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Cell Biology International (2009) 33, 49–56 (Printed in Great Britain)
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Dual role of HIF-1α in delivering a survival or death signal in hypoxia exposed human K562 erythroleukemia cells
Viviana di Giacomoab, Monica Rapinod, Sebastiano Misciaa, Camillo Di Giulioc and Amelia Cataldiab*
aDipartimento di Biomorfologia, Università G. d' Annunzio, Chieti-Pescara, Italy
bCattedra di Anatomia Umana, Facoltà di Farmacia, Università G. d' Annunzio, Chieti-Pescara, Italy
cDipartimento di Scienze Mediche di Base ed Applicate, Università G. d' Annunzio, Chieti-Pescara, Italy
dIstituto di Genetica Molecolare del CNR, Unità di Chieti, Italy

Abstract

Hypoxia (reduced oxygen tension) is a critical stimulus which switches on a cell rapid response, determining damage and death in some cells, and adaptation and survival in others. Here we report that K562 erythroleukemia cells exposed to hypoxia, proliferated more slowly and the percentage of dead cells increased after 22h. In parallel HIF (Hypoxia Inducible Factor)-1α and Bax level increased, as well as the PKC (Protein Kinase C) δ/Erk (Extracellular Signal Regulated Kinase) pathways being activated. The low level of ROS after 5h of hypoxia did not modify cell cycle progression or affect cell death, whereas HIF-1α/CBP (CREB Binding Protein) co-immunoprecipitation and MAPK (Mitogen Activated Protein Kinase)/CREB (c-AMP Response Element Binding) protein signalling pathway activation determined the adaptive survival response. We suggest a dual role for HIF-1α in providing a survival or death signal, based on hypoxia duration, and consider the nuclear transcription factor, CREB, to be a possible target for hypoxic therapy against leukemia disease.

Keywords: HIF-1α, CREB, p38MAPK, Hypoxia, K562 erythroleukemia cells.

*Corresponding author at: Dipartimento di Biomorfologia, Università G. d' Annunzio, Via dei Vestini 6, 66100 Chieti, Italy. Tel.: +39 0871 3554508; fax: +39 0871 574361.

1 Introduction

Mammalian cells require O2 to perform their essential and physiological functions [Lahiri et al., 2006]. To counteract possible changes in O2 levels, hypoxia or hyperoxia, the organism has developed a series of mechanisms both at subcellular and systemic levels (glycolysis, erythropoiesis, and angiogenesis increase in respiratory volume) [Iyer et al., 1998]. Moreover, under O2 level modifications pathological situations such as solid tumour growth, diabetic retinopathy and rheumatoid arthritis also occur [Ikeda, 2005]. Two main biochemical events are involved in the molecular mechanisms at the basis of hypoxia: Reactive Oxygen Species (ROS) production, which implies the activation of signalling pathways regulating in turn cell proliferation, differentiation, death and adaptive response; and HIF (Hypoxia Inducible Factor) family protein expression [Harris, 2002]. The low oxygen tension determines mitochondrial production of ROS which must be detoxified [Finkel and Holbrook, 2000]. ROS induce serious oxidative damage to cells and tissues [Zhang et al., 2005] and act as important mediators for intracellular signalling in a variety of models leading to change in gene expression. HIF-1, composed of 2 subunits, HIF-1α and HIF-1β, is identified as a transcriptional factor which, by binding to HRE (Hypoxia Responsible Element) gene regulates the cellular response to hypoxia [Semenza, 1999, 2000, 2002, 2003]. Under normoxia the intracellular level of HIF-1α is kept low by rapid ubiquitylation and subsequent proteasomal degradation, which depends on the hydroxylation of proline residues by PHD2, while under hypoxia the intracellular level and the transcriptional activity of HIF-1α increase as result of suppressed PHD2 and HIF activities [Berra et al., 2003].

Although hypoxic therapy is applied to solid tumour (Brown, 2007) and in melanoma derived cells treatment [Winnard et al., 2008], leukemia has been scarcely investigated [Desplat et al., 2002; Giuntoli et al., 2007]. We have investigated the molecular events related to human K562 erythroleukemia cell response to different times (5 and 22h) of hypoxia exposure, followed by 1 and 24h of reoxygenation, which is a more potent enhancer of ROS production and cell death than hypoxia alone [Kang et al., 2000]. K562 is mycoplasma-free human erythroleukemia cells, derived from a patient with chronic myelogenous leukemia in blastic crisis [Fuchs et al., 1995]. In particular, the role of HIF-1α and related proteins in sending a survival or death signal and nuclear transcription factor CREB phosphorylative state and intracellular molecular events involving Erk, PKC δ, p38MAPK proteins have been pointed out. CREB is a 43–46kDa nuclear transcription factor, which has been recognized by our group to be involved in the response to ionising radiation/etoposide combined treatment [Cataldi et al., 2006], and in the events linked to differentiation [Di Pietro et al., 2007] in the same experimental cell model. CREB recognizes the highly conserved sequence known as c-AMP responsive element (CRE), 5′-TGACGTCA-3′, which can be phosphorylated on serine-133 (Ser-133) such as PKC, Erk/MAPK and ATM (Ataxia Telangiectasia mutated Protein Kinase) [Yamamoto et al., 1988; Pearson et al., 2001; Stork and Schmitt, 2002; Blois et al., 2004].

2 Materials and methods

2.1 Cell culture and hypoxia exposure

K562 erythroleukemia cells were grown in suspension in HEPES-buffered RPMI 1640, supplemented with 10% FCS (Foetal Calf Serum), glutamine, penicillin/streptomycin in controlled atmosphere. Hypoxia exposure was performed by flushing the cells with a mixture containing 5% CO2/10% O2/85% N2 for 2min in a plexiglass sealed chamber. Once the oxygen tension in the medium reached a steady-state level, cells were maintained in the chamber for 5 and 22h in a CO2 incubator. Reoxygenation for 1 and 24h was done maintaining the cells in 5% CO2/21% O2/74%N2. Cell viability and death were assessed by trypan blue dye exclusion 24h after hypoxia.

For TUNEL and western blotting analyses, cells were recovered at 5 and 22h of hypoxia exposure plus 1h of re-oxygenation, while for ROS determination and cell cycle analysis were recovered after 5 and 2h of hypoxia exposure plus 1 and 24h of reoxygenation.

2.2 Cell cycle analysis

About 5×105 cells per experimental condition were harvested, fixed in 70° cold ethanol and kept at 4°C overnight. Cells were resuspended in 20μg/ml PI and 100μg/ml RNAse. Cell cycle profiles (1×104 cells) were analyzed in an EPICS-XL cytometer using the EXPO32 software (Beckmann Coulter, FL, USA). The percentage of dead cells was determined by gating low fluorescence events (low DNA content). Data were analyzed with Multicycle software (Phoenix Flow Systems, CA, USA).

2.3 Tunel analysis

Cytospinned cells were fixed in paraformaldehyde (4% v/v in PBS, pH 7.4) for 30min at room temperature and permeabilised in 0.1% Triton, 0.1% sodium citrate for 2min on ice. DNA strand breaks, characteristic of apoptotic cells, were identified by labelling the free 3′-OH nucleotide termini with fluorescein–dUTP with In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany). Slides were mounted in glycerol and analyzed under fluorescent microscope (Leica Microscopy System, Heidelberg, Germany). Negative and positive controls were performed according to manufacturer's protocol.

2.4 ROS detection

The production of ROS was determined with an EPICS-XL cytometer by monitoring the increase in green fluorescence of the flow cytometer after labelling the cells (5×105) with 1μM dihydrorhodamine 123 (Eugene, Oregon, USA) for 1h at RT. Dead cells were excluded from analysis by PI staining (5μg/ml) (Sigma–Aldrich, St. Louis, Missouri, USA).

2.5 Protein analysis

For immunoprecipitation, whole cell lysate (500μg) was incubated in the presence of 50μl of the suspended IP matrix (Exacta CRUZ, Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) for 30min at 4°C. Matrix was pelleted for 30min at 4°C and 50μl of suspended IP matrix, 3μg of mouse HIF-1α monoclonal antibody; 500μl of PBS were added to the supernatant and incubated at 4°C on a rotator for 1h. Matrix was pelleted and washed twice with 500μl of PBS. HIF-1α antibody–IP matrix complex was incubated with the lysate at 4°C on a rotator overnight. Matrix containing the immunoprecipitated sample was pelleted and washed 3 times with RIPA buffer. Samples were boiled and stored at −80°C.

Total cell lysates (20μg) or immunoprecipitates were electrophoresed and transferred to nitrocellulose membrane. Nitrocellulose membranes, blocked in 5% non-fat milk, 10mmol/l Tris–HCl pH 7.5, 100mmol/l NaCl, 0.1% (v/v) Tween-20, were probed with mouse HIF-1α, CREB, p-CREB (Ser1-33), p-Erk (Tyr204), p38MAPK, p-PKC δ (Ser-657), PKC δ, Bax, PARP cleaved fragment and p300 monoclonal antibodies (Santa Cruz, Santa Cruz Biotechnology, CA, USA) and then incubated in the presence of specific enzyme conjugated IgG horseradish peroxidase. Samples were normalized by incubation in the presence of mouse β tubulin monoclonal antibody. Immunoreactive bands were detected by ECL detection system (Amersham Intl., Buckinghamshire, UK) and analyzed by densitometry.

2.6 Image processing and analysis

Quantitative analysis involved a Sony videocamera connected to a Leica Quantimet 500 plus software (Leica Cambridge Ltd., Cambridge, UK) determining the change in Integrated Optical Intensity (IOI) using ISO transmission density Kodak CAT 152-3406 (Eastman Kodak Company, Rochester, USA) as standard. Statistical analysis was performed using the analysis of variance (ANOVA). Probability of null hypothesis of 0.1% (p<0.05) was considered statistically significant.

3 Results

3.1 Effects of hypoxia on cell cycle and apoptosis occurrence

K562 erythroleukemia cells were exposed to low oxygen tension (10% O2) for 5 and 22h followed by 1 and 24h of reoxygenation at 7×105/ml, the concentration being chosen as the more suitable, since the exposure of different cell suspension concentrations (2×105/ml, 4×105/ml, 7×105/ml) to hypoxia alone had previously shown no variation in terms of cell cycle profile and dead cells percentage (data not shown).

A high percentage of G1 cells was recorded along with a reduced number of G2 entering cells after 5 and 22h hypoxia plus 1h reoxygenation. These results were paralleled by a higher percentage of dead and apoptotic cells followed by a G0/G1 phase amplification and G2/M phase reduction. There was a reduction in dead and apoptotic cells after 24h compared to unexposed samples (Table 1). TUNEL analysis showed the early stages of apoptosis and even single DNA strand breaks, as noted by the highest level of apoptosis at 22±1h of reoxygenation (Fig. 1).

Table 1.

Flow cytometry analysis of cell cycle after exposure of K562 erythroleukemia cells to low oxygen tension (10% O2) for different time intervals (5 and 22 h) followed by 1 and 24 h of reoxygenation. Results represent the mean percentage ± SD of three different experiments.

Fig. 1

TUNEL detection of apoptosis in K562 erythroleukemia cells exposed to low oxygen tension (10% O2) for different time intervals (5 and 22h) followed by 1h of reoxygenation (r). Five slides were examined for sample. The extent of DNA fragmentation was quantified by direct visual counting of fluorescent labeled nuclei at light microscopy. Arrows indicate evident green fluorescence which accounts for a wide damage at DNA level (A–E). Negative control has been performed by omitting primary antibody (F). In phase contrast light microscopy pictures morphological features of apoptosis are evidenced by arrows (G–N). Magnification: 40×.

G0/G1SG2/MApoptoticDead
u24.8 ± 4.561.5 ± 2.113.7 ± 3.52.2 ± 0.213.6 ± 2.0
5 h24.1 ± 3.859.2 ± 0.416.7 ± 2.12.0 ± 0.115.3 ± 2.2
5h+1 h24.0 ± 4.459.2 ± 1.616.8 ± 2.21.5 ± 0.315.3 ± 2.7
5h+24 h23.9 ± 3.662.3 ± 3.913.8 ± 1.6nd14.7 ± 1.0
22 h27.2 ± 0.9*62.9 ± 1.19.9 ± 0.1*4.2 ± 0.8*26.2 ± 3.3*
22h+1 h27.3 ± 3.5*63.8 ± 2.98.9 ± 2.9*3.6 ± 0.619.6 ± 2.5
22h+24 h30.4 ± 4.9*59.7 ± 4.79.9 ± 1.8*1.2 ± 0.216.7 ± 0.8

3.2 Effects of hypoxia on ROS formation

As reported by others [Kang et al., 2000; Prabhakar et al., 2001], reoxygenation, by inducing formation of ROS (Reactive Oxygen Species) in the attempt to restore normal O2 levels, is a more potent enhancer of cell death than hypoxia alone. Thus, when we checked the level of ROS production, it was unaltered after 5h of hypoxia exposure, whereas it had dramatically accumulated after 22h of hypoxia followed by 1h of reoxygenation (Fig. 2).

Fig. 2

Flow cytometry analysis of Reactive Oxygen Species (ROS) production in K562 erythroleukemia cells exposed to low oxygen tension (10% O2) for different time intervals (5 and 22h) followed by 1 and 24h of reoxygenation. Discontinuous line represents unexposed sample. In the table quantitative evaluation of the data (±SD) is reported.

3.3 Effect of hypoxia on HIF-1α expression and activation

ROS production activates a number of molecular signalling pathways which lead to the cell apoptotic and death response. HIF-1α expression dramatically increased at first after both time intervals of hypoxia exposure (Fig. 3A, B). In normoxic conditions, HIF-1α is rapidly degraded in the cytoplasm via the ubiquitin proteasome pathway [Bàrdos and Ashcroft, 2005] and accumulates in the nucleus under hypoxic conditions, where it forms a DNA-binding heterodimer with the aryl hydrocarbon receptor nuclear translocator and recruits the general coactivator CBP/p300 to initiate transcription at hypoxia-responsive elements [Dames et al., 2002]. In our experimental model, HIF-1α/p300 co-immunoprecipitation was observed after 5h exposure to hypoxia, thus determining HIF-1α activation and induction to interact with target genes, and finally producing rescue proteins (Fig. 3C) [Kallio et al., 1998; Gradin et al., 2002]. In the other samples this effect was not seen, supposedly because of HIF-1α stabilization.

Fig. 3

HIF-1α expression in K562 erythroleukemia cells exposed to low oxygen tension for different time intervals (5 and 22h) followed by 1h of reoxygenation (r). (A) Western blotting is the most representative out of 3 different consistent experiments and (B) densitometric analysis performed on 3 different consistent experiments (±SD). As shown, samples were normalized by incubating membranes in the presence of β tubulin monoclonal antibody. (C) Co-immunoprecipitation of HIF-1α and p300. Immunoprecipitated HIF-1α was probed against rabbit p300 polyclonal antibody and reprobed against mouse HIF-1α monoclonal antibody. Note that HIF-1α/p300 complex is present only in unexposed and in 5h hypoxia exposed samples. u: unexposed.

3.4 Effect of hypoxia on apoptotic signalling events

Concomitantly to HIF-1α increased expression, both Bax and PARP cleaved fragment levels, which indicate apoptotic death occurrence, increased after 22+1h of reoxygenation, (Fig. 4). Moreover, at the same experimental times, Erk1/2 phosphorylation and the p-PKC δ/PKC δ ratio also increased (Fig. 5), suggesting that these proteins belong to the cell death signalling pathway.

Fig. 4

Bax and PARP full length and cleaved fragment expression in K562 erythroleukemia cells exposed to low oxygen tension (10% O2) for different time intervals (5 and 22h) followed by 1h of reoxygenation (r). (A) Western blotting is the most representative out of 3 different consistent experiments and (B) densitometric analysis performed on 3 different consistent experiments (±SD). As shown, samples were normalized by incubating membranes in the presence of β tubulin monoclonal antibody. u: unexposed. *22h+r Bax vs unexposed Bax: p<0.05; *22h+rPARP c.f. vs unexposed PARP c.f.: p<0.05.

Fig. 5

Erk and PKC δ expression and phosphorylation in K562 erythroleukemia cells exposed to low oxygen tension (10% O2) for different time intervals (5 and 22h) followed by 1h of reoxygenation (r). (A) Western blotting is the most representative out of 3 different consistent experiments and (B) densitometric analysis performed on 3 different consistent experiments (±SD). As shown, samples were normalized by incubating membranes in the presence of β tubulin monoclonal antibody. u: unexposed. *p-Erk 22h+r vs p-Erk; u: p<0.05; p-PKC δ/PKC δ 22h+r vs p-PKC δ/PKC δ u: p<0.05.

3.5 Effect of hypoxia on survival signalling events

In parallel, since the cells exposed to 5h and 5h+1h of reoxygenation appear to survive to hypoxic challenge and continue to proliferate (Table 1), we determined the involvement of CREB nuclear transcription factor by measuring the p-CREB/CREB ratio. This showed a dramatic increase in this ratio after 5h hypoxia, but which was lower at the other experimental times (Fig. 6).

Fig. 6

CREB expression and phosphorylation in K562 erythroleukemia cells exposed to low oxygen tension (10% O2) for different time intervals (5 and 22h) followed by 1h of reoxygenation (r). (A) Western blotting is the most representative out of 3 different consistent experiments and (B) densitometric analysis performed on 3 different consistent experiments (±SD). As shown, samples were normalized by incubating membranes in the presence of β tubulin monoclonal antibody. u: unexposed. *p-CREB/CREB 5h vs p-CREB/CREB u: p<0.05.

Concomitantly, p-p38/p38 ratio increased at 5h±r (Fig. 7), decreasing after 22h±reoxygenation, allowing to hypothesize the attempt of the cells to recover from the oxidative stress. The low p-p38/p38 ratio could support p38 failure in stabilizing HIF-1α which, in this way, can produce rescue proteins able to counteract hypoxia (not shown).

Fig. 7

p38MAPK expression and phosphorylation in K562 erythroleukemia cells exposed to low oxygen tension (10% O2) for different time intervals (5 and 22h) followed by 1h of reoxygenation. (A) Western blotting is the most representative out of 3 different consistent experiments and (B) densitometric analysis performed on 3 different consistent experiments (±SD). As shown, samples were normalized by incubating membranes in the presence of β tubulin monoclonal antibody. u: unexposed. *p-p38/p38 5h+r vs p-p38/p38 u: p<0.05.

4 Discussion

Hypoxia is a critical stimulus which switches on an immediate response by the organism. Under hypoxia some cells are irreversibly damaged and die, whereas others can adapt to the stressful environment and survive. In fact, the core of solid tumours, which is poorly vascularized, is quite hypoxic (frequently less than 1% O2) and the tolerance to hypoxia and resistance to apoptosis have been suggested to be mechanisms by which these tumours become more malignant [Nordsmark et al., 1996]. Although the role of hypoxia in solid tumours has been widely investigated [Brown and Giaccia, 1998; Brown, 2007], few studies have been reported regarding the possible effects of hypoxia on leukemia cells [Giuntoli et al., 2007; Winnard et al., 2008]. Thus the study of the signalling mechanisms which drive such response in K562 erythroleukemia cells could allow the identification of molecular targets for setting up hypoxic therapy in the treatment of leukemia diseases. In particular, not hypoxia alone, but hypoxia followed by reoxygenation, determining ROS production [Kang et al., 2000; Duranteau et al., 1998] induces the occurrence of apoptosis. Elevated ROS levels, in fact, observed after 22h of hypoxia exposure followed by 1h of reoxygenation, determined the reduction of cell proliferation and the increase of dead cells percentage up to 26%, while low ROS levels, produced after 5h of hypoxia exposure, exerted no modifications in cell cycle and in dead cell percentage. Moreover HIF-1α is considered a master regulator of O2 variations by governing adaptive patterns of gene expression [Chandel and Schumacker, 2000], whereas in normoxic conditions is continuously expressed and degraded by the ubiquitin proteasome system [Jewell et al., 2001], under hypoxia is not degraded, migrates to the nucleus and rescue proteins are produced by the cell. In addition, depending on the intracellular HIF-1α activity [Ikeda, 2005; Bàrdos and Ashcroft, 2005] – even though HIF-1α level increases after 5 and 22h of hypoxia exposure – it delivers a survival or death signal based on the duration of hypoxia in our experimental model. In agreement with recent studies showing that HIF-1α is phosphorylated by MAP kinases as well by Akt [Richard et al., 1999, Beitner-Johnson et al., 2001], HIF-1α activation (due to co-immunoprecipitation with CBP, a transcriptional coactivating protein known to interact with a number of constitutively active or inducible DNA-binding transcription factors [Shikama et al., 1997; Arany et al., 1996]) seems to occur after 5h hypoxia exposure in parallel to p38 MAPK mediated CREB activation, thus delivering a survival signal. Since nuclear localization per se is insufficient for transcriptional activation of HIF-1α, interaction between HIF-1α and CBP induces a functional activation of HIF-1α which, in this way, produces rescue proteins. HIF-1α requires hypoxia dependent activation of its terminal C domain within the nucleus to generate a functional form that recruits the CREB binding protein CBP/p300 coactivator protein [Quinn, 1993; de Groot et al., 1993], which shows that the mechanism of signal transduction by HIF-1 is a multistep process [Dames et al., 2002; Kallio et al., 1998; Sang et al., 2003]. The high HIF-1α level persisting until 22h of hypoxia is paralleled by PKC δ/Erk pathway activation and with high levels of Bax protein, which explain the increased number of apoptotic and dead cells, thus allowing us to hypothesize that ROS elevated level production, by switching on this signalling pathway, leads to apoptosis and cell death in this kind of leukemia. This is further supported by the response disclosed by these cells when Erk specific inhibitor is previously added in culture. This inhibitor enhances cell “survival”, i.e. reduces the number of dead cells (not shown). Since in vivo exposure to hypoxia has inhibited infiltration of leukemic cells in peripheral blood, bone marrow, spleen and liver in an Acute Myeloid Leukemia (AML) mouse model [Liu et al., 2006] and could represent a benefit for human patients with AML, the dual role suggested here for HIF-1α in delivering a survival or apoptotic signal, based on hypoxia duration, and the identification of nuclear transcription factor CREB as a molecular target, could provide important evidence for initiating hypoxic therapy in leukemia.

Acknowledgments

This work has been supported by FIRB 2001 Grant Project, cod.RBAU01EN5W-001: “Interazioni tra radiazioni ionizzanti e fattori di trascrizione della famiglia CREB/CREM” and 60% MIUR Grant 2006 Prof A Cataldi.

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Received 18 March 2008/23 July 2008; accepted 13 October 2008

doi:10.1016/j.cellbi.2008.10.014
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