REVIEW ARTICLE Intermittent hypoxia is a key regulator of cancer cell and endothelial cell interplay in tumours S Toffoli and C Michiels Laboratory of Biochemistry and Cellular Biology (URBC), University of Namur – FUNDP, Belgium Keywords apoptosis; cancer; chemoresistance; endothelial cell; hypoxia-inducible factor-1; intermittent hypoxia; radioresistance; reactive oxygen species; reoxygenation; tumor cell Correspondence C Michiels, Laboratory of Biochemistry and Cellular Biology (URBC), University of Namur – FUNDP, 61 rue de Bruxelles, 5000 Namur, Belgium Fax: +32 81 72 41 35 Tel: +32 81 72 41 31 E-mail: carine.michiels@fundp.ac.be Solid tumours are complex structures in which the interdependent relationship between tumour and endothelial cells modulates tumour development and metastasis dissemination The tumour microenvironment plays an important role in this cell interplay, and changes in its features have a major impact on tumour growth as well as on anticancer therapy responsiveness Different studies have shown irregular blood flow in tumours, which is responsible for hypoxia and reoxygenation phases, also called intermittent hypoxia Intermittent hypoxia induces transient changes, the impact of which has been underestimated for a long time Recent in vitro and in vivo studies have shown that intermittent hypoxia could positively modulate tumour development, inducing tumour growth, angiogenic processes, chemoresistance, and radioresistance In this article, we review the effects of intermittent hypoxia on tumour and endothelial cells as well as its impacts on tumour development (Received March 2008, accepted April 2008) doi:10.1111/j.1742-4658.2008.06454.x Introduction Hypoxia is increasingly perceived as one of the tumour microenvironment features favouring tumour cell survival, and also resistance to chemotherapy and radiotherapy Hypoxia is defined as a decrease in oxygen level within the tissue However, recent studies have shown that the time frame within which this decrease occurs and, more importantly, its duration may vary greatly from one tumour to another, or even from one area to another within the same tumour These observations have led to the definition of two kinds of hypoxia: chronic hypoxia and intermittent hypoxia Intermittent and chronic hypoxia in solid tumours Chronic hypoxia in tumours, first described in 1955 [1,2], results from limitation of the diffusion of oxygen Oxygen diffuses to a distance of 100–150 lm from blood vessels in normal and malignant tissues At a greater distance, the oxygen tension becomes close to zero, and cells become hypoxic [1] In parallel with chronic hypoxia, it was suggested in 1979 that transient hypoxia or intermittent hypoxia could also appear in tumours, due to the temporary ‘closure’ of blood vessels [3] The existence of acute hypoxia events in tumours was shown a few years later, with the Abbreviations AP-1, activator protein-1; ARNT, aryl hydrocarbon receptor nuclear translocator; EPR, electron paramagnetic resonance; HIF-1, hypoxiainducible factor-1; NF-jB, nuclear factor kappaB; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor FEBS Journal 275 (2008) 2991–3002 ª 2008 The Authors Journal compilation ª 2008 FEBS 2991 Intermittent hypoxia in cancer S Toffoli and C Michiels demonstration that intermittent hypoxia resulted from transient changes in blood flow [1,4,5] Histological analysis of tumour blood vessels showed that structural abnormalities were responsible for this irregular blood flow Indeed, tumour blood vessels are often tortuous and dilated, with excessive branching and numerous dead ends [6] Moreover, compression of these vessels by tumour cells, associated with the immaturity of the tumour vascular network, which is characterized by an absence of or a loose association with mural cells, pericytes and vascular smooth muscle cells, could also play a role in the heterogeneity of the blood flow [7–9] The blood flow stop periodicity, depending on the architectural complexity and maturation level of the tumour vascular network, is very variable from one tumour to another, and also within the same tumour [10,11] Therefore, a precise duration for blood flow interruption in tumours cannot be given However, studies of murine and human tumours have shown that the blood flow fluctuations observed in these tumours could vary from several minutes to more than h in duration [10–16] These blood flow irregularities in tumours can be demonstrated by different methods Direct real-time measurement in vivo of tumour blood flow fluctuations can be performed by the use of microprobes that are directly implanted in tumours Different microprobe systems can be used to study the blood flow fluctuations One of the most used microprobes is the Eppendorf polarographic needle electrode, which allows measurement of the oxygen partial pressure (po2) within tissues [12,17] Polarographic oxygen microelectrode functioning is based on reduction of oxygen at the surface of a cathode by applying a negative voltage between the cathode and the anode The reduction current measured with this kind of electrode is proportional to the number of oxygen molecules being reduced, and diminishes when blood flow is decreased or interrupted [18,19] Other microprobes, such as the OxyLite laser Doppler probe, allow monitoring of tumour blood perfusion [14] These probes illuminate the tissue under observation with single-frequency light from optical fibres coupled to a sensor Mobile red blood cells scatter the monochromatic light and generate a signal that is proportional to the mean erythrocyte velocity multiplied by the number of moving erythrocytes within the sampling volume [20,21] This signal decreases when the blood flow diminishes or stops, and vice versa However, the spatial resolution of these techniques is low, and the use of polarographic or laser Doppler microprobes is restricted to easily accessible tumours [22] For less accessible 2992 neoplasms, the direct real-time measurement in vivo of oxygen tension is performed by the use of imaging techniques [18,22,23], most of which are based on magnetic resonance Blood oxygen level-dependent magnetic resonance imaging and electron paramagnetic resonance (EPR) oxymetry are examples of such techniques [22–24] Blood flow modifications observed with blood oxygen level-dependent magnetic resonance imaging are based on the oxygenation status of endogenous haemoglobin This becomes paramagnetic when it is deoxygenated, and it is then detectable by magnetic resonance imaging Changes in blood flow modify the blood concentration of paramagnetic deoxyhaemoglobin and hence induce variations in the magnetic resonance signal [22,23,25,26] On the other hand, EPR oxymetry is based on the broadening of the resonance spectrum of a paramagnetic material by oxygen [27] Modifications in the EPR signal are directly correlated with the oxygen concentration, which is linked to the blood flow [18] One injection of a paramagnetic agent, such as India ink or charcoal, directly into a tumour is sufficient to allow repeated measurements to be performed over a relatively long period [18,24] Indirect measurements in vivo of tumour po2 fluctuation can also be performed by the use of a double hypoxia marker technique [11,28–30] 2-Nitroimidazoles (e.g misonidazole, EF5, CCI-103F, and pimonidazole) are commonly used as hypoxia markers These molecules are reduced by cellular nitroreductases at po2 levels below 10 mmHg to intermediates that covalently bind to cellular macromolecules [31–33] Hypoxic markers are administered in vivo separately at different times according to a pre-established timing schedule Tumour areas stained only by one marker show transient changes in hypoxia during the time interval between the injections of the two hypoxia markers [22,29] Reduced 2-nitroimidazoles can be detected by immunohistochemistry or immunofluorescence staining after the tumour resection Using radiolabelled 2-nitroimidazoles (e.g [18F]fluoromisonidazole), transiently hypoxic areas can also be detected in vivo by positron emission tomography, which is based on the detection of electromagnetic radiation emitted indirectly by the positron-emitting radioisotope [22,34] Modifications in blood flow are shown by performing scans after each hypoxia marker injection [18,22,23,35] The use of these techniques and their combination allow a better understanding of spatial and temporal changes in hypoxia in solid tumours, and also allow the linkage of these changes with other tumour microenvironmental parameters [18,22,23,35] FEBS Journal 275 (2008) 2991–3002 ª 2008 The Authors Journal compilation ª 2008 FEBS S Toffoli and C Michiels Hypoxia-inducible factor-1 (HIF) a-subunit stabilization and HIF-1 activation under intermittent hypoxia Hypoxia induces numerous changes in gene expression in normal and tumour cells [36] This adaptive response to hypoxia is orchestrated by a family of transcription factors induced by hypoxia The most important and best-studied member of this family is hypoxia-inducible factor-1 (HIF-1) HIF-1 is a heterodimeric transcription factor composed of the HIF-1a (120 kDa) and aryl hydrocarbon receptor nuclear translocator (ARNT, 94 kDa; also called HIF-1b) subunits These two subunits belong to the Per-ARNT-Sim basic–helix–loop helix family [37,38] HIF-1a and ARNT are constitutively expressed [39], but the formation of HIF-1 transcription factor in the nucleus depends on HIF-1a stabilization, which is principally O2-dependent [40] Under normoxia, HIF-1a is hydroxylated on proline 402 in the N-terminal domain and proline 564 in the C-terminal domain by prolyl-4-hydroxylases [41] These hydroxylations allow the binding of von Hippel–Lindau tumour suppressor protein on the oxygen-dependent degradation (ODD) domain of HIF-1a [42] von Hippel–Lindau tumour suppressor protein acts as the substrate recognition protein of the E3 ubiquitin ligase complex [43], and induces the ubiquitination of HIF-1a on its N-terminal and C-terminal domains (amino acids 390–417 and 549–582, respectively) [41] This ubiquitination targets HIF-1a for proteasomal degradation On the other hand, under hypoxic conditions, the prolyl hydroxylase activity decreases and the degradation pathway described above is interrupted [44] HIF-1a therefore rapidly accumulates and translocates into the nucleus, where, after dimerization with ARNT, it induces the transcription of target genes involved, notably, in glycolysis (e.g the glyceraldehyde-3-phosphate dehydrogenase gene) and angiogenesis [e.g the vascular endothelial growth factor (VEGF) gene] [45], thus allowing cells to adapt to hypoxia [46] The stabilization of HIF-1a and activation of HIF-1 have been widely studied under chronic hypoxia The new interest in intermittent hypoxia in recent years has led us to consider again this point: can the succession of short hypoxia and reoxygenation phases, typical of intermittent hypoxia, also stabilize HIF-1a and activate HIF-1? In the absence of oxygen, HIF-1a is rapidly stabilized, and short, intermittent hypoxia periods can be sufficient to induce HIF-1 Indeed, Yuan et al showed, in vitro, HIF-1a stabilization during intermittent hypoxia (cycles of 30 s of hypoxia followed by of reoxygenation) [47] This increase in HIF-1a abun- Intermittent hypoxia in cancer dance was dependent on the number of intermittent hypoxia cycles The kinetics used by Yuan et al undoubtedly demonstrate that short hypoxia–reoxygenation cycles can induce HIF-1a stabilization However, considering these kinetics, the increase in abundance of HIF-1a during intermittent hypoxia cycles could be due to an accumulation of HIF-1a subunit during each cycle, and not to an increase in its stabilization Indeed, although HIF-1a may be extremely rapidly degraded when cells are reoxygenated, its degradation after of reoxygenation was not assayed by Yuan et al Furthermore, Berra et al showed that HIF-1a could still be detected after of reoxygenation in HeLa cells incubated for h or or h under hypoxia They showed that the half-life of HIF-1a is inversely proportional to the duration of hypoxic stress [48], suggesting that long hypoxia periods could decrease HIF-1a stability Other recent studies have also shown an increase in HIF-1a abundance in the course of intermittent hypoxia cycles, using longer cycles of h of hypoxia followed by 30 of reoxygenation [49,50] The times used in these studies allowed the demonstration of complete HIF-1a degradation after each cycle of 30 of reoxygenation, showing that HIF-1a had not accumulated in the course of intermittent hypoxia cycles, and therefore that it is its stabilization that is increased in these conditions [50] HIF-1a stabilization does not always translate into HIF-1 activity One can therefore ask whether hypoxia periods interrupted by reoxygenation periods can be sufficient to induce the transcription of HIF-1 target genes HIF-1a degradation after each reoxygenation makes HIF-1 inactive In these circumstances, HIF-1 can only be transcriptionally active during the hypoxia phases, which can be short Reporter assays showed a significant gradual increase in hypoxia response element (HRE) promoter activity in PC12 cells incubated under intermittent hypoxia, in the course of hypoxia–reoxygenation cycles [47] Interestingly, with the same incubation time, HIF-1 transcriptional activity observed under intermittent hypoxia was almost equal to HIF-1 transcriptional activity observed under chronic hypoxia [50] Moreover, with the same incubation time under hypoxia (duration of reoxygenation under intermittent hypoxia was not considered in this case), Yuan et al showed higher HIF-1 transcriptional activity in comparison to chronic hypoxia [47] Although intermittent hypoxia can induce, like chronic hypoxia, HIF-1a stabilization as well as HIF-1 transcriptional activity, some differences can be seen between these two kinds of hypoxia It was shown in PC12 cells and EAhy926 endothelial cells that, under FEBS Journal 275 (2008) 2991–3002 ª 2008 The Authors Journal compilation ª 2008 FEBS 2993 Intermittent hypoxia in cancer S Toffoli and C Michiels Fig Effects of intermittent hypoxia and chronic hypoxia on HIF-1a stabilization and HIF-1 target gene transcription transient hypoxia, extracellular signal-related kinase ⁄ mitogen-activated protein kinases and phosphoinositide-3-kinase are not required for HIF-1 stabilization and transcriptional activity [47,50], whereas the inhibition of these kinases under chronic hypoxia impaired HIF-1 target gene expression [51,52] On the other hand, at least in endothelial cells, protein kinase A is involved in HIF-1a phosphorylation under intermittent hypoxia but not under chronic hypoxia, and protein kinase A inhibition decreased the transcription of HIF-1 target genes [50] These results suggest that the pathways regulating HIF-1 activity under chronic or intermittent hypoxia are different Figure shows a brief comparison of HIF-1a stabilization and HIF-1 activation under intermittent hypoxia and chronic hypoxia Tumour resistance induced by intermittent hypoxia The effects of chronic hypoxia have been extensively studied, and it has been clearly demonstrated that chronic hypoxia protects tumour cells from apoptosis induced by radiotherapy and chemotherapy [53–60] Recent studies have shown that intermittent hypoxia could also protect tumour cells from anticancer treatments Martinive et al showed, in vivo, a decrease in tumour cell apoptosis in transplantable liver tumour implanted in mice subjected to cycles of intermittent hypoxia before irradiation (10 Gy) with respect to mice kept under normoxia [49] This inhibition of apoptosis under transient hypoxia was also observed in vitro in 2994 FsaII fibrocarcinoma cells and B16 melanoma cells [49] Moreover, Dong & Wang demonstrated the possibility of death-resistant cell selection by the repetition of hypoxia episodes [61] Such selected cells were shown to be resistant to cell death induced by different types of molecules, such as azide, cisplatin and staurosporine [61] Transient hypoxia could also render tumours more invasive Cairns et al observed a highly significant increase in the number of lung micrometastases in KHT tumour-bearing mice exposed to 12 cycles per day (for 8–15 days) of 10 of hypoxia followed by 10 of reoxygenation, in comparison to control mice Interestingly, no increase in lung micrometastasis was observed in mice exposed to chronic hypoxia [15], suggesting again that intermittent hypoxia has different effects from chronic hypoxia In addition, it was shown by Durand & AquinoParsons that blood flow decreases could transiently arrest the division of tumour cells in S-phase [62] These cells are the main targets of chemotherapy, and the arrest of their cell cycle during S-phase reduces considerably their sensitivity to antiproliferative drugs, but it also implies a more rapid initiation of tumour cell repopulation when the blood flow restarts [62] More generally, the transient cessation of tumour blood flow reduces tumour cell exposure to the most highly diffusible anticancer agents, but also reduces their sensitivity to radiotherapy because of the decrease in oxygen supply during tumour irradiation [57,62] The protection against antitumour treatment can also be linked to a particular phenotype acquired by the cells in the course of intermittent hypoxia phases An FEBS Journal 275 (2008) 2991–3002 ª 2008 The Authors Journal compilation ª 2008 FEBS S Toffoli and C Michiels example of such acquired resistance is described by Dong & Wang [61]: upregulation of Bcl-XL has been observed in immortalized rat kidney epithelial cells exposed to repeated periods of hypoxia It was shown in these cells that Bcl-XL could directly interact with the proapoptotic molecule Bax at the mitochondrial level, impeding Bax oligomerization and cytochrome c release, and hence preventing cell apoptosis [61] Genetic instability due to abnormal DNA metabolism linked to impaired activity of enzymes such as topoisomerases, helicases and ligases is often observed in hypoxic tumours [63] Strand breaks, translocations, transversions and other chromosomal rearrangements observed in these conditions can also be responsible for tumour resistance Reynolds et al showed that hypoxia could induce a 3–4-fold elevation in mutation frequency, and higher levels of mutagenesis were observed in cells exposed multiple times to hypoxia [63], suggesting that exposure of cells to transient hypoxia could also induce resistance to antitumour treatments by this mechanism Moreover, oxidative injuries generated by reoxygenation in the course of intermittent hypoxia phases can also be responsible for DNA damage through an increase in 8-oxoguanine, which has been shown to miscode for A and lead to C:G to A:T transversions [64] Reactive oxygen species (ROS) generated during the reoxygenation periods can also play an important role, modifying gene expression through the regulation of the activity of some transcription factors, such as activator protein-1 (AP-1) or nuclear factor kappaB (NF-jB) AP-1 is known to play a pivotal role in tumorigenesis, regulating the expression and function of cell cycle regulators such cyclin D1, p53, p21, p19, and p16 Moreover, its activity was shown to increase in multiple human tumour types, and its inhibition can block tumour promotion, transformation, progression, and invasion [65] AP-1 activation was shown in PC12 cells under intermittent hypoxia, and was clearly associated with ROS production and, more particularly, with superoxide (O2Ỉ–) anion generation [66] Furthermore, it was shown that AP-1 activation involved c-fos, the activation of which persisted for several hours after the intermittent hypoxia ‘stimulus’ [66] Deregulation of c-fos and c-jun proteins can induce transformation in vivo [67], and c-fos upregulation was shown in tumour formation and, more particularly, in liver tumour development In this kind of tumour, AP-1 and c-fos were shown to be able to downregulate tumour suppressor genes and favour angiogenesis and tumour invasiveness [68] Therefore, AP-1 activation under intermittent hypoxia, associated Intermittent hypoxia in cancer with c-fos upregulation, could promote tumour development NF-jB can also be activated by ROS [69] ROS production during the reoxygenation periods [70] might also be able to activate NF-jB Ryan et al showed in HeLa cells and bovine aortic endothelial cells that transient hypoxia activated NF-jB in a number of hypoxia–reoxygenation cycles in an ROS-dependent manner [71] Despite the potential production of ROS during reoxygenation concomitant with NF-jB activation, these authors suggested that NF-jB activation under intermittent hypoxia was not linked to ROS production, because no decrease in NF-jB activation in the presence of the ROS scavenger N-acetyl-l-cysteine was observed [71] However, inhibition of NF-jB activation by N-acetyl-l-cysteine has been shown to occur not through ROS-dependent mechanisms, but rather through inhibition of tumour necrosis factorstimulated signal transduction by lowering tumour necrosis factor receptor affinity [72,73] or through inhibition of its DNA-binding activity [74] Therefore, involvement of ROS in NF-jB activation under intermittent hypoxia cannot be completely excluded It has to be noted that NF-jB activation by ROS is extremely cell type-dependent Beyond the question of the regulation mechanisms of NF-jB, its activation under intermittent hypoxia remains a critical point, because NF-jB plays an important role in tumour development through its ability to induce the transcription of genes coding for apoptosis inhibitor factors (cIAPs, Bcl-XL, FLICE), proproliferation molecules (interleukin-2, G1 cyclins), proangiogenic factors (VEGF, interleukin-8), and enzymes that lead to extracellular matrix degradation (matrix metalloproteases) [75–78] In addition, NF-jB activation was reported as an early event in malignant transformation in vitro [79], and continuous activation of NF-jB was also shown in many kinds of solid tumours [80] Conversely, NF-jB inhibition impairs tumour development NF-jB inhibition in prostate cancer in mice led to a marked reduction in the growth of tumour, demonstrating again the important role played by this transcription factor in tumour development [80] HIF-1 activation in tumours is often associated with a poor prognosis It allows the tumour cells to survive in the absence of oxygen, regulating their cell metabolism and inducing the production of prosurvival molecules, but also inducing the formation of new blood vessels, favouring metastasis [81] HIF-1 activation is always associated with hypoxia, but it was shown that the production of ROS under normoxia was also able to stabilize HIF-1a subunit and contribute to HIF-1 activation [82] The production of ROS during the FEBS Journal 275 (2008) 2991–3002 ª 2008 The Authors Journal compilation ª 2008 FEBS 2995 Intermittent hypoxia in cancer S Toffoli and C Michiels reoxygenation periods under intermittent hypoxia could then also influence HIF-1 activity However, HIF-1a subunit degradation is always observed during reoxygenation after incubation under chronic or transient hypoxia Therefore, HIF-1 activation during reoxygenation after a hypoxia period should be impaired in this case Paradoxically, it was shown that reoxygenation could stimulate HIF-1 signalling Increases in the translation of HIF-1 target genes and HRE–green fluorescent protein construction transcripts were observed after reoxygenation, despite the complete degradation of HIF-1a [83,84] This peculiar observation was explained by Moeller et al., who showed that reoxygenation could enhance downstream HIF-1 signalling by depolymerizing stress granules They showed that a pool of HIF-1-regulated transcripts were kept untranslated in the course of hypoxia in stress granules that were depolymerized during reoxygenation, allowing the rapid translation of sequestrated transcripts under normoxia [83] Interestingly, Moeller et al also observed stress granule formation in tumour cells under hypoxia as well as their degradation during reoxygenation Hence, they suggested that this post-transcriptional regulation process could help cancer cells to recover from a hypoxic shock and prepare the cells for a future insult This mechanism, the regulation of which could involve ROS, as suggested again by Moeller et al., could also explain, at least in part, the cancer cell resistance to anticancer treatment observed under intermittent hypoxia It would be interesting to investigate the involvement of stress granules in the gradual increase in the abundance of HIF-1a observed after each hypoxia step in the course of hypoxia–reoxygenation cycles Effects of intermittent hypoxia on tumour vasculature Tumour blood vessel formation is essential for tumour development As well as comprising a tumour cell dissemination pathway in the body, tumour blood vessels supply to cancer cells the oxygen and nutrients essential for their survival and proliferation In the absence of angiogenesis and new blood vessel formation, tumour growth is restricted, and the tumour size remains ‘microscopic’, generally not increasing beyond 0.5 mm, even in the case of a highly proliferative tumour, in which cell division is balanced by cell apoptosis induced by unfavourable survival conditions [85– 88] In these circumstances, in situ tumours can remain dormant ‘indefinitely’ in the absence of angiogenesis [89] Indeed, antiangiogenic treatments were shown to be able to impair or slow down tumour development 2996 and to reduce the volume of some solid tumours [90– 92] One of the main targets of these treatments comprises the endothelial cells One endothelial cell can control the survival of approximately 50–100 tumour cells [93] Therefore, the destruction of a few endothelial cells may induce the death of a large number of tumour cells Moreover, it was shown that endothelial cell suppression could also mediate apoptosis in drugresistant tumour cells [94,95] The role played by the tumour vascular network is thus critical in the development of a tumour, and therefore the effects of the tumour microenvironment on the cells constituting this network, i.e the endothelial cells, must also be considered Indeed, the tumour environment induces faster endothelial cell proliferation than in normal tissue, and the turnover of endothelial cells in tumours was estimated to be 20–2000 times faster [96] Moreover, significant differences have been shown in the transcriptome of tumour endothelial cells in comparison to endothelium in surrounding normal tissue [97–99] In addition, tumour cells can favour endothelial cell survival within tumours by the production of VEGF, and particularly after irradiation [100] As described previously in this review, intermittent hypoxia influences tumour cell behaviour Transient hypoxia also affects endothelial cells It was shown in vivo that intermittent hypoxia had a proangiogenic effect An increase in capillary density in mouse brains was observed after the repetition of cycles of of hypoxia followed by of reoxygenation for weeks [101] Moreover, in vitro, an increase in endothelial cell migration and formation of tubes was also reported under intermittent hypoxia [49] Therefore, transient hypoxia could increase angiogenic processes also in tumours Furthermore, it was observed that endothelial cells become, like tumour cells, radioresistant after an intermittent hypoxia preconditioning In vitro, an increase in the survival of endothelial cells was observed after irradiation (2 Gy) when intermittent hypoxia preconditioning was performed [49] This protective effect of intermittent hypoxia against radiotherapy on endothelial cells was shown to be HIF-1-dependent Indeed, a decrease in endothelial cell survival after a low level of irradiation (2 Gy) on cells previously incubated under intermittent hypoxia was shown when HIF-1a was silenced by small interfering RNA [49] Endothelial cell radioprotection through the repetition of hypoxia–reoxygenation cycles was also observed in vivo Terminal dUTP nick-end labelling assays showed a decrease in the number of apoptotic cells in the vasculature of transplantable liver tumour borne by mice when rodents were submitted to three cycles of h of hypoxia followed by 30 of FEBS Journal 275 (2008) 2991–3002 ª 2008 The Authors Journal compilation ª 2008 FEBS S Toffoli and C Michiels reoxygenation before tumour irradiation (10 Gy) [49] Interestingly, chronic hypoxia incubation before the irradiation did not protect endothelial cells against apoptosis: in contrast to intermittent hypoxia, it even drastically increased cell apoptosis [49] Moeller et al also showed in vivo a radioprotective effect of hypoxia ⁄ reoxygenation in endothelial cells after irradiation [83] They suggested that this radioprotection was induced by the secretion of endothelial cell-radioprotective cytokines by tumour cells after reoxy- Intermittent hypoxia in cancer genation They showed that tumour cell-conditioned medium recovered after incubation under hypoxia followed by reoxygenation was more radioprotective for endothelial cells than conditioned medium from tumour cells incubated under normoxia, normoxia with radiation, or hypoxia without reoxygenation [83] Moreover, it was shown that this endothelial cell radioprotection mediated by tumour cells after hypoxia–reoxygenation was also HIF-1-dependent Indeed, no significant endothelial cell radioprotective Fig Schematic representation of the effects of intermittent hypoxia on cancer cells and endothelial cells within a tumour Fig Schematic representation of the effects of HIF-1 activation under intermittent hypoxia on cancer cells and endothelial cells within a tumour FEBS Journal 275 (2008) 2991–3002 ª 2008 The Authors Journal compilation ª 2008 FEBS 2997 Intermittent hypoxia in cancer S Toffoli and C Michiels effect was observed when conditioned medium was taken from HIF-1-incompetent tumour cells [83] Therefore, these results suggest that intermittent hypoxia protects endothelial cells in a direct manner by acting directly on the endothelial cell phenotype, as observed by Martinive et al in vitro [49], and also by indirect pathways involving secreted molecules released from tumour cells, as suggested by Moeller et al [83] Conclusion Until now, most attention has been paid to chronic hypoxia However, during the last few years, a new concept has arisen, showing first that changes in po2 level are not always sustained in tumours but that they can be transient, and second that intermittent hypoxia can exert effects that are different from those induced by chronic hypoxia Both tumour cells and endothelial cells are affected by intermittent hypoxia, which can be perceived as the consequence of different stresses resulting from repeated combinations of hypoxia and reoxygenation periods, which may induce different cell responses In contrast, chronic hypoxia causes a prolonged and unique modification of the cell environment Figures and schematically summarize the effects of intermittent hypoxia The major conclusion drawn from these observations is the intricate interplay between tumour cells and endothelial cells, each favouring the survival of the other This delicate ballet has to be understood in detail in order to allow the design of new therapies targeting these processes Acknowledgements ´ ´ ´ Sebastien Toffoli is recipient of a FNRS-Televie grant Carine Michiels is research director of FNRS (Fonds National de la Recherche Scientifique, Belgium) This article presents results of the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming The responsibility is assumed by its authors References Brown JM (1990) Tumor hypoxia, 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In the absence... hypoxia in cancer S Toffoli and C Michiels Fig Effects of intermittent hypoxia and chronic hypoxia on HIF- 1a stabilization and HIF-1 target gene transcription transient hypoxia, extracellular signal-related... or intermittent hypoxia are different Figure shows a brief comparison of HIF- 1a stabilization and HIF-1 activation under intermittent hypoxia and chronic hypoxia Tumour resistance induced by intermittent