Carcinogenesis vol.25 no.12 pp.2325--2335, 2004 doi:10.

1093/carcin/bgh274

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

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

Department of Pathology, Department of Oncology, Department of Medical Biochemistry and 4The University of Calgary and Calgary Laboratory Services, Calgary, Alberta, Canada, T2N 4N1
5 To whom correspondence should be addressed Email: demetric@ucalgary.ca 1 2 3

Flow cytometric analysis of fibroblasts, normal breast epithelial cells and breast or other cancer cell lines identified variation in the abilities of cell lines to undergo cell cycle arrest as a response to hypoxia. Human mammary epithelial cells (HMEC), normal fibroblasts (Hs68 and WI38), HeLa cervical carcinoma and HTB-30 breast carcinoma cells arrest in G1/S in response to severe hypoxia. Hep3B hepatocellular carcinoma cells did not exhibit orderly G1/S arrest in response to severe hypoxia. We found a general decrease in p16INK4a (p16) mRNA levels, with an associated decrease in p16 protein levels in both normal cells and in cancer cells, regardless of their cell cycle response to hypoxia. p27 protein levels did not correlate with the cell lines ability to enter a hypoxic G1/S arrest. Furthermore, cell lines that underwent G1/S arrest showed decreased expression of hypoxia inducible factor 1 (HIF-1a) and at least one member of INK4 or Sdi cell cycle kinase inhibitors families after 12--24 h of hypoxia. Conversely, Hep3B, which did not exhibit orderly hypoxiaassociated G1/S arrest, also did not show decreased HIF1a, INK4 or Sdi protein levels in hypoxia. Furthermore, Hep3B showed constitutive activating phosphorylation of Akt and inhibitory phosphorylation of GSK3b, which was the opposite pattern to that exhibited by the cell lines showing the G1/S arrest phenotype. Inhibition of GSK3b by lithium chloride treatment of HeLa cells converted the HIF-1a, p16 and p27 loss to levels unchanged by hypoxic exposure. Our results suggest that regulation of the cell cycle during hypoxia in either normal or cancer cells is not simply due to up-regulation of cell cycle kinase inhibitors. Furthermore, decreased protein expression of HIF-1a, p16 and p27 was associated with both a hypoxiainduced G1/S arrest phenotype and increased GSK3b activity. Introduction Tumour hypoxia is an important mitigating factor in the treatment of many forms of cancer. As a tumour mass grows, it experiences extended periods of low O2 levels due to the size of the mass and the lack of an appropriate blood supply (1).
Abbreviations: CDK, cyclin-dependent kinase; CDKI, cell cycle kinase inhibitors; HIF-1a, hypoxia inducible factor 1; HMEC, human mammary epithelial cells; p15, p15INK4a; p16, p16INK4a; p18, p18INK4a; p19, p19INK4a; p21, p21WAF1; p27, p27Kip1; p57, p57Kip2; pRb, retinoblastoma protein; RPA, Ribonuclease Protection assay; TS, tumour suppressor. Carcinogenesis vol.25 no.12 # Oxford University Press 2004; all rights reserved.

In order for a tumour to metastasize, it must survive this period long enough for vascularization to occur (2). Even after vascularization, the new blood vessels are often too poorly formed to supply adequate O2 perfusion, resulting in a continually hypoxic tumour environment. Hypoxic tumours are more aggressive and invasive than their normoxic counterparts and exhibit a greater tolerance to many chemo- and radiotherapies, probably due to the importance of the enhancing effects of O2 and/or blood perfusion in these treatments (3--5). The most characterized molecular response to hypoxia is the stabilization and activation of the transcription factor known as hypoxia inducible factor 1 (HIF-1) (6,7). HIF-1 is a heterodimer composed of the HIF-1a subunit, which is a member of the basic helix--loop--helix--PAS protein family, and the HIF1b subunit, which was later identified to be ARNT (8). Although both subunits of HIF-1 are constitutively expressed, HIF-1a is rapidly degraded in the presence of cellular O2 via the ubiquitin-mediated proteosome pathway (9,10). A shift from aerobic metabolism to glycolytic metabolism during hypoxia, in a major part mediated by HIF-1, is accomplished by the up-regulation of various metabolic genes, such as glucose transporters (11), aldolase, lactate dehydrogenase (12) and pyruvate kinase (13). HIF-1 also up-regulates vascular endothelial growth factor (14,15), presumably in order to induce the formation of new blood vessels, and erythropoietin (7), which accelerates red blood cell maturation and extends lifespan to increase blood O2 carrying capacity. Another major consequence of cellular hypoxia is a proliferation arrest of cells (16,17). Cells subject to severe hypoxia will arrest in G1 or early S phase. Cells in late S, G2 or M phase will finish cell division and arrest in G1 (16), unless the hypoxia is severe (18). Progression of cells through the cell cycle is controlled by a series of periodically expressed proteins known as the cyclins, and their associated partners, the cyclin-dependent kinases (CDK) (reviewed in ref. 19). The major proteins associated with the G1/S boundary are the cyclin D family (D1, D2 and D3), their respective CDKs, CDK4 and CDK6, and cyclin E, with its respective CDK, CDK2 (reviewed in ref. 20). The G1/S cyclin/CDKs function to control the phosphorylation of the retinoblastoma protein (pRb) (reviewed in ref. 21). p16INK4a (p16) was identified and found to bind to the catalytic component of the cyclin D/CDK4 complexes, thereby preventing their ability to phosphorylate pRb (22). This protein was also found to be inactivated in multiple tumour cell lines, suggesting that it was a tumour suppressor (TS) gene (23--25). p16 knockout mice were shown to spontaneously develop tumours early on in life (26), providing further evidence that it is a putative TS. Three other members of the INK4 gene family were identified based upon their sequence similarity to p16. p15INK4a (p15), p18INK4a (p18) and p19INK4a (p19) were all identified as members of the INK4 gene family and shared with p16 an ability to bind to CDK4/6 to cause a G1 arrest (27,28). p16 has 2325

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been implicated in the induction of cellular senescence, or withdrawal from normal cell cycle (29) and for contact inhibition of confluent cells (30). p15 is induced by and mediates cell cycle arrest in response to transforming growth factor beta (TGF-b) (27). p18 and p19 expression varies throughout the cell cycle, reaching maximum levels at the G1/S transition (28), and it has been suggested that they function in cell cycle arrest coupled to specific differentiation pathways (reviewed in ref. 31). Furthermore, data from transgenic mouse studies suggests that the INK4 gene family may have non-overlapping functions (32). Another group of CDK inhibitors, the Sdi family is represented by the prototype p21WAF1 (p21) (33--35), p27Kip1 (p27) (36) and p57Kip2 (p57) (37). These proteins have inhibitory effects on several CDKs, and p27 has been shown to be a likely TS gene. Studies examining the effects of hypoxia on cell cycle components have yielded conflicting results. Initial experiments have shown decreases in CDK4 and CDK2 kinase activities, and accumulation of pRb in its hypophosphorylated state (38--40). Analysis of CKIs in hypoxia has shown an increase in p27 protein levels (41), which has been suggested as the cause of the hypoxic G1/S arrest. Recently, this idea has come under fire (18), with evidence showing that p27 is not necessary for hypoxic arrest. Analysis of the INK4 gene family in hypoxia has been inconclusive, with p16 levels initially reported to be below detection levels (40), although a study using monkey CV1P cells showed hypoxia-induced up-regulation of p16 and association with cdk4 (42). Plainly, any of the INK4 or Sdi cell cycle kinase inhibitors (CDKI) may be good candidates to mediate G1 arrest in response to hypoxia. With both CDK4 and CDK2 kinase activities reduced in hypoxia, it may be that a coordinated up-regulation of both CDKI families, INK4 and Cip/Kip, is necessary for hypoxiainduced cell cycle arrest. p15 and p27 have been documented to cooperate in TGF-b-induced cellular arrest (43), supporting this idea of coordination. Also, there is evidence that induction of p16 causes a rearrangement of p27 and p21 from cyclinD/CDK4 to cyclinE/CDK2, effectively inhibiting both G1/S kinase complexes (44--47). Therefore, hypoxic cell cycle arrest may not be solely attributed to one G1/S kinase pathway, and may require the coordinated up-regulation of multiple CDKIs. In this study, we explore the effects of hypoxia on a number of different cell lines in order to determine the potential inducers of the hypoxiaassociated G1/S arrest. Materials and methods
Tissue culture WI-38, Hs68 (human fibroblasts), Hep3B (hepatocellular carcinoma), HeLa (cervical carcinoma) and HTB-30 (also known as breast carcinoma, SK-BR-3) cell lines (ATCC) were seeded on 175 cm2 dishes at 105 cells/ml in 20 ml of recommended growth media (Invitrogen). Primary human mammary epithelial cells (HMECs) were seeded and grown according to suppliers recommended conditions (Clonetics). All cells were incubated at 5% CO2/37 C for 48 h to 50--60% confluency prior to exposure to hypoxic conditions. Hypoxia was established by incubating cells in the GasPak anaerobic incubator (BD Bioscience), which reduces O2 levels while maintaining CO2 levels at ~5%. O2 levels were monitored by a methylene blue indicator strip, which bleaches white at 0.1--0.5% O2 levels, following which timing of hypoxia was measured. Control cells were incubated alongside hypoxic samples under a normal tissue culture gas mixture (20% O2/5% CO2). For treatment with lithium chloride (LiCl, Sigma), cells were prepared as above, with the addition of 50 mM (final) LiCl to the culture media immediately preceding hypoxia.

Cell cycle analysis For ploidy analysis, cells were prepared as follows, with all steps carried out at 4 C unless noted. Media was removed from the plates and the cells were rinsed with 10 ml of ice-cold phosphate-buffered saline (PBS). The cells were lifted by incubation with 2.5 ml trypsin and neutralized by the addition of 5 ml cell culture media with FBS (Invitrogen). 1 106 cells were counted, spun down at 800 g and re-suspended in 1 ml of FBS-free media containing 1 mM EDTA to prevent clumping. An equal volume of 100% ethanol was added drop wise and the cells were stored at 4 C for 24 h. After storage, the cells were pelleted at 800 g, aspirated and re-suspended in 1 mg/ml RNase A (Invitrogen) in PBS and incubated for 30 min at room temperature. 0.5 ml of 50 mg/ml propidium iodide (Sigma-Aldrich) was added after RNase digestion and incubated for an additional 30 min in the dark. Cells were then analysed for DNA content using a Becton-Dickinson FACScan to determine cell cycle phase. RNA extraction and preparation Total cellular RNA was isolated using TRIzol LS (Invitrogen) according to manufacturers protocol, which was modified as follows: RNA was precipitated using 0.5 vol of isopropanol and 0.5 vol of 1.2 M NaCl/0.8 M sodium citrate, to prevent proteoglycan contamination. RNA was re-suspended in DEPC-treated H2O, quantified, and used immediately or precipitated and then stored in 100% ethanol at 80 C for later use. Northern blot analysis and ribonuclease protection assay For northern blot analysis, 60 mg of total RNA was loaded onto a 0.8% formaldehyde agarose gel and transferred overnight to nitrocellulose using standard procedures (48). Radiolabelled probe was synthesized by nick translation with 32P-CTP from full-length p16 cDNA cloned into pBluescript. Membranes were incubated overnight with 2 106 c.p.m. of radiolabelled probe and exposed to film for 2--7 days. Transcriptional analysis was performed using the Ribonuclease Protection assay (RPA). Total cellular RNA (10 ug) was analysed using the Human cell cycle regulator probe set (BD BioSciences) following the manufacturers protocol. Gels were exposed to autoradiography film overnight on a Kodak intensifying screen, or for up to 5 days for weaker signals. Western blot analysis For analysis of cell cycle proteins, hypoxic and normoxic tissue culture cells were prepared as described above, placed on ice and rinsed with 10 ml of icecold PBS. The cells were lysed on the plate by the addition of 1 ml of NP-40 lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 1% NP-40, 10 uM sodium vanadate, 10 uM PMSF, 10 ug/ml aprotinin, 5 ug/ml leupeptin). Protein concentration was determined using the Bichronate assay (Pierce). Seventy micrograms of cell lysate was loaded onto an SDS--PAGE gel and transferred to nitrocellulose (for detection of HIF-1) or 0.1 m PVDF (Millipore) (for detection of proteins 530 kDa) by blotting overnight at 30 V/60 mA. Membranes were blocked for 1 h at room temp in 10% (w/v) powdered non-fat milk/Tris-buffered saline with 0.1% (v/v) Tween-20 (Sigma). Western blotting was carried out overnight at 4 C using the following antibodies: HIF-1a (# 610959; 1:250), p27 (# 25020; 1:2000) (Transduction Labs), Pan actin (Ab-5; 1:2000), p16 (Ab-1; 1:250), p15 (Ab-6; 1:200), p18 (Ab-3; 1:250) (Neomarkers), p19 (M-167; 1:500), p21 (F-5; 1:1000) and PARP (F-2; 1:2000) (Santa Cruz). Detection of protein phosphorylation by western blotting was performed using the conditions listed above, with the following modifications: after transfer of proteins to nitrocellulose, membranes were incubated in 5% (w/v) bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline with 0.1% (v/v) Tween-20 and 0.1% (v/v) NP-40. Western blotting was performed overnight at 4 C using the following antibodies: GSK3b (#610201; 1/2000) (BD Transduction Labs), Phospho-GSK3a/b Ser 21/9 (#9331S; 1/2000), Phospho-Akt Ser 473 (#9271S; 1/2000), Akt (#9272; 1/2000) (Cell Signaling Technologies).

Results Differing cell types exhibit differing arrest profiles in response to hypoxia We determined the cell cycle phenotype of a variety of cell lines responding to hypoxia. As shown in Figure 1, HTB-30, HeLa and HMEC exhibited hypoxia-associated G1/S arrest occurring at 6--12 h after initial exposure to hypoxic conditions. In comparison, Hep3B, a hepatocellular carcinoma cell line, lacked an observable G1/S arrest (defined as an increase in the G0/G1 peak with a decrease in the S peak). All cell lines, however, showed a lack of proliferation at 24 h in comparison

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Cell cycle regulators in hypoxic cell cycle arrest

Fig. 1. Cell cycle analysis of selected cell lines in hypoxia. Cells were incubated in a normoxic (20% O2; UT) or hypoxic (50.5% O2; T) atmosphere for 2 or 24 h, fixed in ethanol, stained with propidium iodide and analysed for DNA content to determine G1 and S phase cell populations. (A) Graphical representation of flow cytometric analysis of the HTB-30 cell line shows an increase in G0/G1 and a decrease in S phase, indicating cell cycle arrest in response to hypoxia. (B) The HeLa cell line also shows hypoxia-induced cell cycle arrest. (C) Hep3B cells do not exhibit a significant increase in G0/G1 or decrease in S phase, indicating a lack of cell cycle arrest, even after 24 h of hypoxia. (D) Flow cytometry histograms of primary HMEC cells show a gradual decrease in S phase and M phase starting at ~6 to 12 h after initial exposure to low O2, with a parallel increase in percentage of cells in G0/G1. (E) Primary WI38 fibroblasts exhibit a decrease in cells transiting through S phase.

with untreated controls (not shown), indicating that the severe hypoxia did cause proliferation arrest (probably due to insufficient cellular ATP for cell division), but only Hep3B failed to undergo block at the G1/S boundary. p16 RNA and protein levels are reduced by hypoxia As p16 was a potential mediator of G1/S arrest seen in the normal response to hypoxia, we evaluated its expression

in the treated and untreated cell lines. While p16 expression is usually associated with cell cycle arrest, RPA analysis of the diploid fibroblast cell line WI-38 revealed a reduction in p16 mRNA levels after 12 and 24 h exposure to hypoxic growth conditions (Figure 2A). This reduction in p16 mRNA was confirmed by northern blot analysis (Figure 2B). Hep3B cells did not show a reduction in p16 mRNA levels (data not shown). As well, HeLa p16 transcript levels 2327

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Fig. 2. p16 mRNA levels are reduced in diploid fibroblasts WI-38. p16 mRNA levels were analysed by RPA or northern blot in cell lines incubated in a normoxic (20% O2; UT) or hypoxic (50.5% O2; T) atmosphere for the times indicated. Levels of L32 transcript were used as a loading control for RPA and 28S rRNA was used in northern blot analysis. Identically treated whole cell lysates were prepared for immunoblot analysis. (A) WI-38 fibroblast cells, which undergo cell cycle arrest, reveal a reduction in p16 mRNA levels starting at 12 h (12H T) after exposure to hypoxia, as measured by RPA. (B) Northern blots of p16 mRNA levels confirm p16 mRNA reductions in WI-38 fibroblasts treated with hypoxia for 24 h (24H T). (C) p16 mRNA levels correlate with a reduction in p16 protein levels in WI-38 fibroblasts. (D) HMEC and HTB-30 cell lines exhibit a similar reduction in p16 mRNA levels, as measured by RPA, while L32 ribosomal protein mRNA loading standards are unchanged.

showed no reliable changes in response to hypoxia (data not shown). Both WI-38 and Hs68, another normal diploid fibroblast cell line, showed a reduction in p16 protein levels after 24 h in a low oxygen environment (Figure 2C), in agreement with the RPA data, as did both HMEC and HTB-30 cells (Figure 2D). In HMEC and HeLa cell lysates, p16 protein levels again were reduced after 12--24 h in hypoxia (Figure 3C). Hep3B, which did not appear to undergo G1/S arrest in hypoxia, did not show any variation in p16 protein level. Cip/Kip and INK4 proteins are reduced in hypoxia We chose to evaluate the expression of Cip/Kip and the p16related proteins to determine if the relative levels of the remaining CDKIs were modulated in response to hypoxia. As shown in Figure 3, all cell lines used in this study exhibited down-regulation of at least one of the G1/S CDKIs in response 2328

to hypoxia. HMEC, HTB-30 and HeLa showed loss of p16 protein, while Hep3B, the cell line most resistant to hypoxiainduced cell cycle arrest, showed no loss of p16 protein after 24 h of treatment. There was also loss of p19 protein expression, which occurred in Hep3B, the breast cancer cell line HTB-30, and HeLa cells (p19 was undetectable in HMEC cells). Hep3B and HMEC exhibited a loss of p21, again after 12--24-h exposure to hypoxia, while p21 levels were undetectable in both HTB-30 and HeLa cells. Interestingly, HeLa was the only cell line to show a drastic reduction in all detectable CDKIs. In all cells, there was at least one instance of loss of a CDKI of both the INK4 or Cip/Kip families. The loss of CDKI expression does not appear to be due to a general reduction in protein translation or stability, since levels of actin are stable, and usually other related but less abundant proteins, such as p15 or p18, were not affected. Analysis of p57Kip2 and Cyclin D1 also showed a decreased level of these proteins associated

Cell cycle regulators in hypoxic cell cycle arrest

Fig. 3. Western blot analysis of cell cycle regulators reveals loss of at least one CDK inhibitor. 75 ug/lane of protein extracts were loaded onto 8% (for HIF-1 and actin) or 12% acrylamide SDS--PAGE gels and transferred to the appropriate membrane. Pan actin blots were performed for all cell lines to normalize proteinloading amounts. (A) HTB-30 cells lines exhibited a loss of p16 protein, similar to WI-38 normal fibroblasts (previous figure) and also exhibited loss of another INK4 family member, p19. HTB-30 also showed an induction of p27 protein levels late in the hypoxic response. (B) Hep3B exhibited an induction of p27 late in the hypoxic response, but also showed loss of p19 and p21, similar to the observed reduction in the HTB-30 cell line. Hep3B p16 levels, however, were invariant in hypoxia. (C) HeLa cells showed reductions in all CDKIs measured, including p27, although they still underwent hypoxia-induced cell cycle arrest. (D) Primary breast epithelial cells (HMEC) showed dramatic reductions in p21 levels and p16, starting ~12 h after initial exposure to hypoxia. Other CDKI levels (p15, p18, p19) were below detection thresholds in this cell strain.

with hypoxia, but with no correlation to cell cycle arrest phenotype (data not shown). Although all cell lines showed a reduction of at least one CDKI protein level, neither the number of proteins reduced, nor the identity of the CDKI lost, correlated with the ability of the cell line to enter a hypoxia-induced G1/S arrest, although Hep3B, which is resistant to hypoxic cell cycle arrest was the only cell line to not show a reduction in either p27 or p16 levels. Of course, the data only indicate average results for the cell lines, where cells could show either a generalized change in protein level, or more drastic changes in a subclone of cells, with less change in another subclone. p27 is induced by hypoxia, but induction does not correlate with G1/S arrest It has been reported previously that hypoxia-induced G1/S arrest was due to the induction of p27 (41), but other research into p270 s involvement has been contradictory. Measurement of p27 mRNA levels in our cell lines by RPA indicated that only HTB-30 experienced a hypoxia-induced increase in p27 mRNA while the other cell lines, HMEC, HeLa and Hep3B, did not show hypoxic modulation of p27 transcripts (data not shown). Protein levels of p27 were increased in both HTB-30

and Hep3B (Figure 3A and B), but p27 was unchanged in HMEC (Figure 3D) and reduced in HeLa cells (Figure 3C). The most important observation was that induction of p27 could not be correlated with the ability of the cell line to enter a hypoxia-induced G1/S arrest. Both HTB-30 and Hep3B showed increased amounts of p27 protein, but only HTB-30 exhibited a G1/S arrest. In addition, HMECs, which could arrest during exposure to hypoxic conditions, did not show any change in p27 levels and HeLa cells actually experienced a reduction in p27 while still arresting at the G1/S boundary. Western blotting for pan-actin protein levels confirmed that overall cellular protein levels were constant. Considering the above data, we have determined that p27 transcript or protein levels are unlikely to determine the hypoxia-induced G1/S arrest of our cell lines. Lack of apoptotic induction in all cell lines It has been noted that induction of apoptosis in neuronal cells is preceded by the loss of CDKIs, specifically p16 and p21 (49). Considering this evidence, we decided to determine if the loss of the CDKIs we have observed in hypoxia was due to activation of an apoptotic pathway and/or cell loss. Flow cytometric analysis did not reveal significant numbers of 2329

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Fig. 4. Western blots indicate a lack of PARP cleavage during hypoxia time course. Cell lines were incubated in a normoxic (20% O2; UT) or hypoxic (50.5% O2; T) atmosphere for the times indicated and whole cell lysates were prepared for immunoblot analysis. Pan actin protein levels were used as a protein loading control. Analysis of PARP in HeLa, Hep3B, HTB-30 and HMEC cells showed that PARP was maintained at its full-length size of ~119 kDa, indicating a lack of caspase activity and subsequent apoptosis. No faster migrating versions of PARP were detected.

cells with sub-G1 content, indicating that significant apoptosis was unlikely to have occurred (e.g. Figure 1D). We also decided to evaluate PARP cleavage as an indicator of apoptosis. PARP is cleaved from its full-length size of ~119 kDa, via apoptotically activated caspases, to a truncated form of ~85 kDa. As shown in Figure 4, there is no detectable cleavage of PARP at any time point during exposure to hypoxia, in any of the cell lines. Therefore, flow cytometry and PARP cleavage assays do not indicate that significant apoptosis is occurring in any of the four cell lines, even after 24 h of severe hypoxia. The loss of CDKI proteins, therefore, does not appear to be associated with markers of apoptosis. Akt/GSK3b signalling is correlated with the ability to enter G1/S arrest in hypoxia Recent studies have indicated that Akt signalling through GSK3b is involved in decreasing HIF-1a expression during hypoxia (50). Using two epithelial cell lines from our studies, HeLa cells, which arrested in response to hypoxia, and Hep3B cells, which did not exhibit a G1/S arrest in hypoxia, we examined if the loss of HIF-1 and CDKI expression was associated with Akt/GSK3b status. As shown in Figure 5, HeLa cells show an initial increase in Akt phosphorylation between 2 and 6 h in hypoxia, but at 12--24 h there is a drastic decrease in both phospho-Akt and total Akt levels in hypoxia. This down-regulation of Akt levels is correlated with the loss of HIF-1 and CDKI expression and cell cycle arrest. In addition, as the level of phospho-Akt decreases, there is a reciprocal loss of the inhibitory phosphorylation of serine 9 on GSK3b, indicating a decrease in Akt activity and a probable increase in GSK3b activity. In contrast, Hep3B cells 2330

Fig. 5. Phospho-specific western blots reveal differential AKT/GSK3b activation in cells with and without an intact hypoxia-induced G1/S arrest. Whole cell lysates were prepared from cells incubated in normoxia (20% O2; UT) or hypoxic (50.5% O2; T) atmosphere for 2, 6, 12 and 24 h. Western blotting of Akt and GSK3b showed that in hypoxia-treated HeLa cells (A), Akt exhibited activating phosphorylation, but phosphoprotein and total protein levels were lost between 6 and 12 h post-hypoxia. Concomitantly, inhibitory phosphorylation on GSK3b was lost. (B) Hep3B cells did not exhibit modulation of Akt and GSK3b phosphorylation with hypoxia treatment.

Cell cycle regulators in hypoxic cell cycle arrest

Fig. 6. Inhibition of GSK3b activity prevents degradation of HIF-1 and CDKI proteins in hypoxia. HeLa cells were pre-treated with 50 mM LiCl (final), added directly to the tissue culture media, immediately prior to incubation in a normoxic (20% O2; UT) or hypoxic (50.5% O2; T) atmosphere for the times indicated (2, 6, 12 and 24 h). Whole cell lysates were prepared and protein levels were determined by immunoblotting. Pan actin levels were used to control for total protein loading. (A) Normoxic HeLa cells were treated in the presence or absence of 50 mM LiCl for 12 or 24 h. Cell extracts were electrophoresed, blotted and detected with antibodies to HIF-1a, p16, p27 and actin. (B) Hypoxic (T) or normoxic (UT) HeLa cells were treated in the presence of 50 mM LiCl for 12 or 24 h. Cell extracts were electrophoresed, blotted and detected with antibodies to HIF-1a, p16, p27 and actin. Over time, no decrease in HIF-1a, p16 or p27 was noted in the hypoxic cells treated with LiCl, in sharp contrast to their expression when LiCl was not present (Figure 3).

maintained high levels of phospho-Akt, irrespective of hypoxia, and maintained GSK3b inhibition, as indicated by high levels of inhibitory serine 9 phosphorylation. Associated with a constitutively phosphorylated Akt is maintenance of high levels of HIF-1 expression and a lack of a hypoxia induced G1/S arrest. To evaluate the effect of GSK3b on the CDKI proteins levels, HeLa and Hep3B were treated with the GSK3b inhibitor LiCl and the results are shown in Figure 6. In the presence of LiCl, HeLa levels of HIF-1a and p16 do not change appreciably with the 12- and 24-h hypoxia-treatment time points, in sharp contrast to their expression in the absence of LiCl (Figure 3), although p27 levels were slightly reduced with 50 mM LiCl treatment in non-hypoxic cells, as compared with untreated cells. Hep3B showed no effect of LiCl treatment on the levels of these three proteins in hypoxia. Essentially, LiCl treatment converted the hypoxia-associated HeLa expression of HIF-1a, p16 and p27 proteins to a similar pattern shown by Hep3B, reversing the low levels noted in nonlithium-treated HeLa cells. This indicates that GSK3b activity may be responsible for the observed hypoxic reduction of these proteins in HeLa and, perhaps, the other cell lines analysed. Treatment of HeLa cells with hypoxia in the presence or absence of 50 mM LiCl did not prevent a reduction in cells transiting the S phase and arresting in G1 (Figure 6C). Although there was a decrease in G1 cell arrest (as measured by percent cells in G1 associated with LiCl at the 24-h hypoxia time point) compared with untreated cells, there was actually an increase as compared with normoxic cells treated for 24 h

with LiCl. Interestingly, HeLa cells pre-treated with LiCl, but incubated in normoxia, showed an increase in the number of cells entering the S phase (~2-fold) over untreated normoxic HeLa cells, perhaps implying an overall stimulatory effect on the cell cycle from LiCl. Discussion Hypoxia has been long known to initiate a profound, reversible cell cycle arrest in many different cell types and tumour cell lines (16,51). Cells in late S, G2 and M phase will finish the cycle and arrest in G1, unless the hypoxia is severe (18). Cells in G1 and early S phase will remain there (16). Arresting in early S phase is easy to rationalize as due to a dependency on cell energy levels for the completion of DNA synthesis. Finishing the cell cycle and arresting at G1 is typical of cells with down-regulation of pRb phosphorylation. With initial research identifying reductions in CDK2 and CDK4 activity, along with hypophosphorylation of the pRb, it was therefore believed that the hypoxia-induced cell cycle arrest was probably mediated by one of the specific inhibitors of the G1 kinases from the INK4 or Cip/Kip families, as is usually the case (reviewed in ref. 52). Sequential modification of Rb by two separate cyclin/ cdk complexes is necessary for cell cycle progression, and coordinated up-regulation of p27 and p15 is essential for TGFb-mediated cell cycle arrest (43,53). Accordingly, simultaneous measurement of expression of both the INK4 and Cip/ Kip gene families may be necessary in order to elucidate the mechanism of hypoxia-induced G1/S arrest. 2331

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Response to hypoxia is cell line dependent While generally confirming the hypoxia-induced cell cycle arrest phenomenon, the panel of cell lines we analysed exhibited variability in their ability to undergo arrest. Normal diploid cell strains, such HMECs, exhibited a coordinated arrest, with a gradual decrease in S phase and an increase in G0/G1 phase. HTB-30, a breast carcinoma cell line, and HeLa a cervical carcinoma cell line also exhibited similar cell cycle findings and followed essentially the same inhibition kinetics as normal cells, showing peak arrest at ~12 h after exposure to low atmospheric O2. Conversely, Hep3B cells, a line that is commonly used in hypoxia studies, did not undergo a coordinated, hypoxia-associated cell cycle arrest even after 24 h in hypoxic culture. While the levels of G1 increase/S phase decrease are not pronounced, it is likely that our severe hypoxia conditions precluded the normal completion of the cell cycle in many of the cells, including Hep3B (18); however, the ability to undergo regulated G1/S blockade was the important observation rather than the magnitude of blockade. We expect that titration of hypoxia using more elaborate equipment may identify a higher O2 level correlating with an increase in the magnitude of the G1/S blockade by providing enough oxygen for cells to complete the cell cycle to the G1/S arrest point. One could certainly argue that there are many differences between these cell lines. Both HTB-30 and Hep3B have mutated p53 (54,55), while HeLa p53 is down-regulated by HPV E6. Hep3B lacks functional Rb (55), while HeLa pRb function is down-regulated by HPV E7. HTB-30 has amplified the HER2 gene. There does not appear to be a clear correlation with the cell cycle arrest phenotype of these cells and p53, pRb or HER2 status. p16 expression is reduced under hypoxic conditions p160 s importance as a TS has been widely established in a number of tumour types (23--25) but previous studies of the effects of hypoxia on p16 have yielded inconclusive results (40,42). We evaluated p16 levels under hypoxia in the normal diploid cells, WI-38 and HMECs, both of which exhibit a normal G1/S arrest under hypoxia (Figure 1D and E). Ribonuclease protection assay and northern blot analysis of WI-38 cells indicated a reduction in p16 mRNA, which was concordant with a decrease in p16 protein levels. The same phenomenon was observed in HMECs and HTB-30 cells at both the mRNA and protein levels and at the protein level in HeLa cells. Hep3B cells, which did not show a hypoxia-induced arrest, did not have an appreciable reduction in p16 mRNA or protein levels. It is unclear why p16 transcript and/or protein levels are reduced in hypoxia. A recent paper reported that cerebral ischaemia causes a reduction in INK4 gene family expression as a precursor to apoptosis (49). Although our flow cytometry analysis did not show a population of cells with a sub-G1 DNA content at 24 h, or cleavage of PARP, further studies, using other methods of analysis, may reveal if there is a correlation between initiation of apoptosis and the loss of p16 expression. p16 is constitutively regulated in cycling cells (56,57) and is thought to be a relatively long lived protein (48 h) (58) although we cannot rule out that a decrease in transcript level may be responsible for low levels of the protein. HeLa, which did not appear to show a reproducible decrease in p16 transcript with hypoxia, did show reduced protein levels, implying 2332

there is probably a change in the p16 protein lifespan in hypoxia. p27 is induced by hypoxia, but does not appear to mediate hypoxia-induced G1/S arrest Recently, there have been a number of conflicting papers concerning the importance of p27 in hypoxia. It has been reported that p27 is induced in mouse embryonic fibroblasts at both the mRNA and protein levels (41). The induction of p27 was not HIF-1 dependent, as determined by promoter analysis. However, MEFs derived from p27/p21 double knockout mice are still able to initiate a G1/S arrest, indicating that neither p27 nor p21 is essential for hypoxia-induced arrest (18). In our study, HTB-30 alone experienced an increase in p27 transcript levels (data not shown). p27 protein levels were elevated in HTB-30 and Hep3B cells, but this up-regulation did not correlate with induction of a G1/S arrest, as Hep3B cells are resistant to hypoxia-induced arrest. HeLa cells exhibit a reduction in p27 protein levels while retaining the ability to induce a cell cycle arrest. HMECs, a primary, non-immortalized cell, exhibit no change in either p27 mRNA or protein levels while undergoing arrest. Considering this data, we conclude that an increased level of p27, although induced in some cell lines by hypoxia, is not likely to be the main effector of the observed G1/S arrest. Loss of CDKI expression in hypoxia does not correlate with hypoxia-induced G1/S arrest or apoptosis Much of the recent research into hypoxia-induced arrest has focused solely upon one or two of the CDKI family members. Our study, evaluating all CDKI gene products, showed that there is a rapid (~12 h) loss of one or more CDKIs from both of the Cip/Kip and INK4 gene families after exposure to low (50.5%) oxygen. The most commonly lost CDKIs were p21 and p19, although the pattern of CDKI loss could not be correlated to either the cell line tissue origin, or the cells ability to undergo hypoxic cell cycle arrest. Actin blotting of hypoxic cell lysates reveals that general protein levels are stable in hypoxia. It has been suggested that CDKIs may confer a survival advantage to ischaemic neurons, and that loss of their expression precedes induction of apoptosis (49). Lack of sub-G1 DNA content in cytometric measurements and the presence of intact PARP even after 24 h of exposure to extreme hypoxia indicate that these cells are not undergoing typical apoptosis. Recent research into hypoxia-induced signalling effects may give clues as to the mechanism of the phenomena that we have described. Hypoxia may be associated with up-regulation of the PI3K/Akt signalling pathway (59--61), which results in up-regulation of HIF-1 via a variety of mechanisms (50,62,63), although other investigators have reported that PI3K/Akt signalling does not seem to be involved in the hypoxic response (64,65). It was found recently that short-term hypoxia (5 h) was associated with an increase in Akt activity and increased protein levels of HIF-1 (50). However, long-term hypoxia (up to 16 h) was associated with inhibition of Akt function, and activation of GSK3b. GSK3b activation was associated with a reduction in HIF-1 protein levels. GSK3b activity was also associated with loss of p21 (66) and cyclin D1 (67), with p21 being a direct target for GSK3b (66). Furthermore, GSK3b expression is increased in an expression microarray

Cell cycle regulators in hypoxic cell cycle arrest

analysis of hypoxia (68). The effect of GSK3b on INK4 family regulation has not yet been reported. In our study, we usually observed an initial activation of Akt, as measured by protein phosphorylation, followed by a loss of Akt phosphorylation and inhibitory GSK3b phosphorylation late (412 h) in the hypoxic response. Correlated with this was a reduction in HIF-1 protein levels even in the presence of hypoxic conditions and a reduction in CDKI expression. Interestingly, the loss of Akt/GSK3b phosphorylation and HIF-1 expression levels was correlated with the ability of the cell line to enter a hypoxic G1/S arrest. HeLa cells, which were permissive to hypoxia-induced G1/S arrest, exhibited modulation of the Akt/GSK3b signalling pathway and loss of HIF-1 and CDKI expression. Hep3B, which appeared to have constitutively activated Akt and consequently continually inhibited GSK3b, was resistant to hypoxia-induced G1/S arrest and had stable levels of HIF-1 and CDKI proteins throughout the hypoxia time course. When GSK3b activity in HeLa cells was inhibited with LiCl, there was no reduction in HIF-1 or CDKI protein levels resulting in a pattern similar to that of hypoxic cell cycle arrest. Although incubation with LiCl prevented the observed reduction in HIF-1 and CDKI protein levels, LiCl did not prevent hypoxic cell cycle arrest. It appears that LiCl may have a stimulatory effect on the HeLa cell cycle in general (compare LiCl-treated versus -untreated normoxic cells), thus while there is a cell cycle arrest observed between normoxic versus hypoxic HeLa cells treated with LiCl, this effect is much less pronounced in hypoxic cells treated with LiCl than in untreated hypoxic cells, at the 24-h time point. The complexity of these findings makes it difficult to interpret the real effect of LiCl on the cell cycle in HeLa. This result is of no great surprise, as LiCl is well known to have pleotropic effects on a variety of cell pathways in addition to serving as an inhibitor of GSK3b (69,70), and these effects could affect the cell cycle in many unpredictable ways. It is intriguing to postulate that the same mechanism by which GSK3b activity is associated with HIF-1, cyclin D1 and p21 degradation may also mediate INK4 loss. In our cell lines, which exhibited hypoxia-induced cell cycle arrest, the reduction in CDKI expression was mirrored by a reduction in HIF-1 protein levels. Interestingly, inhibition of PI3K, an upstream activator of Akt, with the chemical inhibitor LY294002 resulted in the down-regulation of p15, p16, p19 and p21 protein levels but an increase in p27 protein levels (71). As well, we have noticed that a consensus GSK3b phosphorylation site (S/T)XXXP(S/T) is present in p16 (Ser12), and another potential site (S/T)XXXS is found in both p15 (Ser14) and p19 (Ser76). A short motif (TPLE) identical to that phosphorylated by GSK3b on p21 (66) is also present within p19 (Thr141) and five residues upstream of it is an aspartic acid residue that could serve as a surrogate for substrate priming by prior phosphorylation (72), exactly as seems to occur with p21. Other potential GSK3b sites can be found in both p27 (Thr187, Ser110, Thr142, Ser161) and p57 (Thr310, Ser32, Ser84, Ser258). Interestingly, the Thr187 GSK3b consensus site of p27 is identical to the Thr310 site in p57. The significance of any of these is unknown, but the potential for the CDKIs to be direct targets of GSK3b phosphorylation and degradation is worthy of study. After evaluating our data, we propose that the observed loss of CDKIs in hypoxia is downstream and due to the inhibition of PI3K/Akt signalling in these cell lines (Figure 7). The loss

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

of HIF-1 expression in our cells, we believe, is also due to the hypoxic inactivation of Akt as noted above and subsequent up-regulation of GSK3b. How down-regulation of Akt is mediated in hypoxia is unknown, but upstream regulators of Akt, such as PTEN (73), may be involved. The association of resistance to hypoxia-induced cell cycle arrest and resistance to hypoxia-associated degradation of p21 and INK4 proteins may be associated with abnormally increased PI3K/Akt activity. It is tempting to speculate that such hypoxia-resistance phenotypes may be selected in cancer cells with dysregulated PTEN function, in a manner similar to that proposed for p53 (74). Studies into the molecular events surrounding hypoxiainduced cell cycle arrest have been conflicting. Many reports have utilized tumorigenic cell lines in which the major cell cycle pathways are altered due to inactivating mutations in key cell cycle regulators, such as p53, pRb and p16, leading to inconclusive results (40). In addition, many of these studies use animal-derived cell lines, which are capable of undergoing spontaneous immortalization. Our study focused on the similarities and differences between a normal diploid cell strain (HMEC) and cancerous cell lines (HeLa, HTB-30, Hep3B) in their cell cycle responses to hypoxia, as well as their expression of cell cycle kinase inhibitors. While we still have not yet identified the precise mediators of the hypoxia-induced cell 2333

A.H.Box and D.J.Demetrick

cycle arrest phenotype, we have accumulated evidence for the involvement of the PI3K/Akt/GSK3b pathway in regulating this response. Elucidation of the precise mediators will involve study of the expression of other cell cycle regulatory proteins such as CDKs, cyclins or phosphatases and/or post-translational modifications of the CDKIs or other cell cycle proteins. Acknowledgements
We would also like to thank undergraduate students Jason Morin, Eli Akbari and Carol Yuen for their valuable help with the western blots, and Philip Berardi, Chris Nichols and Alan Box for helpful discussions and/or review of the manuscript. The authors wish to acknowledge the operating support of the Cancer Research Society for making this work possible. Salary support for D.J.D. was provided by Calgary Laboratory Services. A.H.B. is supported by a stipend from the Alberta Cancer Board Strategic Training Program in Translational Cancer Research.

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