Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest inhuman cancer cell lines

Adrian Harold Box1--3 and Douglas James Demetrick1--5

1Department of Pathology, 2Department of Oncology, 3Department ofMedical Biochemistry and 4The University of Calgary and CalgaryLaboratory Services, Calgary, Alberta, Canada, T2N 4N1

5To whom correspondence should be addressedEmail: demetric@ucalgary.ca

Flow cytometric analysis of fibroblasts, normal breastepithelial cells and breast or other cancer cell lines identi-fied variation in the abilities of cell lines to undergo cellcycle arrest as a response to hypoxia. Human mammaryepithelial cells (HMEC), normal fibroblasts (Hs68 andWI38), HeLa cervical carcinoma and HTB-30 breast car-cinoma cells arrest in G1/S in response to severe hypoxia.Hep3B hepatocellular carcinoma cells did not exhibitorderly G1/S arrest in response to severe hypoxia. Wefound a general decrease in p16INK4a (p16) mRNA levels,with an associated decrease in p16 protein levels in bothnormal cells and in cancer cells, regardless of their cellcycle response to hypoxia. p27 protein levels did not corre-late with the cell line’s ability to enter a hypoxic G1/Sarrest. Furthermore, cell lines that underwent G1/S arrestshowed decreased expression of hypoxia inducible factor 1(HIF-1a) and at least one member of INK4 or Sdi cell cyclekinase inhibitors families after 12--24 h of hypoxia.Conversely, Hep3B, which did not exhibit orderly hypoxia-associated G1/S arrest, also did not show decreased HIF-1a, INK4 or Sdi protein levels in hypoxia. Furthermore,Hep3B showed constitutive activating phosphorylation ofAkt and inhibitory phosphorylation of GSK3b, which wasthe opposite pattern to that exhibited by the cell linesshowing the G1/S arrest phenotype. Inhibition of GSK3bby lithium chloride treatment of HeLa cells converted theHIF-1a, p16 and p27 loss to levels unchanged by hypoxicexposure. Our results suggest that regulation of the cellcycle during hypoxia in either normal or cancer cells isnot simply due to up-regulation of cell cycle kinaseinhibitors. Furthermore, decreased protein expression ofHIF-1a, p16 and p27 was associated with both a hypoxia-induced G1/S arrest phenotype and increased GSK3bactivity.

Introduction

Tumour hypoxia is an important mitigating factor in the treat-ment of many forms of cancer. As a tumour mass grows, itexperiences extended periods of low O2 levels due to the sizeof the mass and the lack of an appropriate blood supply (1).

In order for a tumour to metastasize, it must survive this periodlong enough for vascularization to occur (2). Even after vas-cularization, the new blood vessels are often too poorly formedto supply adequate O2 perfusion, resulting in a continuallyhypoxic tumour environment. Hypoxic tumours are moreaggressive and invasive than their normoxic counterparts andexhibit 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 ashypoxia inducible factor 1 (HIF-1) (6,7). HIF-1 is a hetero-dimer composed of the HIF-1a subunit, which is a member ofthe basic helix--loop--helix--PAS protein family, and the HIF-1b 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 viathe 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 accom-plished by the up-regulation of various metabolic genes, suchas glucose transporters (11), aldolase, lactate dehydrogenase(12) and pyruvate kinase (13). HIF-1 also up-regulates vascu-lar endothelial growth factor (14,15), presumably in order toinduce the formation of new blood vessels, and erythropoietin(7), which accelerates red blood cell maturation and extendslifespan to increase blood O2 carrying capacity.Another major consequence of cellular hypoxia is a prolif-

eration arrest of cells (16,17). Cells subject to severe hypoxiawill arrest in G1 or early S phase. Cells in late S, G2 or M phasewill finish cell division and arrest in G1 (16), unless thehypoxia is severe (18). Progression of cells through the cellcycle is controlled by a series of periodically expressed pro-teins 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 thecyclin 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 functionto 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, therebypreventing their ability to phosphorylate pRb (22). This proteinwas 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 developtumours early on in life (26), providing further evidence thatit is a putative TS.Three other members of the INK4 gene family were identi-

fied based upon their sequence similarity to p16. p15INK4a

(p15), p18INK4a (p18) and p19INK4a (p19) were all identifiedas members of the INK4 gene family and shared with p16 anability to bind to CDK4/6 to cause a G1 arrest (27,28). p16 has

Abbreviations: CDK, cyclin-dependent kinase; CDKI, cell cycle kinaseinhibitors; HIF-1a, hypoxia inducible factor 1; HMEC, human mammaryepithelial 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. 2325

Carcinogenesis vol.25 no.12 pp.2325--2335, 2004doi:10.1093/carcin/bgh274

been implicated in the induction of cellular senescence, orwithdrawal from normal cell cycle (29) and for contact inhibi-tion of confluent cells (30). p15 is induced by and mediates cellcycle arrest in response to transforming growth factor beta(TGF-b) (27). p18 and p19 expression varies throughout thecell cycle, reaching maximum levels at the G1/S transition(28), and it has been suggested that they function in cellcycle arrest coupled to specific differentiation pathways(reviewed in ref. 31). Furthermore, data from transgenicmouse studies suggests that the INK4 gene family may havenon-overlapping functions (32). Another group of CDK inhi-bitors, 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, andp27 has been shown to be a likely TS gene.Studies examining the effects of hypoxia on cell cycle

components have yielded conflicting results. Initial experi-ments have shown decreases in CDK4 and CDK2 kinaseactivities, and accumulation of pRb in its hypophosphory-lated state (38--40). Analysis of CKIs in hypoxia has shownan increase in p27 protein levels (41), which has beensuggested as the cause of the hypoxic G1/S arrest.Recently, this idea has come under fire (18), with evidenceshowing that p27 is not necessary for hypoxic arrest. Ana-lysis of the INK4 gene family in hypoxia has been incon-clusive, with p16 levels initially reported to be belowdetection levels (40), although a study using monkey CV-1P cells showed hypoxia-induced up-regulation of p16 andassociation with cdk4 (42). Plainly, any of the INK4 or Sdicell cycle kinase inhibitors (CDKI) may be good candidatesto mediate G1 arrest in response to hypoxia. With bothCDK4 and CDK2 kinase activities reduced in hypoxia, itmay be that a coordinated up-regulation of both CDKIfamilies, INK4 and Cip/Kip, is necessary for hypoxia-induced cell cycle arrest. p15 and p27 have been documen-ted to cooperate in TGF-b-induced cellular arrest (43),supporting this idea of coordination. Also, there is evidencethat induction of p16 causes a rearrangement of p27 andp21 from cyclinD/CDK4 to cyclinE/CDK2, effectively inhi-biting both G1/S kinase complexes (44--47). Therefore,hypoxic cell cycle arrest may not be solely attributed toone G1/S kinase pathway, and may require the coordinatedup-regulation of multiple CDKIs. In this study, we explorethe effects of hypoxia on a number of different cell lines inorder to determine the potential inducers of the hypoxia-associated 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 ofrecommended growth media (Invitrogen). Primary human mammary epithelialcells (HMECs) were seeded and grown according to supplier’s recommendedconditions (Clonetics). All cells were incubated at 5% CO2/37

�C for 48 h to50--60% confluency prior to exposure to hypoxic conditions. Hypoxia wasestablished by incubating cells in the GasPak anaerobic incubator (BDBioscience), which reduces O2 levels while maintaining CO2 levels at ~5%.O2 levels were monitored by a methylene blue indicator strip, which bleacheswhite at 0.1--0.5%O2 levels, following which timing of hypoxia was measured.Control cells were incubated alongside hypoxic samples under a normal tissueculture 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.

Cell cycle analysis

For ploidy analysis, cells were prepared as follows, with all steps carried out at4�C unless noted. Media was removed from the plates and the cells were rinsedwith 10 ml of ice-cold phosphate-buffered saline (PBS). The cells were liftedby incubation with 2.5 ml trypsin and neutralized by the addition of 5 ml cellculture media with FBS (Invitrogen). 1� 106 cells were counted, spun down at800 g and re-suspended in 1 ml of FBS-free media containing 1 mM EDTA toprevent clumping. An equal volume of 100% ethanol was added drop wise andthe cells were stored at 4�C for 24 h. After storage, the cells were pelleted at800 g, aspirated and re-suspended in 1 mg/ml RNase A (Invitrogen) in PBS andincubated for 30 min at room temperature. 0.5 ml of 50 mg/ml propidiumiodide (Sigma-Aldrich) was added after RNase digestion and incubated for anadditional 30 min in the dark. Cells were then analysed for DNA content usinga Becton-Dickinson FACScan to determine cell cycle phase.

RNA extraction and preparation

Total cellular RNA was isolated using TRIzol LS (Invitrogen) according tomanufacturer’s protocol, which was modified as follows: RNA was precipi-tated using 0.5 vol of isopropanol and 0.5 vol of 1.2 M NaCl/0.8 M sodiumcitrate, to prevent proteoglycan contamination. RNA was re-suspended inDEPC-treated H2O, quantified, and used immediately or precipitated andthen 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 usingstandard procedures (48). Radiolabelled probe was synthesized by nick trans-lation with 32P-CTP from full-length p16 cDNA cloned into pBluescript.Membranes were incubated overnight with 2 � 106 c.p.m. of radiolabelledprobe and exposed to film for 2--7 days.

Transcriptional analysis was performed using the Ribonuclease Protectionassay (RPA). Total cellular RNA (10 ug) was analysed using the Human cellcycle regulator probe set (BD BioSciences) following the manufacturer’sprotocol. Gels were exposed to autoradiography film overnight on a Kodakintensifying screen, or for up to 5 days for weaker signals.

Western blot analysis

For analysis of cell cycle proteins, hypoxic and normoxic tissue culture cellswere prepared as described above, placed on ice and rinsed with 10 ml of ice-cold PBS. The cells were lysed on the plate by the addition of 1 ml of NP-40lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1.5 mMMgCl2, 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 concentrationwas determined using the Bichronate assay (Pierce). Seventy micrograms ofcell 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 proteins530 kDa) by blotting overnight at 30 V/60 mA. Membranes were blocked for1 h at room temp in 10% (w/v) powdered non-fat milk/Tris-buffered salinewith 0.1% (v/v) Tween-20 (Sigma). Western blotting was carried out overnightat 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 performedusing the conditions listed above, with the following modifications: aftertransfer 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 at4�C using the following antibodies: GSK3b (#610201; 1/2000) (BD Transduc-tion Labs), Phospho-GSK3a/b Ser 21/9 (#9331S; 1/2000), Phospho-Akt Ser473 (#9271S; 1/2000), Akt (#9272; 1/2000) (Cell Signaling Technologies).

Results

Differing cell types exhibit differing arrest profiles inresponse to hypoxia

We determined the cell cycle phenotype of a variety of celllines responding to hypoxia. As shown in Figure 1, HTB-30,HeLa and HMEC exhibited hypoxia-associated G1/S arrestoccurring at 6--12 h after initial exposure to hypoxic condi-tions. In comparison, Hep3B, a hepatocellular carcinoma cellline, lacked an observable G1/S arrest (defined as an increasein 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

A.H.Box and D.J.Demetrick

2326

with untreated controls (not shown), indicating that the severehypoxia did cause proliferation arrest (probably due to insuffi-cient cellular ATP for cell division), but only Hep3B failed toundergo 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 inthe normal response to hypoxia, we evaluated its expression

in the treated and untreated cell lines. While p16 expressionis usually associated with cell cycle arrest, RPA analysis ofthe diploid fibroblast cell line WI-38 revealed a reduction inp16 mRNA levels after 12 and 24 h exposure to hypoxicgrowth conditions (Figure 2A). This reduction in p16mRNA was confirmed by northern blot analysis (Figure 2B).Hep3B cells did not show a reduction in p16 mRNAlevels (data not shown). As well, HeLa p16 transcript levels

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 for2 or 24 h, fixed in ethanol, stained with propidium iodide and analysed for DNA content to determine G1 and S phase cell populations. (A) Graphicalrepresentation 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 arrestin 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 cellsshow 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 cellsin G0/G1. (E) Primary WI38 fibroblasts exhibit a decrease in cells transiting through S phase.

Cell cycle regulators in hypoxic cell cycle arrest

2327

showed no reliable changes in response to hypoxia (data notshown).Both WI-38 and Hs68, another normal diploid fibroblast cell

line, showed a reduction in p16 protein levels after 24 h in alow oxygen environment (Figure 2C), in agreement with theRPA data, as did both HMEC and HTB-30 cells (Figure 2D).In HMEC and HeLa cell lysates, p16 protein levels again werereduced after 12--24 h in hypoxia (Figure 3C). Hep3B, whichdid not appear to undergo G1/S arrest in hypoxia, did not showany 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 p16-related proteins to determine if the relative levels of theremaining CDKIs were modulated in response to hypoxia. Asshown in Figure 3, all cell lines used in this study exhibiteddown-regulation of at least one of the G1/S CDKIs in response

to hypoxia. HMEC, HTB-30 and HeLa showed loss of p16protein, while Hep3B, the cell line most resistant to hypoxia-induced cell cycle arrest, showed no loss of p16 protein after24 h of treatment. There was also loss of p19 protein expres-sion, which occurred in Hep3B, the breast cancer cell lineHTB-30, and HeLa cells (p19 was undetectable in HMECcells). Hep3B and HMEC exhibited a loss of p21, again after12--24-h exposure to hypoxia, while p21 levels were undetect-able in both HTB-30 and HeLa cells. Interestingly, HeLa wasthe only cell line to show a drastic reduction in all detectableCDKIs. In all cells, there was at least one instance of loss of aCDKI of both the INK4 or Cip/Kip families. The loss of CDKIexpression does not appear to be due to a general reduction inprotein translation or stability, since levels of actin are stable,and usually other related but less abundant proteins, such asp15 or p18, were not affected. Analysis of p57Kip2 and CyclinD1 also showed a decreased level of these proteins associated

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 ina 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 forRPA 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 levelscorrelate 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.

A.H.Box and D.J.Demetrick

2328

with hypoxia, but with no correlation to cell cycle arrestphenotype (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 ofthe cell line to enter a hypoxia-induced G1/S arrest, althoughHep3B, which is resistant to hypoxic cell cycle arrest was theonly cell line to not show a reduction in either p27 or p16levels. Of course, the data only indicate average results for thecell lines, where cells could show either a generalized changein 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 correlatewith G1/S arrest

It has been reported previously that hypoxia-induced G1/Sarrest was due to the induction of p27 (41), but other researchinto p270s involvement has been contradictory. Measurementof p27 mRNA levels in our cell lines by RPA indicated thatonly HTB-30 experienced a hypoxia-induced increase in p27mRNA while the other cell lines, HMEC, HeLa and Hep3B,did not show hypoxic modulation of p27 transcripts (data notshown). Protein levels of p27 were increased in both HTB-30

and Hep3B (Figure 3A and B), but p27 was unchanged inHMEC (Figure 3D) and reduced in HeLa cells (Figure 3C).The most important observation was that induction of p27could not be correlated with the ability of the cell line toenter a hypoxia-induced G1/S arrest. Both HTB-30 andHep3B showed increased amounts of p27 protein, but onlyHTB-30 exhibited a G1/S arrest. In addition, HMECs, whichcould arrest during exposure to hypoxic conditions, did notshow any change in p27 levels and HeLa cells actually experi-enced a reduction in p27 while still arresting at the G1/Sboundary. Western blotting for pan-actin protein levels con-firmed that overall cellular protein levels were constant. Con-sidering the above data, we have determined that p27 transcriptor protein levels are unlikely to determine the hypoxia-inducedG1/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 ispreceded by the loss of CDKIs, specifically p16 and p21 (49).Considering this evidence, we decided to determine if the lossof the CDKIs we have observed in hypoxia was due toactivation of an apoptotic pathway and/or cell loss. Flowcytometric analysis did not reveal significant numbers of

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 andactin) or 12% acrylamide SDS--PAGE gels and transferred to the appropriate membrane. Pan actin blots were performed for all cell lines to normalize protein-loading 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 anotherINK4 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 inthe 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 inhypoxia. (C) HeLa cells showed reductions in all CDKIs measured, including p27, although they still underwent hypoxia-induced cell cycle arrest. (D) Primarybreast 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.

Cell cycle regulators in hypoxic cell cycle arrest

2329

cells with sub-G1 content, indicating that significant apoptosiswas unlikely to have occurred (e.g. Figure 1D). We alsodecided to evaluate PARP cleavage as an indicator of apopto-sis. PARP is cleaved from its full-length size of ~119 kDa, viaapoptotically activated caspases, to a truncated form of ~85kDa. As shown in Figure 4, there is no detectable cleavage ofPARP at any time point during exposure to hypoxia, in any ofthe cell lines. Therefore, flow cytometry and PARP cleavageassays do not indicate that significant apoptosis is occurring inany of the four cell lines, even after 24 h of severe hypoxia.The loss of CDKI proteins, therefore, does not appear to beassociated with markers of apoptosis.

Akt/GSK3b signalling is correlated with the ability to enterG1/S arrest in hypoxia

Recent studies have indicated that Akt signalling throughGSK3b is involved in decreasing HIF-1a expression duringhypoxia (50). Using two epithelial cell lines from our studies,HeLa cells, which arrested in response to hypoxia, and Hep3Bcells, which did not exhibit a G1/S arrest in hypoxia, weexamined if the loss of HIF-1 and CDKI expression wasassociated with Akt/GSK3b status. As shown in Figure 5,HeLa cells show an initial increase in Akt phosphorylationbetween 2 and 6 h in hypoxia, but at 12--24 h there is a drasticdecrease in both phospho-Akt and total Akt levels in hypoxia.This down-regulation of Akt levels is correlated with the lossof HIF-1 and CDKI expression and cell cycle arrest. In addi-tion, as the level of phospho-Akt decreases, there is a reci-procal loss of the inhibitory phosphorylation of serine 9 onGSK3b, indicating a decrease in Akt activity and a probableincrease in GSK3b activity. In contrast, Hep3B cells

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 aprotein 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.

Fig. 5. Phospho-specific western blots reveal differential AKT/GSK3bactivation 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. Westernblotting of Akt and GSK3b showed that in hypoxia-treated HeLa cells (A),Akt exhibited activating phosphorylation, but phosphoprotein and totalprotein levels were lost between 6 and 12 h post-hypoxia. Concomitantly,inhibitory phosphorylation on GSK3b was lost. (B) Hep3B cells did notexhibit modulation of Akt and GSK3b phosphorylation with hypoxiatreatment.

A.H.Box and D.J.Demetrick

2330

maintained high levels of phospho-Akt, irrespective ofhypoxia, and maintained GSK3b inhibition, as indicated byhigh levels of inhibitory serine 9 phosphorylation. Associatedwith a constitutively phosphorylated Akt is maintenance ofhigh levels of HIF-1 expression and a lack of a hypoxiainduced G1/S arrest.To evaluate the effect of GSK3b on the CDKI proteins

levels, HeLa and Hep3B were treated with the GSK3b inhi-bitor LiCl and the results are shown in Figure 6. In the presenceof LiCl, HeLa levels of HIF-1a and p16 do not change appre-ciably with the 12- and 24-h hypoxia-treatment time points, insharp contrast to their expression in the absence of LiCl (Fig-ure 3), although p27 levels were slightly reduced with 50 mMLiCl treatment in non-hypoxic cells, as compared withuntreated cells. Hep3B showed no effect of LiCl treatmenton the levels of these three proteins in hypoxia. Essentially,LiCl treatment converted the hypoxia-associated HeLa expres-sion of HIF-1a, p16 and p27 proteins to a similar patternshown by Hep3B, reversing the low levels noted in non-lithium-treated HeLa cells. This indicates that GSK3b activitymay be responsible for the observed hypoxic reduction of theseproteins in HeLa and, perhaps, the other cell lines analysed.Treatment of HeLa cells with hypoxia in the presence orabsence of 50 mM LiCl did not prevent a reduction in cellstransiting the S phase and arresting in G1 (Figure 6C).Although there was a decrease in G1 cell arrest (as measuredby percent cells in G1 associated with LiCl at the 24-h hypoxiatime point) compared with untreated cells, there was actuallyan increase as compared with normoxic cells treated for 24 h

with LiCl. Interestingly, HeLa cells pre-treated with LiCl, butincubated in normoxia, showed an increase in the number ofcells entering the S phase (~2-fold) over untreated normoxicHeLa cells, perhaps implying an overall stimulatory effect onthe cell cycle from LiCl.

Discussion

Hypoxia has been long known to initiate a profound, reversiblecell cycle arrest in many different cell types and tumour celllines (16,51). Cells in late S, G2 and M phase will finish thecycle and arrest in G1, unless the hypoxia is severe (18). Cellsin G1 and early S phase will remain there (16). Arresting inearly S phase is easy to rationalize as due to a dependency oncell energy levels for the completion of DNA synthesis. Fin-ishing the cell cycle and arresting at G1 is typical of cells withdown-regulation of pRb phosphorylation. With initial researchidentifying reductions in CDK2 and CDK4 activity, along withhypophosphorylation of the pRb, it was therefore believed thatthe hypoxia-induced cell cycle arrest was probably mediatedby one of the specific inhibitors of the G1 kinases from theINK4 or Cip/Kip families, as is usually the case (reviewed inref. 52). Sequential modification of Rb by two separate cyclin/cdk complexes is necessary for cell cycle progression, andcoordinated up-regulation of p27 and p15 is essential forTGFb-mediated cell cycle arrest (43,53). Accordingly, simul-taneous measurement of expression of both the INK4 and Cip/Kip gene families may be necessary in order to elucidate themechanism of hypoxia-induced G1/S 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), addeddirectly 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 totalprotein 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, blottedand 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 for12 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, p16or p27 was noted in the hypoxic cells treated with LiCl, in sharp contrast to their expression when LiCl was not present (Figure 3).

Cell cycle regulators in hypoxic cell cycle arrest

2331

Response to hypoxia is cell line dependent

While generally confirming the hypoxia-induced cell cyclearrest phenomenon, the panel of cell lines we analysed exhib-ited variability in their ability to undergo arrest. Normaldiploid cell strains, such HMECs, exhibited a coordinatedarrest, with a gradual decrease in S phase and an increase inG0/G1 phase. HTB-30, a breast carcinoma cell line, and HeLaa cervical carcinoma cell line also exhibited similar cell cyclefindings and followed essentially the same inhibition kineticsas normal cells, showing peak arrest at ~12 h after exposure tolow atmospheric O2. Conversely, Hep3B cells, a line that iscommonly used in hypoxia studies, did not undergo a coordi-nated, hypoxia-associated cell cycle arrest even after 24 h inhypoxic culture.While the levels of G1 increase/S phase decrease are not

pronounced, it is likely that our severe hypoxia conditionsprecluded the normal completion of the cell cycle in many ofthe cells, including Hep3B (18); however, the ability toundergo regulated G1/S blockade was the important observa-tion rather than the magnitude of blockade. We expect thattitration of hypoxia using more elaborate equipment mayidentify a higher O2 level correlating with an increase in themagnitude of the G1/S blockade by providing enough oxygenfor 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 havemutated p53 (54,55), while HeLa p53 is down-regulated byHPV E6. Hep3B lacks functional Rb (55), while HeLa pRbfunction is down-regulated by HPV E7. HTB-30 has amplifiedthe HER2 gene. There does not appear to be a clear correlationwith the cell cycle arrest phenotype of these cells and p53, pRbor HER2 status.

p16 expression is reduced under hypoxic conditions

p160s importance as a TS has been widely established in anumber of tumour types (23--25) but previous studies of theeffects of hypoxia on p16 have yielded inconclusive results(40,42). We evaluated p16 levels under hypoxia in the normaldiploid cells, WI-38 and HMECs, both of which exhibit anormal G1/S arrest under hypoxia (Figure 1D and E). Ribonu-clease protection assay and northern blot analysis of WI-38cells indicated a reduction in p16 mRNA, which was concor-dant with a decrease in p16 protein levels. The same phenom-enon was observed in HMECs and HTB-30 cells at both themRNA and protein levels and at the protein level in HeLacells. Hep3B cells, which did not show a hypoxia-inducedarrest, did not have an appreciable reduction in p16 mRNAor protein levels.It is unclear why p16 transcript and/or protein levels are

reduced in hypoxia. A recent paper reported that cerebralischaemia causes a reduction in INK4 gene family expressionas a precursor to apoptosis (49). Although our flow cytometryanalysis did not show a population of cells with a sub-G1 DNAcontent at 24 h, or cleavage of PARP, further studies, usingother methods of analysis, may reveal if there is a correlationbetween initiation of apoptosis and the loss of p16 expression.p16 is constitutively regulated in cycling cells (56,57) and isthought to be a relatively long lived protein (48 h) (58)although we cannot rule out that a decrease in transcript levelmay be responsible for low levels of the protein. HeLa, whichdid not appear to show a reproducible decrease in p16 tran-script with hypoxia, did show reduced protein levels, implying

there is probably a change in the p16 protein lifespan inhypoxia.

p27 is induced by hypoxia, but does not appear to mediatehypoxia-induced G1/S arrest

Recently, there have been a number of conflicting papersconcerning the importance of p27 in hypoxia. It has beenreported that p27 is induced in mouse embryonic fibroblastsat both the mRNA and protein levels (41). The induction ofp27 was not HIF-1 dependent, as determined by promoteranalysis. However, MEFs derived from p27/p21 doubleknockout mice are still able to initiate a G1/S arrest, indicatingthat neither p27 nor p21 is essential for hypoxia-inducedarrest (18).In our study, HTB-30 alone experienced an increase in p27

transcript levels (data not shown). p27 protein levels wereelevated in HTB-30 and Hep3B cells, but this up-regulationdid not correlate with induction of a G1/S arrest, asHep3B cells are resistant to hypoxia-induced arrest. HeLacells exhibit a reduction in p27 protein levels while retainingthe ability to induce a cell cycle arrest. HMECs, a primary,non-immortalized cell, exhibit no change in either p27 mRNAor protein levels while undergoing arrest. Considering thisdata, we conclude that an increased level of p27, althoughinduced in some cell lines by hypoxia, is not likely to be themain effector of the observed G1/S arrest.

Loss of CDKI expression in hypoxia does not correlate withhypoxia-induced G1/S arrest or apoptosis

Much of the recent research into hypoxia-induced arrest hasfocused solely upon one or two of the CDKI family members.Our study, evaluating all CDKI gene products, showed thatthere is a rapid (~12 h) loss of one or more CDKIs from both ofthe Cip/Kip and INK4 gene families after exposure to low(50.5%) oxygen. The most commonly lost CDKIs were p21and p19, although the pattern of CDKI loss could not becorrelated to either the cell line tissue origin, or the cell’sability to undergo hypoxic cell cycle arrest. Actin blotting ofhypoxic cell lysates reveals that general protein levels arestable in hypoxia. It has been suggested that CDKIs mayconfer a survival advantage to ischaemic neurons, and thatloss of their expression precedes induction of apoptosis (49).Lack of sub-G1 DNA content in cytometric measurements andthe presence of intact PARP even after 24 h of exposure toextreme hypoxia indicate that these cells are not undergoingtypical apoptosis.Recent research into hypoxia-induced signalling effects may

give clues as to the mechanism of the phenomena that we havedescribed. Hypoxia may be associated with up-regulationof the PI3K/Akt signalling pathway (59--61), which results inup-regulation of HIF-1 via a variety of mechanisms (50,62,63),although other investigators have reported that PI3K/Aktsignalling does not seem to be involved in the hypoxicresponse (64,65).It was found recently that short-term hypoxia (5 h) was

associated with an increase in Akt activity and increasedprotein 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 associatedwith a reduction in HIF-1 protein levels. GSK3b activity wasalso associated with loss of p21 (66) and cyclin D1 (67), withp21 being a direct target for GSK3b (66). Furthermore,GSK3b expression is increased in an expression microarray

A.H.Box and D.J.Demetrick

2332

analysis of hypoxia (68). The effect of GSK3b on INK4 familyregulation 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 ofAkt phosphorylation and inhibitory GSK3b phosphorylationlate (412 h) in the hypoxic response. Correlated with this wasa reduction in HIF-1 protein levels even in the presence ofhypoxic conditions and a reduction in CDKI expression. Inter-estingly, the loss of Akt/GSK3b phosphorylation and HIF-1expression levels was correlated with the ability of the cell lineto enter a hypoxic G1/S arrest. HeLa cells, which were permis-sive to hypoxia-induced G1/S arrest, exhibited modulation ofthe Akt/GSK3b signalling pathway and loss of HIF-1 andCDKI expression. Hep3B, which appeared to have constitu-tively activated Akt and consequently continually inhibitedGSK3b, was resistant to hypoxia-induced G1/S arrest andhad stable levels of HIF-1 and CDKI proteins throughout thehypoxia time course. When GSK3b activity in HeLa cells wasinhibited with LiCl, there was no reduction in HIF-1 or CDKIprotein levels resulting in a pattern similar to that of hypoxiccell cycle arrest.Although incubation with LiCl prevented the observed

reduction in HIF-1 and CDKI protein levels, LiCl did notprevent hypoxic cell cycle arrest. It appears that LiCl mayhave a stimulatory effect on the HeLa cell cycle in general(compare LiCl-treated versus -untreated normoxic cells), thuswhile there is a cell cycle arrest observed between normoxicversus hypoxic HeLa cells treated with LiCl, this effect ismuch less pronounced in hypoxic cells treated with LiCl thanin untreated hypoxic cells, at the 24-h time point. The com-plexity of these findings makes it difficult to interpret the realeffect of LiCl on the cell cycle in HeLa. This result is of nogreat surprise, as LiCl is well known to have pleotropic effectson a variety of cell pathways in addition to serving as aninhibitor of GSK3b (69,70), and these effects could affectthe 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 D1and p21 degradation may also mediate INK4 loss. In our celllines, which exhibited hypoxia-induced cell cycle arrest, thereduction in CDKI expression was mirrored by a reduction inHIF-1 protein levels. Interestingly, inhibition of PI3K, anupstream activator of Akt, with the chemical inhibitorLY294002 resulted in the down-regulation of p15, p16, p19and p21 protein levels but an increase in p27 protein levels(71). As well, we have noticed that a consensus GSK3b phos-phorylation 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 thatphosphorylated by GSK3b on p21 (66) is also present withinp19 (Thr141) and five residues upstream of it is an asparticacid residue that could serve as a surrogate for substrate prim-ing by prior phosphorylation (72), exactly as seems to occurwith p21. Other potential GSK3b sites can be found in bothp27 (Thr187, Ser110, Thr142, Ser161) and p57 (Thr310,Ser32, Ser84, Ser258). Interestingly, the Thr187 GSK3b con-sensus site of p27 is identical to the Thr310 site in p57. Thesignificance of any of these is unknown, but the potential forthe CDKI’s to be direct targets of GSK3b phosphorylation anddegradation is worthy of study.After evaluating our data, we propose that the observed loss

of CDKIs in hypoxia is downstream and due to the inhibitionof PI3K/Akt signalling in these cell lines (Figure 7). The loss

of HIF-1 expression in our cells, we believe, is also due to thehypoxic inactivation of Akt as noted above and subsequentup-regulation of GSK3b. How down-regulation of Akt ismediated in hypoxia is unknown, but upstream regulators ofAkt, such as PTEN (73), may be involved. The association ofresistance to hypoxia-induced cell cycle arrest and resistanceto hypoxia-associated degradation of p21 and INK4 proteinsmay be associated with abnormally increased PI3K/Akt activ-ity. It is tempting to speculate that such hypoxia-resistancephenotypes may be selected in cancer cells with dysregula-ted PTEN function, in a manner similar to that proposed forp53 (74).Studies into the molecular events surrounding hypoxia-

induced cell cycle arrest have been conflicting. Many reportshave utilized tumorigenic cell lines in which the major cellcycle pathways are altered due to inactivating mutations in keycell cycle regulators, such as p53, pRb and p16, leading toinconclusive results (40). In addition, many of these studiesuse animal-derived cell lines, which are capable of undergoingspontaneous immortalization. Our study focused on the simi-larities and differences between a normal diploid cell strain(HMEC) and cancerous cell lines (HeLa, HTB-30, Hep3B) intheir cell cycle responses to hypoxia, as well as their expres-sion of cell cycle kinase inhibitors. While we still have not yetidentified the precise mediators of the hypoxia-induced cell

Fig. 7. A proposed model outlining an Akt/GSK3b-dependent mechanismfor INK4 and Cip/Kip down-regulation late in the hypoxic response.

Cell cycle regulators in hypoxic cell cycle arrest

2333

cycle arrest phenotype, we have accumulated evidence for theinvolvement of thePI3K/Akt/GSK3bpathway in regulating thisresponse.Elucidationof theprecisemediatorswill involve studyof the expression of other cell cycle regulatory proteins suchas CDKs, cyclins or phosphatases and/or post-translationalmodifications of the CDKIs or other cell cycle proteins.

Acknowledgements

We would also like to thank undergraduate students Jason Morin, Eli Akbariand Carol Yuen for their valuable help with the western blots, and PhilipBerardi, Chris Nichols and Alan Box for helpful discussions and/or review ofthe manuscript. The authors wish to acknowledge the operating support of theCancer Research Society for making this work possible. Salary support forD.J.D. was provided by Calgary Laboratory Services. A.H.B. is supported by astipend from the Alberta Cancer Board Strategic Training Program in Transla-tional Cancer Research.

References

1.Dewhirst,M.W., Ong,E.T., Rosner,G.L., Rehmus,S.W., Shan,S.,Braun,R.D., Brizel,D.M. and Secomb,T.W. (1996) Arteriolar oxygenationin tumour and subcutaneous arterioles: effects of inspired air oxygencontent. Br. J. Cancer Suppl., 27, S241--246.

2.Brizel,D.M., Scully,S.P., Harrelson,J.M., Layfield,L.J., Bean,J.M.,Prosnitz,L.R. and Dewhirst,M.W. (1996) Tumor oxygenation predictsfor the likelihood of distant metastases in human soft tissue sarcoma.Cancer Res., 56, 941--943.

3.Gatenby,R.A., Kessler,H.B., Rosenblum,J.S., Coia,L.R., Moldofsky,P.J.,Hartz,W.H. and Broder,G.J. (1988) Oxygen distribution in squamous cellcarcinoma metastases and its relationship to outcome of radiation therapy.Int. J. Radiat. Oncol. Biol. Phys., 14, 831--838.

4.Schwickert,G., Walenta,S., Sundfor,K., Rofstad,E.K. and Mueller-Klieser,W. (1995) Correlation of high lactate levels in human cervicalcancer with incidence of metastasis. Cancer Res., 55, 4757--4759.

5.Tomida,A. and Tsuruo,T. (1999) Drug resistance mediated by cellularstress response to the microenvironment of solid tumors. Anticancer DrugDes., 14, 169--177.

6.Wang,G.L. and Semenza,G.L. (1993) Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia.J. Biol. Chem., 268, 21513--21518.

7.Semenza,G.L. and Wang,G.L. (1992) A nuclear factor induced by hypoxiavia de novo protein synthesis binds to the human erythropoietin geneenhancer at a site required for transcriptional activation. Mol. Cell. Biol.,12, 5447--5454.

8.Wang,G.L., Jiang,B.H., Rue,E.A. and Semenza,G.L. (1995) Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulatedby cellular O2 tension. Proc. Natl Acad. Sci. USA, 92, 5510--5514.

9.Salceda,S. and Caro,J. (1997) Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome systemunder normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J. Biol. Chem., 272, 22642--22647.

10.Huang,L.E., Gu,J., Schau,M. and Bunn,H.F. (1998) Regulation ofhypoxia-inducible factor 1alpha is mediated by an O2-dependentdegradation domain via the ubiquitin-proteasome pathway. Proc. NatlAcad. Sci. USA, 95, 7987--7992.

11.Chen,C., Pore,N., Behrooz,A., Ismail-Beigi,F. and Maity,A. (2001)Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interactionbetween H-ras and hypoxia. J. Biol. Chem., 276, 9519--9525.

12.Semenza,G.L., Jiang,B.H., Leung,S.W., Passantino,R., Concordet,J.P.,Maire,P. and Giallongo,A. (1996) Hypoxia response elements in thealdolase A, enolase 1 and lactate dehydrogenase A gene promoters containessential binding sites for hypoxia-inducible factor 1. J. Biol. Chem., 271,32529--32537.

13.Semenza,G.L., Roth,P.H., Fang,H.M. and Wang,G.L. (1994)Transcriptional regulation of genes encoding glycolytic enzymes byhypoxia-inducible factor 1. J. Biol. Chem., 269, 23757--23763.

14.Liu,Y., Cox,S.R., Morita,T. and Kourembanas,S. (1995) Hypoxiaregulates vascular endothelial growth factor gene expression in endothelialcells. Identification of a 50 enhancer. Circ. Res., 77, 638--643.

15.Forsythe,J.A., Jiang,B.H., Iyer,N.V., Agani,F., Leung,S.W., Koos,R.D.and Semenza,G.L. (1996) Activation of vascular endothelial growth factor

gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol., 16,4604--4613.

16.Amellem,O. and Pettersen,E.O. (1991) Cell inactivation and cell cycleinhibition as induced by extreme hypoxia: the possible role of cell cyclearrest as a protection against hypoxia-induced lethal damage. Cell Prolif.,24, 127--141.

17.Krtolica,A. and Ludlow,J.W. (1996) Hypoxia arrests ovarian carcinomacell cycle progression, but invasion is unaffected. Cancer Res., 56,1168--1173.

18.Green,S.L., Freiberg,R.A. and Giaccia,A.J. (2001) p21 (Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessaryfor hypoxia-induced arrest. Mol. Cell. Biol., 21, 1196--1206.

19.Swanton,C. (2004) Cell-cycle targeted therapies. Lancet Oncol., 5, 27--36.20.Ekholm,S.V. and Reed,S.I. (2000) Regulation of G (1) cyclin-dependent

kinases in the mammalian cell cycle. Curr. Opin. Cell. Biol., 12, 676--684.21.Bartek,J., Bartkova,J. and Lukas,J. (1996) The retinoblastoma protein

pathway and the restriction point. Curr. Opin. Cell. Biol., 8, 805--814.22.Serrano,M., Hannon,G.J. and Beach,D. (1993) A new regulatory motif in

cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature,366, 704--707.

23.Okamoto,A., Demetrick,D.J., Spillare,E.A. et al. (1994) Mutations andaltered expression of p16INK4 in human cancer. Proc. Natl Acad. Sci.USA, 91, 11045--11049.

24.Nobori,T., Miura,K., Wu,D.J., Lois,A., Takabayashi,K. and Carson,D.A.(1994) Deletions of the cyclin-dependent kinase-4 inhibitor gene inmultiple human cancers. Nature, 368, 753--756.

25.Kamb,A., Gruis,N.A., Weaver-Feldhaus,J., Liu,Q., Harshman,K.,Tavtigian,S.V., Stockert,E., Day,R.S. 3rd, Johnson,B.E. andSkolnick,M.H. (1994) A cell cycle regulator potentially involved ingenesis of many tumor types. Science, 264, 436--440.

26.Serrano,M., Lee,H., Chin,L., Cordon-Cardo,C., Beach,D. andDePinho,R.A. (1996) Role of the INK4a locus in tumor suppression andcell mortality. Cell, 85, 27--37.

27.Hannon,G.J. and Beach,D. (1994) p15INK4B is a potential effector ofTGF-beta-induced cell cycle arrest. Nature, 371, 257--261.

28.Hirai,H., Roussel,M.F., Kato,J.Y., Ashmun,R.A. and Sherr,C.J.(1995) Novel INK4 proteins, p19 and p18, are specific inhibitors of thecyclin D-dependent kinases CDK4 and CDK6. Mol. Cell. Biol., 15,2672--2681.

29.Alcorta,D.A., Xiong,Y., Phelps,D., Hannon,G., Beach,D. and Barrett,J.C.(1996) Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a)in replicative senescence of normal human fibroblasts. Proc. Natl Acad.Sci. USA, 93, 13742--13747.

30.Wieser,R.J., Faust,D., Dietrich,C. and Oesch,F. (1999) p16INK4 mediatescontact-inhibition of growth. Oncogene, 18, 277--281.

31.Roussel,M.F. (1999) The INK4 family of cell cycle inhibitors in cancer.Oncogene, 18, 5311--5317.

32.Latres,E., Malumbres,M., Sotillo,R., Martin,J., Ortega,S., Martin-Caballero,J., Flores,J.M., Cordon-Cardo,C. and Barbacid,M. (2000)Limited overlapping roles of P15 (INK4b) and P18 (INK4c) cell cycleinhibitors in proliferation and tumorigenesis. EMBO J., 19, 3496--3506.

33.El-Diery,W.S., Tokino,T., Velculescu,V.E., Levy,D.B., Parsons,R.,Trent,J.M., Lin,D., Mercer,W.E., Kinzler,K.W. and Vogelstein,B. (1993)WAF1, a potential mediator of p53 tumour suppression. Cell, 75, 817--825.

34.Xiong,Y., Hannon,G.J., Zhang,H., Casso,D., Kobayashi,R. and Beach,D.(1993) p21 is a universal inhibitor of cyclin kinases. Nature, 366, 701--704.

35.Gu,Y., Turck,C.W. and Morgan,D.O. (1993) Inhibition of cdk2 activity invivo by an associated 20K regulatory subunit. Nature, 366, 707--710.

36.Polyak,K., Lee,M.H., Erdjument-Bromage,H., Koff,A., Roberts,J.M.,Tempst,P. and Massague,J. (1994) Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellularantimitogenic signals. Cell, 78, 59--66.

37.Matsuoka,S., Edwards,M.C., Bai,C., Parker,S., Zhang,P., Baldini,A.,Harper,J.W. and Elledge,S.J. (1995) p57KIP2, a structurally distinctmember of the p21CIP1 Cdk inhibitor family, is a candidate tumorsuppressor gene. Genes Dev., 9, 650--662.

38.Krtolica,A., Krucher,N.A. and Ludlow,J.W. (1999) Molecular analysis ofselected cell cycle regulatory proteins during aerobic and hypoxicmaintenance of human ovarian carcinoma cells. Br. J. Cancer, 80,1875--1883.

39.Ludlow,J.W., Howell,R.L. and Smith,H.C. (1993) Hypoxic stress inducesreversible hypophosphorylation of pRB and reduction in cyclin Aabundance independent of cell cycle progression. Oncogene, 8, 331--339.

40.Krtolica,A., Krucher,N.A. and Ludlow,J.W. (1998) Hypoxia-induced pRBhypophosphorylation results from downregulation of CDK and upregula-tion of PP1 activities. Oncogene, 17, 2295--2304.

A.H.Box and D.J.Demetrick

2334

41.Gardner,L.B., Li,Q., Park,M.S., Flanagan,W.M., Semenza,G.L. andDang,C.V. (2001) Hypoxia inhibits G1/S transition through regulation ofp27 expression. J. Biol. Chem., 276, 7919--7926.

42.Zygmunt,A., Tedesco,V.C., Udho,E. and Krucher,N.A. (2002) Hypoxiastimulates p16 expression and association with cdk4. Exp. Cell. Res., 278,53--60.

43.Reynisdottir,I., Polyak,K., Iavarone,A. and Massague,J. (1995) Kip/Cipand Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in response toTGF-beta. Genes Dev., 9, 1831--1845.

44.Parry,D., Mahony,D., Wills,K. and Lees,E. (1999) Cyclin D-CDK subunitarrangement is dependent on the availability of competing INK4 and p21class inhibitors. Mol. Cell. Biol., 19, 1775--1783.

45.Grimison,B., Langan,T.A. and Sclafani,R.A. (2000) P16Ink4a tumorsuppressor function in lung cancer cells involves cyclin-dependent kinase2 inhibition by Cip/Kip protein redistribution. Cell Growth Differ., 11,507--515.

46. Jiang,H., Chou,H.S. and Zhu,L. (1998) Requirement of cyclin E-Cdk2inhibition in p16 (INK4a)-mediated growth suppression. Mol. Cell. Biol.,18, 5284--5290.

47.McConnell,B.B., Gregory,F.J., Stott,F.J., Hara,E. and Peters,G. (1999)Induced expression of p16 (INK4a) inhibits both CDK4- and CDK2-associated kinase activity by reassortment of cyclin-CDK-inhibitorcomplexes. Mol. Cell. Biol., 19, 1981--1989.

48.Chomczynski,P. (1993) A reagent for the single-step simultaneousisolation of RNA, DNA and proteins from cell and tissue samples.Biotechniques, 15, 532--534, 536--537.

49.Katchanov,J., Harms,C., Gertz,K. et al. (2001) Mild cerebral ischemiainduces loss of cyclin-dependent kinase inhibitors and activation of cellcycle machinery before delayed neuronal cell death. J. Neurosci., 21,5045--5053.

50.Mottet,D., Dumont,V., Deccache,Y., Demazy,C., Ninane,N., Raes,M. andMichiels,C. (2003) Regulation of hypoxia-inducible factor-1alpha proteinlevel during hypoxic conditions by the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase 3beta pathway in HepG2 cells. J. Biol. Chem.,278, 31277--31285.

51.Taylor,W.G. and Camalier,R.F. (1982) Modulation of epithelial cellproliferation in culture by dissolved oxygen. J. Cell Physiol., 111, 21--27.

52.Sherr,C.J. (2000) The Pezcoller lecture: cancer cell cycles revisited.Cancer Res., 60, 3689--3695.

53.Lundberg,A.S. and Weinberg,R.A. (1998) Functional inactivation of theretinoblastoma protein requires sequential modification by at least twodistinct cyclin-cdk complexes. Mol. Cell. Biol., 18, 753--761.

54.Sugikawa,E., Hosoi,T., Yazaki,N., Gamanuma,M., Nakanishi,N. andOhashi,M. (1999) Mutant p53 mediated induction of cell cycle arrestand apoptosis at G1 phase by 9-hydroxyellipticine. Anticancer Res., 19,3099--3108.

55.Morel,A.P., Unsal,K., Cagatay,T., Ponchel,F., Carr,B. and Ozturk,M.(2000) P53 but not p16INK4a induces growth arrest in retinoblastoma-deficient hepatocellular carcinoma cells. J. Hepatol., 33, 254--265.

56.Soucek,T., Pusch,O., Hengstschlager-Ottnad,E., Wawra,E., Bernaschek,G.and Hengstschlager,M. (1995) Expression of the cyclin-dependent kinaseinhibitor p16 during the ongoing cell cycle. FEBS Lett., 373, 164--169.

57.Hara,E., Smith,R., Parry,D., Tahara,H., Stone,S. and Peters,G. (1996)Regulation of p16CDKN2 expression and its implications for cellimmortalization and senescence. Mol. Cell. Biol., 16, 859--867.

58.Shapiro,G.I., Park,J.E., Edwards,C.D., Mao,L., Merlo,A., Sidransky,D.,Ewen,M.E. and Rollins,B.J. (1995) Multiple mechanisms of p16INK4Ainactivation in non-small cell lung cancer cell lines. Cancer Res., 55,6200--6209.

59.Alvarez-Tejado,M., Naranjo-Suarez,S., Jimenez,C., Carrera,A.C.,Landazuri,M.O. and del Peso,L. (2001) Hypoxia induces the activationof the phosphatidylinositol 3-kinase/Akt cell survival pathway in PC12cells: protective role in apoptosis. J. Biol. Chem., 276, 22368--22374.

60.Beitner-Johnson,D., Rust,R.T., Hsieh,T.C. and Millhorn,D.E. (2001)Hypoxia activates Akt and induces phosphorylation of GSK-3 in PC12cells. Cell Signal., 13, 23--27.

61.Chen,E.Y., Mazure,N.M., Cooper,J.A. and Giaccia,A.J. (2001) Hypoxiaactivates a platelet-derived growth factor receptor/phosphatidylinositol 3-kinase/Akt pathway that results in glycogen synthase kinase-3 inactiva-tion. Cancer Res., 61, 2429--2433.

62.Tang,T.T. and Lasky,L.A. (2003) The forkhead transcription factorFOXO4 induces the down-regulation of hypoxia-inducible factor 1 alphaby a von Hippel-Lindau protein-independent mechanism. J. Biol. Chem.,278, 30125--30135.

63.Arsham,A.M., Howell,J.J. and Simon,M.C. (2003) A novel hypoxia-inducible factor-independent hypoxic response regulating mammaliantarget of rapamycin and its targets. J. Biol. Chem., 278, 29655--29660.

64.Arsham,A.M., Plas,D.R., Thompson,C.B. and Simon,M.C. (2002)Phosphatidylinositol 3-kinase/Akt signaling is neither required for hypoxicstabilization of HIF-1 alpha nor sufficient for HIF-1-dependent target genetranscription. J. Biol. Chem., 277, 15162--15170.

65.Alvarez-Tejado,M., Alfranca,A., Aragones,J., Vara,A., Landazuri,M.O.and del Peso,L. (2002) Lack of evidence for the involvement of thephosphoinositide 3-kinase/Akt pathway in the activation of hypoxia-inducible factors by low oxygen tension. J. Biol. Chem., 277,13508--13517.

66.Rossig,L., Badorff,C., Holzmann,Y., Zeiher,A.M. and Dimmeler,S. (2002)Glycogen synthase kinase-3 couples AKT-dependent signaling to theregulation of p21Cip1 degradation. J. Biol. Chem., 277, 9684--9689.

67.Diehl,J.A., Cheng,M., Roussel,M.F. and Sherr,C.J. (1998) Glycogensynthase kinase-3beta regulates cyclin D1 proteolysis and subcellularlocalization. Genes Dev., 12, 3499--3511.

68.Scandurro,A.B., Weldon,C.W., Figueroa,Y.G., Alam,J. and Beckman,B.S.(2001) Gene microarray analysis reveals a novel hypoxia signaltransduction pathway in human hepatocellular carcinoma cells. Int. J.Oncol., 19, 129--135.

69.Kobayashi,Y., Pang,T., Iwamoto,T., Wakabayashi,S. and Shigekawa,M.(2000) Lithium activates mammalian Naþ/Hþ exchangers: isoformspecificity and inhibition by genistein. Pflugers Arch., 439, 455--462.

70.Patel,S., Yenush,L., Rodriguez,P.L., Serrano,R. and Blundell,T.L. (2002)Crystal structure of an enzyme displaying both inositol-polyphosphate-1-phosphatase and 30-phosphoadenosine-50-phosphate phosphatase activ-ities: a novel target of lithium therapy. J. Mol. Biol., 315, 677--685.

71.Collado,M., Medema,R.H., Garcia-Cao,I., Dubuisson,M.L., Barradas,M.,Glassford,J., Rivas,C., Burgering,B.M., Serrano,M. and Lam,E.W. (2000)Inhibition of the phosphoinositide 3-kinase pathway induces a senescence-like arrest mediated by p27Kip1. J. Biol. Chem., 275, 21960--21968.

72.Doble,B.W. and Woodgett,J.R. (2003) GSK-3: tricks of the trade for amulti-tasking kinase. J. Cell Sci., 116, 1175--1186.

73.Zundel,W., Schindler,C., Haas-Kogan,D. et al. (2000) Loss of PTENfacilitates HIF-1-mediated gene expression. Genes Dev., 14, 391--396.

74.Graeber,T.G., Osmanian,C., Jacks,T., Housman,D.E., Koch,C.J.,Lowe,S.W. and Giaccia,A.J. (1996) Hypoxia-mediated selection of cellswith diminished apoptotic potential in solid tumours [see comments].Nature, 379, 88--91.

Received May 10, 2004; revised August 11, 2004; accepted August 23, 2004

Cell cycle regulators in hypoxic cell cycle arrest

2335