tor of apoptosis in either cell line. It simply fails to induceapoptosis in HeLa cells by itself at dosages that approximatethe cell cycle arrest characteristics of true hypoxia.

Differential activation of the AKT/GSK3B pathway usinghypoxia vs. DFX exposure

Confirming our previous results, HeLa cells showed arapid increase in the activating phosphorylation of AKT atserine 473 (Fig. 3A) after hypoxia treatment. Phospho-AKTlevels were initially elevated 2–6 h after initial hypoxic in-cubation, but long-term exposure to hypoxia resulted in de-creased levels of phospho-AKT, with a complete loss at24 h after initial exposure. In addition, hypoxia resulted in adecrease of total AKT protein levels, with a near completeabsence of AKT noted following 12–24 h of exposure to hy-poxia. Along with the reduction in phospho-AKT, there wasan associated loss in the inhibitory phosphorylation ofGSK3B at serine 9, a widely accepted indicator of AKT ac-tivity. This loss in inhibitory phospho-GSK3B was not dueto overall GSK3B protein loss, as total GSK3B levels re-mained stable during the experiment. These data indicate alikely decrease in AKT activity.

In contrast, exposure of HeLa cells to DFX resulted indifferent kinetics for AKT protein expression. As shown inFig. 3B, incubation of HeLa cells with DFX caused a slowerinduction of phospho-AKT than hypoxia, with a minor ini-tial increase at 6 h and greater levels of phospho-AKT after12 and 24 h of exposure to DFX. In addition, total AKT

protein levels also remained stable after 12 h of DFX treat-ment, in contrast to hypoxia treatment. Associated with theincrease in phospho-AKT, inhibitory pGSK3B Ser-9 levelswere maintained throughout the hypoxia course. Again, totalGSK3B levels remained stable throughout the hypoxiccourse. These data indicate that DFX treatment, in contrastto hypoxia treatment, likely does not upregulate GSK3B ac-tivity, even after 24 h exposure.

As compared with the hypoxia-induced apoptosis-sensitive HeLa cell line, the apoptosis-resistant Hep3B cellline did not show reduced AKT levels in late hypoxia(Fig. 3C). Although phospho-AKT levels differed acrosstime points, total AKT levels remained relatively un-changed. Additionally, phospho or total GSK3B levelsshowed no variance during hypoxic treatment. DFX-treatedHep3B showed similar results, with minimal changes toAKT and GSK3B phosphoprotein and total protein levels(Fig. 3D) over the 24 h course of treatment. Althoughphospho-Ser473 is usually accepted as a proxy for activeAKT, we also evaluated the phosphorylation of Thr308.Figure 4 shows that the pattern of phospho-Thr308 AKT inresponse to hypoxic treatment of HeLa cells (Fig. 4A) andHep3B cells (Fig. 4B) or after treatment with DFX (Fig.4C) is identical to that of phospho-Ser473, showing that thephosphorylation of these residues by different kinases(Sarbassov et al. 2005, 2006) is still coordinately regulatedin both hypoxia- and DFX-treated cells.

We decided to evaluate a nontumour-derived epithelialcell line to determine whether there was a more widespreadcorrelation between apoptosis resistance and AKT stabilityafter various periods of hypoxia treatment. As can be seenin Fig. 5A, ‘‘normal’’ HMECs exhibit significant cell deathafter 24 h of hypoxia. Figure 5B shows the result of evaluat-ing AKT pathway members in these same cells. In HMECs,phospho-Ser473 AKT levels decline after 12 h and are al-most completely absent after 24 h along with functionalloss of AKT (as shown by the absence of phospho-GSK3B),correlating with the appearance of cell death. Thus, in 2 of 2apoptosis-sensitive cell lines (HeLa and HMEC), phospho-AKT and AKT activity is lost in late hypoxia, whereas inour apoptosis-resistant cell line (Hep3B), phospho-AKT andAKT activity is not lost in late hypoxia.

Table 1. Evaluation of hypoxia-induced HeLa cell cycle arrest between various O2 levels and desferoxamine (DFX) con-centrations.

Percentage of cells

Cell treatment Phase UT (0%) 0% O2 UT (1%) 1% O2 UT (2%) 2% O2

Hypoxia G1) 60.40±4.01 69.97±3.63 61.73±2.45 66.95±3.33 64.16±2.08 66.82±5.16S 28.52±2.11 21.55±3.53 31.46±4.84 26.76±1.19 26.89±0.84 22.59±6.29

Percentage of cells

Concn. of DFX (mmol/L)

Phase UT 260 520 780 1040 2080DFX G1 60.21±3.68 77.59±5.46 76.83±5.21 77.84±4.51 77.24±4.57 76.05±3.82

S 29.14±3.62 21.27±5.86 21.95±5.80 21.38±4.96 21.48±5.56 22.75±4.88

Note: Cells were split into control plates or treatment plates at least 48 h prior to treatment to control for serum change. Parallel controlcells were incubated in normoxia (O2, UT (untreated)), or treated with the O2 and DFX concentrations detailed in the Table for 24 h, fixedin ethanol, stained with propidium iodide, and analyzed by flow cytometry to determine G1 and S phase cell populations. Separate un-treated controls were used for each time point to control for potential increases in cell density associated with longer culture times owing totreatment. (n = 3, standard error of the mean (SEM)).

Table 2. Cell cycle analysis of Hep3B cells exposed to hypoxia ordesferoxamine (DFX).

Percentage of cells

Type of cell treatment Phase UT THypoxia G1 58.99±4.66 58.42±2.28

S 24.16±2.86 29.04±3.24DFX G1 60.75±0.79 69.27±1.08

S 28.26±1.88 24.62±0.97

Note: Hep3B cells were incubated in normoxia (20% O2, UT (untreated)),hypoxia (<0.5% O2, T (treated)), or desferoxamine (260 mmol/L, T) for 24 h,fixed in ethanol, stained with propidium iodide, and analyzed for DNA con-tent to determine G1 and S phase cell populations. (n ‡ 3, SEM).

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Loss of AKT protein in hypoxia is not prevented by anumber of protease inhibitors

Since the AKT protein was being lost in hypoxia, we de-cided to determine what protease, if any, was involved inthe observed protein loss. HeLa cells were treated with15 mmol/L MG-132, a 26S proteosome inhibitor. As shownin Fig. 6A, MG-132 failed to stabilize AKT protein levels.HIF-1 protein and cyclin D1 are known targets of proteo-somal degradation and their observed loss in early hypoxiawas significantly alleviated, indicating that MG-132 wasfunctioning as a proteosome inhibitor. MG-132 was less ef-fective in preventing proteolysis of HIF or cyclin D1 after12 h. This may be due to the fact that longer term MG-132treatments can result in apoptosis on its own.

Since we were unable to prevent AKT loss with MG-132,we decided to see if other protease inhibitors would be capa-ble of blocking AKT degradation (Fig. 6B). Pretreatment ofHeLa cells with either the the general caspase (BOC-FMK)inhibitor or the alternate proteosome inhibitor B-lactacystinalso did not abrogate AKT degradation after exposure to hy-

poxia for 24 h. This data indicates that the observed hypoxicreduction of total AKT levels is likely occurring through acaspase- and proteasome-independent mechanism.

Discussion

We were originally interested in comparing the HeLa andHep3B cell signaling responses to hypoxia, since they ex-hibit completely different cell cycle arrest phenotypes. Ourprevious work demonstrated that Hep3B cells were resistantto the associated cell cycle arrest, while HeLa cells weresensitive to cell cycle arrest, and we identified several qual-itative differences between cell cycle or AKT signalingpathway protein expression kinetics (Box and Demetrick2004). Evaluation of apoptosis in our previous work wasbased on the identification of poly ADP ribose polymerase(PARP) cleavage. Since we did not detect this change withhypoxia treatment of either cell line, we concluded that ourconditions did not induce traditional apoptosis.

In our follow-up work, however, we noted that refractile,

Fig. 1. Cell viability indicates cell death only occurs in hypoxia-treated HeLa cells. (A) HeLa and Hep3B cells were incubated under nor-moxia (20% O2, UT), hypoxia (<0% O2), or desferoxamine (DFX, 260 mmol/L) for the indicated times and the percent viability was deter-mined. (n = 3, SEM). HeLa and Hep3B cells were incubated under normoxia, hypoxia, or DFX for the indicated times. Cells were analyzedfor the percentage of annexin-V staining to determine the level of apoptosis. (B) Evaluation of hypoxia- or DFX-induced apoptosis overtime in HeLa cells. A single concentration of DFX (260 mmol/L) was chosen based on a similar cell cycle effect to hypoxia on HeLa. (C)Evaluation of increasing DFX concentrations on apoptosis after 24 h of treatment in HeLa cells. H2O2 (4 mmol/L) was used as a positivecontrol. (D) Evaluation of hypoxia- and DFX-induced apoptosis over time in Hep3B cells (n = 2, SEM). The methodology was identical tothat for (B). UT, untreated.

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floating cells could be observed in HeLa cell cultures afterprolonged hypoxia treatment, but not in Hep3B cell cultures.Figure 1 and Tables 1 and 2 show that we can indeed ob-serve apoptosis in HeLa cultures as measured by annexin-Vstaining, though we could not detect it by PARP cleavage(Box and Demetrick 2004). Apoptosis could be measuredafter approximately 12 h of hypoxia treatment. This timepoint may be a result of the eventual depletion of dissolvedoxygen, resulting in anoxia of the cycling cells (Papandreouet al. 2005), or may be dictated by the timing of essentialsteps within a signaling cascade. We did not observe sig-nificant apoptosis in Hep3B cells, thus the phenotype ofhypoxia-induced apoptosis occurred in HeLa but not inHep3B cells, correlating with cellular arrest.

It has long been known that hypoxia will initiate apopto-sis in cells if the stimulus is protracted and (or) severe. Infact, anoxia may be required (Papandreou et al. 2005). Re-cent research has indicated that hypoxia-induced cell deathmay not be a consequence of a traditional apoptotic path-way. Studies utilizing HeLa cells treated with hypoxia, inan almost identical fashion as our experiments, have shownthat a cell death similar to apoptosis occurs in a caspase-

independent manner (Shinzawa and Tsujimoto 2003). Sinceactivated caspases mediate PARP cleavage in response toapoptosis (Nicholson et al. 1995), caspase-independent celldeath in hypoxia would explain our HeLa cell results. In-deed, recent studies have indicated that an autophagic celldeath, independent of caspase activity, may be responsiblefor hypoxia-induced cell death and may account for our ob-servations (Azad et al. 2008; Tracy et al. 2007).

We were originally motivated to investigate whether DFXtreatment could provide a good model for our investigationof hypoxia-induced cell cycle arrest, since it is a moreexperimentally convenient model system to use than regulat-ing atmospheric oxygen levels. HeLa cells exposed to260 mmol/L DFX did not exhibit a measurable apoptotic re-sponse, even after 24 h of incubation in 2 mmol/L DFX.DFX treatment of HeLa cells did not resultin any significantlevels of protection against hypoxia-induced or hydrogenperoxide induced apoptosis. In Hep3B cells, DFX did not in-duce significant cell death. These observations were surpris-ing, as DFX has been used to induce apoptosis in cardiacmyocytes (Hauck et al. 2002), hematopoetic cells (Alcantaraet al. 2001; Guo et al. 2006; Kim et al. 2002), and neuro-

Fig. 2. Desferoxamine (DFX) does not protect HeLa cells from hypoxia-induced apoptosis. (A) HeLa cells were incubated with normoxia(20% O2, UT), hypoxia (<0.5% O2, T), or hypoxia with DFX added immediately prior to hypoxic exposure (<0.5% O2, 260 mmol/L, T + DFX)for the indicated times. Cells were analyzed for the percentage of annexin-V staining to determine the level of apoptosis. (B) HeLa cells wereincubated without H2O2 (UT), with the indicated concentrations of H2O2 (T), or with H2O2 immediately after the addition of 260 mmol/L DFX(T + DFX) for 4 h. Cells were analyzed for the percentage of annexin-V staining to determine the level of apoptosis. (n = 2, SEM).UT, un-treated; T, treated.

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Fig. 3. Hypoxia and desferoxamine (DFX) have significantly different effects upon AKT/GSK3B signaling proteins. HeLa and Hep3B cellswere exposed to either hypoxia or DFX as in Fig. 2, for the indicated time points and then used to prepare total cell lysates. of Lysates(40 mg/lane) were utilized for Western blotting with anti-phospho-AKT, total AKT, phospho-GSK3B, or total GSK3B. Panel (A) shows theresults using lysates from hypoxia-treated HeLa cells. Panel (B) shows lysates from DFX-treated HeLa cells. Panel (C) Hep3B cells exposedto hypoxia as listed above and blotted for the proteins listed. (D) Hep3B cell lysates from DFX-treated cells, blotted for the proteins indi-cated. Data shown are representative of two independent experiments.

Fig. 4. Phospho-AKT (Thr308 and Ser473) levels are coordinately regulated in hypoxia. HeLa and Hep3B cells were exposed to hypoxia ordesferoxamine (DFX) (HeLa) as in Fig. 3 for the indicated time points and then used to prepare total cell lysates. Lysates were Westernblotted for anti-phosphoThr308-AKT, phosphoSer473-AKT, total AKT, and actin. Panel (A) shows the results using lysates from hypoxia-treated HeLa cells. Panel (B) shows lysates from hypoxia-treated Hep3B cells. Panel (C) shows lysates from DFX-treated HeLa cells. Allwere blotted for the proteins indicated.

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blastoma cells (Fan et al. 2001), although it was suggestedas being protective against hypoxia-associated cytotoxicityin Chinese hamster V79 cells (Herscher et al. 1994). Ourdata show that DFX was not interfering with apoptosis in-duced by either hypoxia or hydrogen peroxide in our epi-thelial cell lines, merely that it was not a good inducer ofcell death. It is possible, or even probable, that there is acell lineage specificity to the ability of DFX to induceapoptosis.

None of the 3 assays (PARP cleavage, vital dye exclu-sion, or annexin-V staining) indicated that any noteworthyapoptosis occurred in hypoxia-treated Hep3B cells up to24 h of treatment, or that relevant apoptosis was occurringin DFX-treated HeLa or Hep3B cells after 24 h of treatment.Our results indicated that Hep3B cells were resistant to bothhypoxia-induced cell cycle arrest and apoptosis, whereasDFX could induce moderate cycle arrest, but not apoptosis.Conversely, hypoxia treatment could induce both pheno-types in HeLa. Since both treatments resulted in initial highlevels of HIF-1a expression, activation of this pathwayalone could not be responsible for mediating apoptosis.

We undertook an expression microarray screen of the hy-poxic transcriptional response in HeLa cells as comparedwith the DFX response.2 Upregulation of known hypoxia-

regulated genes validated the readout (Fig. S2).2 While wedid detect some differences in transcription between the 2treatments (Fig. S3 and Table S3)2 these could not be con-firmed using alternate mRNA analysis methods (Fig. S4).2Furthermore, as also shown by others, we found robustupregulation of genes with proapoptotic function, such asBNIP3, early in the hypoxic response (Fig. S5).2 Thus, notonly could we not identify transcriptional differences thatcould explain the phenotypic differences between hypoxiaand DFX treatment, both of which upregulate HIF-1a, butwe identified upregulation of genes early in hypoxia thatshould have promoted apoptosis in both hypoxia- and DFX-treated cells.

Our previously published work showed that the 12 h timepoint of hypoxia treatment in HeLa and HMEC cells coin-cided with the initiation of a loss of HIF-1a, CDKIs such asp16 or p27 and, perhaps more importantly, AKT signalingas measured by phosphorylation of GSK3B, activating phos-phorylation of AKT, or amount of total AKT. Activation ofAKT is well known to occur in hypoxia (Chen et al. 2001).Phosphorylation of GSK3B, a major target for AKT, hasbeen linked to the degradation of HIF-1 (Mottet et al. 2003;Schnitzer et al. 2005), which was observed by us in HeLacells treated with hypoxia. We previously investigatedGSK3B regulation in the hypoxic response of our cell lines(Box and Demetrick 2004) and found that activating phos-phorylation of AKT and inhibitory phosphorylation ofGSK3B occurred in early hypoxia, but that both phospho-proteins were lost later (after 12 h) in hypoxia. These obser-vations were also confirmed and expanded upon in our morerecent studies that showed a loss of activating AKT phos-phorylation (both Thr308 and Ser473) and (or) total proteinlevels occurring approximately 12 h post-hypoxia treatment.As our data shows, this appears to be the time at whichhypoxia-induced apoptosis starts to occur in hypoxia-treatedHeLa (Fig. 3A) or HMEC (Fig. 6) cells. In contrast, cellsthat did not exhibit significant hypoxic apoptosis also re-tained high levels of total and phospho-AKT levels after12 h of hypoxia treatment.

The phenomenon of loss of phospho-AKT and (or) totalAKT in our Western blots is intriguing. We see this in dif-ferent epithelial cells, HMEC and HeLa, which were suscep-tible to both cellular arrest and apoptosis. Hypoxia has beenshown to inhibit the phosphorylation of Ser473 in the HL-60leukemic cell line, but the mechanism of this loss is still notcharacterized, and the process did not result in a loss of totalAKT protein (Seo et al. 2007). Loss of pAKT could obvi-ously be mediated by loss of AKT. Loss of AKT phosphor-ylation could also be mediated by an increase in proteinphosphatase activity. Although studies have shown that hy-poxia can result in increased protein phosphatase activity(Krtolica et al. 1998), conflicting studies have shown thathypoxia can downregulate protein phosphatases (Comerfordet al. 2006) and protein phosphatase 2A activity (Truttmannet al. 2004) in particular. Since AKT can be negatively regu-lated by PP2A-directed dephosphorylation, it is unclear atthe moment which phosphatase may be the effector in this

Fig. 5. Viability and AKT expression in normal mammary epi-thelial cells treated with hypoxia. (A) Human mammary epithlialcells (HMECs) were incubated in normoxia (20% O2, UT) or hy-poxia (<0% O2) for the indicated times. Cells were counted and thepercent viability was determined. (n = 3, SEM). (B) HMECs wereexposed to normoxia or hypoxia and used to prepare total cell ly-sates. Lysates (40 mg/lane) were used for Western blotting for anti-phospho AKT, total AKT, phospho-GSK3B, or total GSK3B.

2 Supplementary data for this article are available on the journal Web site (bcb.nrc.ca) or may be purchased from the Depository of Unpub-lished Data, Document Delivery, CISTI, National Research Council Canada, Building M-55, 1200 Montreal Road, Ottawa, ON K1A 0R6,Canada. DUD 3824. For more information on obtaining material refer to http://cisti-icist.nrc-cnrc.gc.ca/cms/unpub_e.html.

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instance (Ugi et al. 2004; Van Kanegan et al. 2005; Zuluagaet al. 2007).

Of course, apparent ‘‘loss’’ of physical AKT, includingphospho-AKT species, could be mediated by sequestrationto a different cellular compartment if those compartmentsare not evaluated by the protein extraction techniques. Fi-nally, loss due to degradation of AKT in hypoxia has notbeen studied as of yet, although recent literature has de-scribed the degradation of AKT in response to other cellularstimuli, such as platelet-derived growth factor, insulin-likegrowth factor-1, and TNF-a (Adachi et al. 2003; Medina etal. 2005). Additionally, degradation of AKT in response toH2O2-induced apoptosis has also been reported (Martin etal. 2002). It seems likely that degradation of AKT is a regu-lated process, and we hypothesize that this degradation is atleast partially responsible for regulating apoptosis in re-sponse to hypoxia. Interestingly, we commonly observedmultiple bands at the accepted molecular mass of AKT inmany of our Western blots. This phenomenon has also beenobserved by others (Sarbassov et al. 2005, 2006). The multi-ple bands detected with anti-panAKT antibodies may repre-sent simultaneous expression of the different AKT isoforms,which differ slightly in molecular mass and (or) mobility(Nakatani et al. 1999), or their phosphorylated isoforms(Okano et al. 2000). Unfortunately, these multiple bands donot appear to have been the subject of detailed discussion inthe literature to date. Analysis of total AKT levels in HeLacells after 12–24 h of hypoxic exposure in our study also re-vealed the appearance of a faster migrating band at the timepoints in which total AKT levels were dropping (Fig. 3A).This faster migrating band was not detected at similar timepoints in DFX-treated HeLa cells or in any of the Hep3Bcell time points (hypoxia- or DFX-treated). Treatment ofHeLa cells with inhibitors of proteosome-dependent orcaspase-dependent proteolysis did not block AKT disappear-ance. These results suggests that the mechanism by whichAKT is being degraded is independent of these processes.

AKT activation in hypoxia has been hypothesized as anti-apoptotic (Alvarez-Tejado et al. 2001), deactivating viaphosphorylation proapoptosis proteins such as BAD (Dattaet al. 1997). AKT activation reduces cardiac myocyte apop-tosis in models of ischemia (Matsui et al. 2001). AKT acti-vation prevents hippocampal neurons from apoptosisthrough a p53-dependent mechanism (although that is likelynot relevant in HeLa cells) (Yamaguchi et al. 2001). Re-cently, depletion of AKT by RNAi has been found to pro-mote apoptosis in hypoxia-treated neuroblastoma cells (Liuet al. 2006). AKT levels have been found to be commonlyupregulated in diverse cancers, and upregulation of AKTcould transform NIH3T3 cells (Sun et al. 2001). AKT hasalso been shown to be upregulated by loss of PTEN orupregulation of PDK1 (Lin et al. 2005) and (or) ERBB-2(Tokunaga et al. 2006) in breast cancer. Interestingly, dys-function of PTEN is found in at least 2 breast cancer celllines, HTB-130 and HTB-132, which we have also found tobe hypoxia-resistant (data not shown). This information, inaddition to our data, suggests the possibility that AKT mightbe an important switch regulating the initiation of hypoxia-induced apoptosis in a manner resembling that observed forpRB and the G1/S checkpoint.

Loss of AKT may represent a commitment of the cell tohypoxia-induced cell death. Initial exposure to hypoxia inHeLa cells resulted in an increase in AKT activity, as meas-ured by the increase in AKT phosphorylation. This was ob-served with the addition of DFX to HeLa cells as well,albeit at a slightly delayed time course. The initial activationof AKT in hypoxia has been described in the literature(Alvarez-Tejado et al. 2001). Hypoxic preconditioning ofcells imparts a significant survival advantage during a sub-sequent ischemic event (Cho et al. 2007; Laughner et al.2001; Sasabe et al. 2005). This has been attributed to the in-itial activation of AKT by hypoxia and highlights the impor-tance of hypoxia-induced antiapoptotic signaling. Therefore,elimination of the AKT-signaling network may be critical in

Fig. 6. Loss of AKT owing to hypoxia in HeLa cells is not abrogated by addition of proteosome inhibitor MG-132. HeLa cells were ex-posed to normoxia or hypoxia for the indicated time points. (A) Cells were pretreated with 15 mmol/L of MG-132 for 1 h prior to hypoxicexposure. Lysates (40 mg/lane) were used for Western blotting and detected with the antibodies as indicated. Total actin levels were used tocontrol for total protein loaded. HIF-1 and cyclin D1 levels serve as a control for proteasomal inhibition. (B) HeLa cells were exposed tonormoxia or hypoxia for 24 h. Cells were pretreated with a proteolysis inhibitor at the indicated concentration 1 h before hypoxic exposure.Lysates (40 mg/lane) were used for Western blotting and detected with anti-total AKT and actin as antibodies.

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regulating hypoxia-induced cell death. For example, if thehypoxic exposure was not severe or protracted, AKT signal-ing could ensure survival long enough for metabolic adapta-tion and alleviation of low O2 levels through angiogenesis.This concept is reinforced by the fact that AKT signalingwill enhance HIF-1 protein levels and increase the hypoxictranscriptional response (Laughner et al. 2001). If the hy-poxic growth conditions became overwhelming, removal ofAKT signaling would abrogate its potent antiapoptotic ef-fects and may allow for cell death to occur.

In our model, after cells are exposed to hypoxia, AKT isactivated by phosphorylation. Active AKT then acts as anantagonist to programmed cell death through a variety ofknown mechanisms and (or) perhaps by blocking theBNIP3 pathway, which is initiated by both hypoxia andDFX treatment. At a certain threshold of time and low oxy-gen levels (approximately 12 h in our system) AKT is madeinactive (via several potential mechanisms as discussed pre-viously), resulting in the removal of a blockade to cell deathand subsequent apoptosis.

In summary, while our work shows that both hypoxia andthe hypoxia mimic DFX result in upregulation of HIF-1 andsimilar transcriptional targets, there are differences in thephysiologic and molecular responses between the 2 treat-ments. Treatment of HeLa cells by DFX, while mimickingthe stabilization of HIF-1, upregulation of HIF-signaling,and hypoxia-induced cell cycle arrest, does not mimicAKT-signaling events and other molecular responses foundin cells exposed to true hypoxia. Using these 2 models, wemay now separate apoptosis from cell cycle arrest pheno-types in HeLa, in the context of highly upregulated HIF-1.Furthermore, we confirm and build upon our earlier observa-tions regarding AKT regulation. In cells that are sensitive tohypoxic arrest and (or) apoptosis, phospho-AKT is signifi-cantly decreased at approximately the same time that apop-tosis is observed (~12 h post-treatment). In cells that areresistant to hypoxic arrest and (or) apoptosis, phospho-AKTis not similarly decreased. Plainly, AKT activity is likely nota major regulator of hypoxic cell cycle arrest, as its regula-tion does not appear to be altered with DFX treatment,which does initiate cell cycle arrest in both HeLa andHep3B cells (moderate). This has led us to propose a modelimplicating AKT as a major regulator of the cell death re-sponse of hypoxia-induced, apoptosis-sensitive cells. Furtherexperiments to test this hypothesis are in progress.

AcknowledgementsThe authors wish to acknowledge initial funding from the

Kids Cancer Care Foundation and later funding of this workby the Ruth Barker Foundation. Salary support for DJD wasprovided by Calgary Laboratory Services. AHB was sup-ported by a stipend from the CIHR – Alberta Cancer BoardStrategic Training Program in Translational Cancer Re-search. Steve Robbins was a very helpful source of ideasand reagents for the performance of the experiments. ErinMacRae and Monica Nguyen provided excellent assistance,and Mary Tretiak maintained the growth of the cell lines.We would also like to thank Philip Berardi, Chris Nichols,and Alan Box for helpful discussions and (or) review of ini-tial drafts of the manuscript.

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