Jean-Philippe Cosse

Rommelaere Guillaume

Ninane Noelle

MICHIELS Arnould Thierry

BNIP3 protects HepG2 cells against etoposide-induced cell death under hypoxia by an autophagy-independent pathway

Keywords: apoptosis, autophagy, BNIP3, hypoxia, cancer cells

Tumor hypoxia is a common characteristic of most solid tumors and is correlated with poor prognosis for patients partly because hypoxia promotes resistance to cancer therapy. Hypoxia selects cancer cells that are resistant to apoptosis and allows the onset of mechanisms that promote cancer cells survival including autophagy. Previously, we showed that human hepatoma HepG2 cells were protected under hypoxia against the etoposide-induced apoptosis.

In this study, respective putative contribution of autophagy and BNIP3 in the protection conferred by hypoxia against the etoposide-induced apoptosis was investigated. We report that autophagy is induced by etoposide, a process that is not affected by hypoxic conditions.

Using Atg5 siRNA, we show that etoposide-induced autophagy promotes apoptotic cell death under normoxia but not under hypoxia. Then, we investigated whether the hypoxia-induced protein BNIP3 could explain the different effect of autophagy on cell death under hypoxia or normoxia. We show that the silencing of BNIP3 does not affect autophagy whatever the pO 2 but participates in the protective effect of hypoxia against etoposide-induced apoptosis.

Together, these results suggest that autophagy might be involved in etoposide-induced cell death only under normoxia and that BNIP3 is a major effector of the protective mechanism conferred by hypoxia to protect cancer cells against etoposide-induced apoptotic cell death.

Introduction

Tumor hypoxia is recognized as a poor prognosis factor associated with resistance to cancer therapy such as radiotherapy and chemotherapy [START_REF] Rockwell | Oxygen delivery: implications for the biology and therapy of solid tumors[END_REF][START_REF] Vaupel | Hypoxia and anemia: effects on tumor biology and treatment resistance[END_REF]. Inside the tumor, oxygen supply is compromised by the inadequate blood flow due to the abnormal vasculature network and by the high rate of cancer cell proliferation. Indeed, cancer cell proliferation also contributes to hypoxia by increasing the interstitial pressure and the diffusion distance from the blood vessels. Cancer cells localized in hypoxia areas are more resistant to chemotherapy than cells in well-oxygenated zones because drug delivery in hypoxia areas is limited and because oxygen is required for the cytotoxicity of some drugs [START_REF] Tredan | Drug resistance and the solid tumor microenvironment[END_REF]. Besides direct effects, hypoxia also promotes cancer cell resistance against chemotherapy by inducing adaptive mechanisms to hypoxia [START_REF] Cosse | Tumour hypoxia affects the responsiveness of cancer cells to chemotherapy and promotes cancer progression[END_REF]. Regulation of gene expression, involving HIF-1, is an important component of the initiation of these adaptive responses to hypoxia. HIF-1 regulates the expression of over a hundred of genes and among them are genes encoding proteins involved in the regulation of angiogenesis, erythropoiesis, anaerobic metabolism, drug resistance and proliferation. In addition, cell survival and cell death are both regulated by hypoxia, in part through the regulation of programmed cell death I and II processes named apoptosis and autophagy respectively. The regulation of apoptosis by hypoxia involves the regulation of the expression of genes such as Bid, Bax, Noxa, Survivin and Mcl-1 [START_REF] Erler | Hypoxia-mediated down-regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance[END_REF][START_REF] Kim | BH3-only protein Noxa is a mediator of hypoxic cell death induced by hypoxia-inducible factor 1alpha[END_REF][START_REF] Peng | Cross-talk between epidermal growth factor receptor and hypoxia-inducible factor-1alpha signal pathways increases resistance to apoptosis by up-regulating survivin gene expression[END_REF][START_REF] Piret | Hypoxiainducible factor-1-dependent overexpression of myeloid cell factor-1 protects hypoxic cells against tert-butyl hydroperoxide-induced apoptosis[END_REF]. In addition, hypoxia selects cancer cells with reduced apoptotic potential [START_REF] Graeber | Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours[END_REF]. Autophagy is first a cellular response in which proteins, organelles and portions of cytoplasm are engulfed, digested and recycled to sustain cellular metabolism during stress such as nutrient starvation and hypoxia [START_REF] Zhang | Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[END_REF].

Although stimulation of autophagy might constitute a protective response, extensive and non regulated autophagy may lead to cell death which may be either independent or linked to apoptosis [START_REF] Scarlatti | Does autophagy have a license to kill mammalian cells?[END_REF].

A c c e p t e d M a n u s c r i p t 4 Under hypoxia, the induction of autophagy is mainly due to the hypoxia-inducible protein BNIP3, encoded by a HIF-1 target gene. BNIP3 belongs to the BH3-only Bcl-2 family and contains a BH3 domain and a transmembrane domain that targets BNIP3 to the outer mitochondrial membrane. Unlike other BH3-only proteins, interactions with other proteins as well as its functions depend on the transmembrane domain but not on the BH3 domain [START_REF] Chen | Nix and Nip3 form a subfamily of pro-apoptotic mitochondrial proteins[END_REF].

Before its role in autophagy was discovered, BNIP3 was associated with cell death by multiple mechanisms. Currently, it seems that BNIP3 may participate in cell death by inducing necrosis, apoptosis or autophagy. In some cases, localization of BNIP3 into the outer mitochondrial membrane causes an increase in ROS production, cytochrome c release by PTP opening, loss of m and leads to caspase-dependent cell death [START_REF] Regula | Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes[END_REF]. Bax and Bak seem to be essential for BNIP3-induced cell death characterized by apoptosis features [START_REF] Kubli | Bnip3 mediates mitochondrial dysfunction and cell death through Bax and Bak[END_REF]. Caspaseindependent cell death through the release of endoG but not of cytochrome c was also observed following BNIP3 insertion into the outer mitochondrial membrane [START_REF] Zhang | BNIP3 upregulation and EndoG translocation in delayed neuronal death in stroke and in hypoxia[END_REF]. Finally, BNIP3 can also induce autophagic cell death. However, the precise mechanisms involved are still poorly understood. It seems that BNIP3 could be involved in autophagosome-lysosome fusion or in earlier stages such as Beclin-1 activation and mTOR inhibition [START_REF] Zhang | Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[END_REF][START_REF] Azad | Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3[END_REF][START_REF] Li | Bnip3 mediates the hypoxia-induced inhibition on mammalian target of rapamycin by interacting with Rheb[END_REF].

Finally, BNIP3 has been linked to protective autophagy and cell survival [START_REF] Zhang | Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[END_REF][START_REF] Tracy | Regulation of mitochondrial integrity, autophagy and cell survival by BNIP3[END_REF].

We have previously shown that human hepatoma HepG2 cells could be protected against etoposide-induced apoptosis under hypoxia. Given the potential role of both autophagy and BNIP3 in the regulation of cell death and cell survival, we investigated their putative role in etoposide-induced cell death and the protection provided by hypoxia. supplemented with 1 mM L-glutamine (Sigma, St Louis, USA) with or without etoposide (Sigma, St Louis, USA) at 50 μM, under hypoxia or under normoxia, medium was removed and cells were fixed for 10 min with PBS containing 4% paraformaldehyde and then washed 3 x 5 min in PBS. Cells were then permeabilized with 0.2% Triton X-100 in PBS. After 5 min, 5 µl of DAPI (10 µg/ml) (Sigma, St Louis, USA) were added in well. After 15 min of incubation in dark at 37°C, diluted DAPI was removed and cells were washed with PBS.

Then, the coverslips were mounted in Mowiol (Sigma, St Louis, USA) and observed with a fluorescent microscope.

Western blotting

Transient transfection with LC3-GFP

HepG2 cells were seeded in 75-cm 2 polystyrene flasks (Costar, Lowell, USA) at a density of 6.10 6 on day 0. On day 2, HepG2 cells were transfected with an expression plasmid pEGFP-LC3m encoding GFP-LC3 (from Professor Bassam Janji, LHCE CRP-Santé, Luxembourg) (2µg/ml) and using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) (3 µl/µg DNA) as a transfection agent for 4 h. At the end of this incubation, transfection medium was replaced by fresh medium containing 10% of foetal calf serum. On day 3, cells were trypsinized and 40,000 cells were seeded on glass coverslips in 24-well culture plates. On day 4, medium was replaced by serum-free CO 2 -independent medium (Invitrogen, Carlsbad, USA) supplemented with 1 mM L-glutamine (Sigma, , St Louis, USA) with or without etoposide (Sigma, , St Louis, USA) at 50 μM and cells were incubated under hypoxia or under normoxia during 16h.

At the end of this incubation, medium was removed and cells were fixed for 10 min with PBS containing 4% paraformaldehyde and then washed 3 x 5 min with PBS. To visualize nuclei, cells were incubated at room temperature for 35 min in the presence of TO-PRO-3 (Invitrogen, Carlsbad, USA, 1:80 dilution in PBS + RNase 2 mg/ml). After 3 washes in PBS, the coverslips were mounted in Mowiol (Sigma, St Louis, USA) and observed with a confocal fluorescent microscope (SP2, Leica, Mannheim, Germany). were incubated with transfection medium for 24 h. After this incubation, transfection medium was replaced by fresh medium with 10% of foetal calf serum. DharmaFECT1 was used at a final dilution 1:500 and siRNA were used at 50 nM. siCONTROL RISC free (D-001220-01) siRNA were used as negative control.

Short interfering RNA transfection

Caspase-3 activity assay

The fluorogenic substrate Ac-DEVD-AFC (BD Biosciences, San Diego, USA) was used to measure caspase-3 activity according to Lozano et al [START_REF] Lozano | Cell autonomous apoptosis defects in acid sphingomyelinase knockout fibroblasts[END_REF]. Cell extracts were prepared as described by Wellington et al [START_REF] Wellington | Caspase cleavage of gene products associated with triplet expansion disorders generates truncated fragments containing the polyglutamine tract[END_REF]. Cells were seeded in 6-well plates (250,000 cells/well).

The assay for caspase-3 activity was performed according to Cosse et al [START_REF] Cosse | Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines[END_REF].

LDH release assay

LDH release was measured with the cytotoxicity detection kit from Roche Molecular Biochemical (Basel, Switzerland) according to the manufacturer's protocol.

Real time RT-PCR

At the end of the incubation, total RNA was extracted using the Trizol extraction kit (Invitrogen, Carlsbad, USA). mRNA contained in 2 µg total RNA was reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. Forward and reverse primers for BNIP3 and RPL13A were designed using the Primer Express 1.5 software (Applied Biosystem, Carlsbad, USA) (23kDHBP-F : GCCTACAAGAAAGTTTGCCTATCTG/23kDHBP-R : Green PCR Mastermix (Applied Biosystem, Carlsbad, USA) and primers (Applied Biosystem, Carlsbad, USA) at the optimal concentrations. RPL13A was used as the reference gene for normalization and mRNA expression level was quantified using the threshold cycle method.

Statistical analysis

Statistical analyses were performed using Student's t test using SigmaStat software (Jandel Scientific Software). For each set of data, real triplicates were performed in one experiment.

A c c e p t e d M a n u s c r i p t 10

Results

Hypoxia protects HepG2 cells against etoposide-induced apoptosis

We have previously shown that hypoxia protects HepG2 cells against etoposide-induced apoptosis [START_REF] Cosse | Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines[END_REF][START_REF] Cosse | Hypoxia-induced decrease in p53 protein level and increase in c-jun DNA binding activity results in cancer cell resistance to etoposide[END_REF][START_REF] Sermeus | Hypoxia induces protection against etoposide-induced apoptosis: molecular profiling of changes in gene expression and transcription factor activity[END_REF]. To confirm these results, cells were incubated for 16 h in serum freemedium in the presence or in the absence of etoposide, under normoxia (21% O 2 ) or under hypoxia (1% O 2 ). At the end of the incubation, cells were fixed and nuclei were labelled with DAPI and observed with fluorescent microscopy (Figure 1A). We observed nuclear fragmentation in cells exposed to etoposide under normoxia but not in cells exposed to etoposide under hypoxia nor in cells that were not incubated with etoposide whatever the pO 2 .

In addition, the cleavage of PARP-1 was assessed by Western blot (Figure 1B). Etoposide induced the cleavage of PARP-1 and hypoxia prevented the cleavage of the protein. Hypoxia per se had no effect on the cleavage of PARP-1. In conclusion, as previously shown, etoposide induces apoptosis while hypoxia, a condition that does not affect apoptosis, protects cells against etoposide-induced apoptotic cell death.

Effects of hypoxia and/or etoposide on autophagy

Autophagy is known to be a cellular response to energy-limiting stresses such as serum deprivation. Moreover, it has been described that some chemotherapeutic drugs can also induce autophagy [START_REF] Takeuchi | Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors[END_REF][START_REF] Kanzawa | Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells[END_REF]. In order to evaluate the effect of hypoxia and/or etoposide on autophagy, cells were incubated for 16 h with or without etoposide under normoxia or under hypoxia. At the end of the incubation, the presence of autophagic vacuoles was assessed using transmission electron microscopy (Figure 2A). Despite being incubated 16 hours in serumfree medium, basal autophagy induced by these conditions remained low (Figure 2A, Nx). We observed that etoposide increased the number of autophagosomes in cells treated with etoposide both under normoxia and under hypoxia. In addition, the incorporation of the autofluorescent marker Monodansylcadaverine (MDC) has been assessed (Figure 2B). MDC was first described as a specific marker of autophagic vacuoles [START_REF] Biederbick | Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles[END_REF][START_REF] Munafo | A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation[END_REF]. However, subsequent studies have suggested that MDC is not a specific marker for early autophagosomes [START_REF] Bampton | The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes[END_REF][START_REF] Klionsky | r i p t 25[END_REF].

We observed that the MDC-associated fluorescence was higher in cells treated with etoposide than in control cells. Very low difference was observed between cells under hypoxia and cells under normoxia, independently of the presence of etoposide. To confirm these results, the conversion of LC3-I to LC3-II has been investigated. LC3 is the human homolog of yeast Atg8. LC3-I is obtained by the cleavage of nascent LC3 (proLC3) by Atg4 and is located in cytosol. During autophagy, LC3-I is involved in the formation of autophagosome and is incorporated into the autophagic vacuole membrane. For this, LC3-I must be conjugated to phosphatidylethanolamine (PE) by Atg7. PE-conjugated LC3-I is called LC3-II [START_REF] Ichimura | In vivo and in vitro reconstitution of Atg8 conjugation essential for autophagy[END_REF].

Although the molecular weight of LC3-II is larger than the weight of LC3-I due to the conjugation to PE, LC3-II migrates faster than LC3-I in SDS-PAGE due to its higher hydrophobicity. Therefore, LC3-I and LC3-II are usually detected in SDS-PAGE at a molecular mass around 16 kDa and 14 kDa respectively [START_REF] Mizushima | How to interpret LC3 immunoblotting[END_REF]. Cellular extracts were prepared from cells incubated with or without etoposide, under normoxia or under hypoxia for a period ranging from 2 to 24 hours and the level of both forms of LC3 was assessed by Western blot (Figure 3A). Etoposide induced the conversion of LC3-I to LC3-II under normoxia between 8 and 24 hours. Surprisingly, hypoxia did not induce the conversion of LC3-I to LC3-II. After 16 hours of incubation, the abundance of total LC3 drastically decreased in cells exposed to hypoxia. As a positive control, cells were treated with thapsigargin, a well-known inducer of autophagy. We observed that thapsigargin promoted the conversion of LC3-I to LC3-II and also increased the abundance of total LC3. These results indicate that etoposide is capable of inducing both apoptosis and autophagy. To determine the sequence of apoptotic and autophagic responses induced by etoposide, cleavage of PARP-1 was studied together with normoxia, while it was more diffuse in etoposide-incubated cells under hypoxia as well as in control cells whatever the pO 2 . Under hypoxia, the abundance of total LC3 decreased as previously observed by Western blot. It is known that LC3-II is located both in the inner and the outer membrane of autophagic vacuoles [START_REF] Tanida | Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy[END_REF]. After autophagosome-lysosome fusion, the fraction of LC3-II that is located in the inner membrane is degraded while the fraction of LC3-II located in the outer membrane is released in the cytosol and converted back in LC3-I, so the decrease of total LC3 protein level may be due to a higher autophagic rate. However, we did not observe any differences between autophagy under hypoxia and autophagy under normoxia in the presence of etoposide although the rate of autophagy is difficult to assess through the conversion of LC3 as long term exposure to hypoxia alters the abundance of the protein. In addition, following treatment with bafilomycin A1 which blocks autophagosomelysosome fusion, the abundance of total LC3 was higher in normoxic cells than in hypoxic cells (Figure 3C). This indicates that the difference in the abundance of total LC3 between normoxia and hypoxia was not due to change in the autophagic rate. Therefore, we postulated A c c e p t e d M a n u s c r i p t 13 that this difference was the consequence of the inhibition of the cap-dependent translation under hypoxia. To test this hypothesis, cells were treated with cycloheximide to block protein synthesis (Figure 3D). We observed that the abundance of total LC3 is reduced under normoxia in cells treated with cycloheximide. This confirms our hypothesis that difference in LC3 level between normoxia and hypoxia was not due to a change in autophagic rate between these conditions but to a decrease in protein synthesis under hypoxia.

Differential effect of etoposide-induced autophagy under normoxia compared to hypoxia

We have shown that etoposide induced autophagy while hypoxia had no effect on the etoposide-induced autophagy. We have also evidenced that the resistance of HepG2 cells against etoposide is higher under hypoxia than under normoxia. The fact that the autophagic rate is independent of hypoxia does not exclude the involvement of autophagy in the difference in resistance to etoposide observed between normoxia and hypoxia as autophagy may be protective or may contribute to cell death. To determine the role of etoposide-induced autophagy in the modulation of etoposide-induced cell death under normoxia and under hypoxia, we inhibited autophagy using Atg5 small interfering RNA (siRNA). Atg5 is a component of the Atg12-Atg5 conjugation complex required for the formation of autophagosome. In this process that requires also Atg7 and Atg10, Atg12 is covalently linked to Atg5. The Atg12-Atg5 conjugation occurs prior the LC3-PE conjugation [START_REF] Ichimura | In vivo and in vitro reconstitution of Atg8 conjugation essential for autophagy[END_REF]. Atg5 protein and mRNA levels were reduced by over 80 % in Atg5 siRNA-transfected cells (data not shown). To evaluate the effect of Atg5 silencing on etoposide-induced autophagy, cells were incubated for 16 h with or without etoposide under normoxia or under hypoxia and both the LC3-I and LC3-II abundance was assessed by Western blot. Atg5 siRNA decreased the conversion LC3-I to LC3-II since the LC3-I level was increased and the LC3-II level did not change or was even decreased in these conditions, indicating a slower autophagy (Figure 4A).

It should be noted that the inhibition of autophagy by the silencing of Atg5 did not increase A c c e p t e d M a n u s c r i p t 14 the total LC3 abundance under hypoxia to the total abundance of LC3 under normoxia, a data again supporting the fact that the lower total abundance of LC3 under hypoxia is independent of autophagy. Then, we studied the effect of autophagy inhibition on the etoposide-induced apoptosis by following caspase-3 activity using a fluorogenic substrate (Figure 4B). The cleavage of Ac-DEVD-AFC substrate was increased by etoposide while hypoxia inhibited these effects. Interestingly, the silencing of Atg5 reduced the cleavage of Ac-DEVD-AFC substrate under normoxia. In contrast, Atg5 silencing did not affect the cleavage of Ac-DEVD-AFC substrate under hypoxia. In conclusion, autophagy seems to promote etoposideinduced cell death markers of apoptosis under normoxia. By contrast, autophagy does not seem to affect the protective effect of hypoxia although, even if in some cases, it appears to have a weak protective effect. These results were confirmed by measuring overall viability.

LDH release was investigated in cells incubated for 40 hours with etoposide (Figure 4C).

Etoposide increased LDH release while hypoxia reduced the etoposide-induced LDH release.

The silencing of Atg5 reduced the etoposide-induced LDH release under normoxia and did not modify the etoposide-induced LDH release under hypoxia, indicating that autophagy could promote etoposide-induced cell death under normoxia but would not have any effect under hypoxia. Generally, negative control siRNA had no or little influence on studied parameters. Altogether, these results indicate that autophagy does not modulate the protection against etoposide conferred by hypoxia.

Effect of BNIP3 on the resistance against etoposide-induced apoptosis under hypoxia

To explain why etoposide-induced autophagy does not have the same consequences under normoxia and under hypoxia, we investigated the role of BNIP3, a hypoxia-induced protein for which an involvement in autophagy has been clearly demonstrated [START_REF] Azad | Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3[END_REF]. BNIP3 is a BH3only protein encoded by a HIF-1-target gene [START_REF] Sowter | HIF-1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors[END_REF]. As expected, the protein and mRNA levels of BNIP3 are increased under hypoxia in HepG2 (Figures 5A and5B). In contrast, etoposide A c c e p t e d M a n u s c r i p t 15 seemed to slightly reduce protein and mRNA levels but also to limit the upregulation of BNIP3 by hypoxia. BNIP3 has been associated with cell death by necrosis, apoptosis and autophagy. Moreover, a protective role has been conferred to BNIP3 in part through the upregulation of autophagy [START_REF] Zhang | Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[END_REF]. To determine the putative role of BNIP3 on autophagy and apoptosis in the protection conferred by hypoxia, the expression of BNIP3 was silenced using specific siRNA. A decrease in BNIP3 mRNA of 91 % was observed under hypoxia (data not shown). The impact of the silencing of BNIP3 on autophagy was determined by assessing the abundance of LC3-I and LC3-II by Western blot (Figure 5C). BNIP3 silencing had no effect on the abundance of both forms of LC3, suggesting that it does not influence autophagy in these experimental conditions. The possible involvement of BNIP3 in apoptosis was determined by following caspase-3 activity. We observed that caspase-3 activity was not affected by the silencing of BNIP3 under normoxia (Figure 5D). In contrast, the silencing of BNIP3 induced a weak increase in active caspase-3 protein level in cells incubated in the presence of etoposide under hypoxia. In conclusion, we observed that the silencing of BNIP3 sensitized cells against etoposide-induced apoptosis under hypoxia while it had no effect on the etoposide-induced apoptosis under normoxia. To confirm these results, viability was assessed by measuring LDH release after 40 hours of incubation (Figure 5E). Etoposide increased LDH release and this was attenuated in hypoxic cells showing the protection conferred by hypoxia. There was no significant difference in etoposide-induced LDH release between control cells and cells transfected with BNIP3 siRNA under normoxia. In contrast, the etoposide-induced release is higher in BNIP3 siRNA-transfected cells than in control cells, indicating that BNIP3 is involved in the protective effect conferred by hypoxia.

A c c e p t e d M a n u s c r i p t 16

Discussion

Our recent studies have demonstrated that hypoxia protects human hepatoma HepG2 cells against etoposide-induced apoptosis (Figures 1A and1B) [START_REF] Piret | Hypoxiainducible factor-1-dependent overexpression of myeloid cell factor-1 protects hypoxic cells against tert-butyl hydroperoxide-induced apoptosis[END_REF][START_REF] Cosse | Differential effects of hypoxia on etoposide-induced apoptosis according to the cancer cell lines[END_REF][START_REF] Cosse | Hypoxia-induced decrease in p53 protein level and increase in c-jun DNA binding activity results in cancer cell resistance to etoposide[END_REF][START_REF] Sermeus | Hypoxia induces protection against etoposide-induced apoptosis: molecular profiling of changes in gene expression and transcription factor activity[END_REF]. Hypoxia is an energylimiting stress which can leads to autophagy upregulation. Autophagy starts with the formation of an autophagosome, enclosed within a double membrane that engulfs part of cytoplasm. The formation of autophagosome depends upon Atg proteins [START_REF] Yang | Molecular mechanism and regulation of autophagy[END_REF]. Autophagy can be stimulated by various stresses, including nutrient depletion or hypoxia. In this case, autophagy is used to provide the constituents required to maintain metabolism essential for cell survival or to promote cell death, suggesting a dual role of autophagy [START_REF] Elmore | The mitochondrial permeability transition initiates autophagy in rat hepatocytes[END_REF][START_REF] Clarke | Developmental cell death: morphological diversity and multiple mechanisms[END_REF][START_REF] Bursch | The autophagosomal-lysosomal compartment in programmed cell death[END_REF].

Connections between autophagy and apoptosis have been identified. It has been shown that autophagy can either collaborate with apoptosis either to promote cell death or to prevent apoptotic cell death [START_REF] Lemasters | The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy[END_REF][START_REF] Boya | Inhibition of macroautophagy triggers apoptosis[END_REF][START_REF] Ravikumar | Rapamycin pretreatment protects against apoptosis[END_REF][START_REF] Espert | Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4[END_REF].

Therefore, we sought to evaluate the role of autophagy in the etoposide-induced apoptosis and the protective effect of hypoxia on it. To evaluate the autophagic rate, autophagosomes were observed by transmission electron microscopy and MDC-labelling (Figures 2A and2B). In addition, LC3-I and LC3-II protein level and localization of LC3-GFP were studied (Figures 3A and3B). We observed that autophagy was enhanced by etoposide. The upregulation of autophagy by chemotherapeutic drugs has already been described [START_REF] Abedin | Autophagy delays apoptotic death in breast cancer cells following DNA damage[END_REF][START_REF] Hoyer-Hansen | Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death[END_REF][START_REF] Kanzawa | Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3[END_REF][START_REF] Kaushal | Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity[END_REF][START_REF] Qadir | Macroautophagy inhibition sensitizes tamoxifen-resistant breast cancer cells and enhances mitochondrial depolarization Breast[END_REF][START_REF] Shimizu | Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes[END_REF]. Chemotherapyinduced autophagy may promote cell resistance against it. Doxorubicin-induced DNA damage stimulates cytoprotective autophagy through a PARP-1-dependent pathway [START_REF] Munoz-Gamez | PARP-1 is involved in autophagy induced by DNA damage[END_REF]. Similarly, cisplatin-induced autophagy delays apoptosis in renal tubular epithelial cells [START_REF] Kaushal | Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity[END_REF] and tamoxifen and camptothecin-induced autophagy increases MCF7 breast carcinoma cell line viability [START_REF] Abedin | Autophagy delays apoptotic death in breast cancer cells following DNA damage[END_REF][START_REF] Qadir | Macroautophagy inhibition sensitizes tamoxifen-resistant breast cancer cells and enhances mitochondrial depolarization Breast[END_REF]. Examples of autophagic cell death in response to chemotherapy were also described. Vitamin D analogue, EB1089, kills MCF7 through the induction of massive autophagy [START_REF] Hoyer-Hansen | Vitamin D analog EB1089 triggers dramatic lysosomal changes and Beclin 1-mediated autophagic cell death[END_REF] while arsenic trioxide induces autophagic cell death in human malignant A c c e p t e d M a n u s c r i p t 17 glioma cell lines [START_REF] Kanzawa | Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3[END_REF]. Autophagic cell death is also an alternative cell death pathway to apoptosis when it is no longer functional. Indeed, etoposide-induced autophagic cell death was described in apoptosis-deficient mouse embryonic fibroblasts [START_REF] Shimizu | Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes[END_REF]. We then investigated the impact of hypoxia on the etoposide-induced apoptosis. The results showed that hypoxia had no effect by itself on autophagy and did not modify the etoposide-induced autophagy (Figures 3A and3B). This is surprising and inconsistent with the literature in which the regulation of autophagy by hypoxia is well documented. Indeed, Papandreou et al. have

shown that hypoxia induces autophagy through the AMPK activation [START_REF] Papandreou | Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3[END_REF] but the main mechanism of induction of autophagy by hypoxia requires HIF-1 and BNIP3 [START_REF] Zhang | Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[END_REF][START_REF] Azad | Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3[END_REF]. The reason of this discrepancy is not known. It is however known that the level of pO 2 may influence the induction of autophagy by hypoxia [START_REF] Rouschop | Regulation of autophagy through multiple independent hypoxic signaling pathways[END_REF].

Although the etoposide-induced autophagy rate under normoxia and under hypoxia was similar, it is conceivable that autophagy plays a role in the protective effect of hypoxia. As mentioned previously, autophagy can lead to cell death, possibly by cooperating with apoptosis, or can promote cell survival by providing the constituents required to maintain metabolism essential for survival. Furthermore, autophagy may antagonize apoptosis through the elimination of depolarized mitochondria to prevent the release of apoptogenic factors such as AIF and cytochrome c from damaged mitochondria [START_REF] Elmore | The mitochondrial permeability transition initiates autophagy in rat hepatocytes[END_REF][START_REF] Lemasters | The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy[END_REF]. To better delineate the role of autophagy in the etoposide-induced cell death and to determine whether or not there were differences between etoposide-induced autophagy under hypoxia and under normoxia, we inhibited autophagy. For this, Atg5 expression was silenced using specific siRNA. Atg5 is a component of the Atg12-Atg5 conjugation system required for the process of autophagosome formation. The effect of Atg5 silencing on autophagy was evaluated by studying the abundance of both forms of LC3. This marker of autophagy is appropriate to study the inhibition of autophagy due to Atg5 silencing since it has been shown that the LC3-I

A c c e p t e d M a n u s c r i p t 18 conversion to LC3-II was downstream the Atg12-Atg5 conjugation and required Atg5 [START_REF] Ichimura | In vivo and in vitro reconstitution of Atg8 conjugation essential for autophagy[END_REF].

The silencing of Atg5 inhibited autophagy as LC3-II level decreased and LC3-I level increased in these conditions (Figure 4A). To determine the effect of autophagy inhibition on etoposide-induced apoptosis, the activity of active caspase-3 as well as cell viability were studied (Figures 4B andC). Atg5 silencing reduced the etoposide-induced apoptosis under normoxia but did not influence it under hypoxia. This means that autophagy contributes to etoposide-induced apoptosis under normoxia and acts upstream of apoptosis. In contrast, autophagy had no effect neither on the etoposide-induced apoptosis under hypoxia, nor on the protection conferred by hypoxia. The inhibition of the expression of Atg5 gene with siRNA has already been reported to promote resistance to chemotherapy [START_REF] Longo | Autophagy inhibition enhances anthocyanin-induced apoptosis in hepatocellular carcinoma[END_REF][START_REF] Yokoyama | Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells[END_REF]. However, it has been reported that Atg5 might be cleaved by calpains and truncated Atg5 could stimulate apoptosis by direct interaction with Bcl-XL in the mitochondrial outer membrane [START_REF] Yousefi | Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis[END_REF]. We can not exclude this possibility.

BNIP3 is a BH3-only Bcl2 family member encoded by a HIF-1 target gene [START_REF] Sowter | HIF-1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors[END_REF]. Many studies have reported that BNIP3 expression induces cell death with features that are both consistent and inconsistent with apoptosis suggesting that BNIP3 induces cell death through multiple pathways including apoptosis, necrosis and autophagy according to cell lines and conditions [START_REF] Regula | Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes[END_REF][START_REF] Kubli | Bnip3 mediates mitochondrial dysfunction and cell death through Bax and Bak[END_REF][START_REF] Zhang | BNIP3 upregulation and EndoG translocation in delayed neuronal death in stroke and in hypoxia[END_REF][START_REF] Kanzawa | Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3[END_REF]. Indeed, BNIP3-induced cell death is related to mitochondrial dysfunction, membrane depolarization, permeability transition pore opening and increased generation of ROS according to the experimental conditions. Both caspase-dependent and caspase-independent forms of cell death have been related to BNIP3. In some cases, BNIP3induced cell death was associated with the appearance of autophagic vacuoles and cellular morphological features of necrosis [START_REF] Velde | BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore[END_REF]. On the other hand, BNIP3-induced autophagy was described as a survival mechanism, providing time and resources for cell adaptation. hypoxia by preventing an increase in ROS levels [START_REF] Zhang | Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[END_REF]. Bcl-2 family pro-survival members inhibit autophagy by sequestering Beclin-1, a protein required for initiating autophagy [START_REF] Pattingre | Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy[END_REF].

Indeed, BNIP3 promotes autophagy by displacing Beclin-1 from Bcl-2 [START_REF] Zhang | Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[END_REF]. The involvement of BNIP3 in other steps of autophagy has also been described. It seems that BNIP3 can induce autophagy by interacting with RheB, resulting in mTOR inhibition [START_REF] Li | Bnip3 mediates the hypoxia-induced inhibition on mammalian target of rapamycin by interacting with Rheb[END_REF]. Moreover, the involvement of BNIP3 in later stages of autophagy, such as autophagosome-lysosome fusion, has also been described [START_REF] Azad | Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3[END_REF]. In this work, we have studied the involvement of BNIP3 in the protective effect of hypoxia against apoptosis for several reasons. First, the expression of BNIP3 is linked to hypoxia and HIF-1 [START_REF] Sowter | HIF-1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors[END_REF]. Secondly, BNIP3 is involved both in cell death as in cell survival [START_REF] Zhang | Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[END_REF][START_REF] Regula | Inducible expression of BNIP3 provokes mitochondrial defects and hypoxia-mediated cell death of ventricular myocytes[END_REF][START_REF] Kubli | Bnip3 mediates mitochondrial dysfunction and cell death through Bax and Bak[END_REF][START_REF] Velde | BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore[END_REF]. Thirdly, BNIP3 is a connection between apoptosis and autophagy [START_REF] Zhang | Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia[END_REF]. To study the role of BNIP3, the expression of BNIP3 was silenced using siRNA. The study of LC3-I and LC3-II proteins level showed that BNIP3 did not affect the etoposide-induced autophagy whatever the pO 2 level (Figure 5C). Moreover, BNIP3 silencing had no effect on etoposide-induced apoptosis under normoxia as caspase-3 activity was not affected by BNIP3 silencing (Figure 5D). In contrast, BNIP3 silencing increased caspase-3 activity under hypoxia, indicating that BNIP3 is involved in the protection conferred by hypoxia against the etoposide-induced apoptosis. In the literature, the role of BNIP3 in cell survival is clearly linked to its function in regulating autophagy. However, in HepG2 cells with the experimental conditions used, we showed that the protective effect of BNIP3 is independent of autophagy. Similarly, a recent report mentioned a protective effect of BNIP3 by a mechanism independent on autophagy. Indeed, Burton et al. have shown that BNIP3 is located in the nucleus and acts as transcriptional repressor of apoptosis-inducing factor (AIF) gene, in association with PSF and HDAC1, in human glioma U251 and U87 cells [START_REF] Burton | BNIP3 (Bcl-2 19 kDa interacting protein) acts as transcriptional repressor of apoptosis-inducing factor expression preventing cell death in human malignant gliomas[END_REF].

Further studies are thus needed to investigate how BNIP3 can inhibit apoptosis under hypoxia.

A c c e p t e d M a n u s c r i p t 20

In conclusion, we observed that hypoxia protects HepG2 cells against etoposide-induced apoptosis by the induction of BNIP3. BNIP3 is shown to be induced by hypoxia by a HIF-1dependent manner and to promote autophagy that can lead to cell death or cell survival according to the experimental conditions. In HepG2 cells, we observed that autophagy is induced by etoposide but not by hypoxia. Moreover, we observed that autophagy has a dual role as it promotes etoposide-induced apoptosis under normoxia but not under hypoxia.

Finally, we showed that BNIP3 does not regulate autophagy and protects HepG2 against etoposide-induced apoptosis by an autophagy-independent mechanism (Figure 6). *Graphical Abstract

A c c e p t e d M a n u s c r i p t 5 2

 5 Material and Methods2.1 Cell culture and hypoxia incubationHuman hepatoma HepG2 cells were maintained in culture in 75-cm2 polystyrene flasks (Costar, Lowell, USA) with 15 ml Dulbecco's modified Eagle's medium liquid (DMEM, Invitrogen, Carlsbad, USA)) and 10% of foetal calf serum (Invitrogen, Carlsbad, USA) and incubated under an atmosphere containing 5% CO 2 . For hypoxia experiments (1 % O 2 ), cells were incubated in serum-free CO 2 -independent medium (Invitrogen, Carlsbad, USA) supplemented with 1 mM L-glutamine (Sigma, St Louis, USA) with or without etoposide (Sigma, St Louis, USA) at 50 μM. Normoxic control cells were incubated in the same conditions but in normal atmosphere (21 % O 2 ). For some experiments, bafilomycin A1 (100 nM) (Sigma, St Louis, USA), thapsigargin (1 µM) (Sigma, St Louis, USA) or cycloheximide (200 µM) (Sigma, St Louis, USA) was added to medium. 2.2 Visualization of DAPI-labelled nucleus 40,000 cells were seeded on glass coverslips in 24-well culture plates. At the end of the incubation in serum-free CO 2 -independent medium (Invitrogen, Carlsbad, USA)

A c c e p t e d M a n u s c r i p t 6 2 . 4

 624 Cells, seeded in 25 cm 2 flasks, were scrapped in 200 μl of lysis buffer (40 mM Tris pH 7.5, 150 mM KCl, 1 mM EDTA, 1% triton X-100) containing a protease inhibitor mixture (« Complete » from Roche Molecular Biochemicals, Bsael, Switzerland, 1 tablet in 2 ml H 2 O, added at a 1: 25 dilution) and phosphatase inhibitors (25 mM NaVO 3 , 250 mM paranitrophenyl phosphate, 250 mM α-glycerophosphate and 125 mM NaF, at a 1 : 25 dilution). Western blot analysis was performed as described in Piret et al. [8] using a mouse anti-PARP-1 monoclonal antibody (#556493 from Pharmingen, BD Biosciences, San Diego, USA), antibodies directed against Caspase-3 (#9664 from Cell Signaling, Danvers, USA), LC3 (0231-100/LC3-5F10 from Nanotools, Teningen, Germany), Atg5 (#2630S from Cell Signaling, Danvers, USAZ), BNIP3 (B7931 from Sigma, St Louis, USA), p62/SQSTM1 (H00008878-M03 from Abnova, Tapei, Taiwan) were used at 1:5000 dilution. Membranes were reprobed with α-tubulin antibody (Sigma, St Louis, USA, final dilution 1/50,000) or with β-actin antibody (Sigma, St Louis, USA, final dilution 1/50,000) for protein loading control. Transmission electron microscopy Cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) during 90 min, postfixed overnight in 1% osmium tetroxide in 0.1 M sodium cacodylate (pH 7.4), dehydrated in a graduated series of ethanol and embedded in LX 112 resin. Blocks were cut on an ultramicrotome (NOVA, LKB, El Cajon, USA) to obtain 40 nm sections. Sections were stained with uranyl acetate and observed on a Tecnaï 10 transmission electron microscope (Tecnaï 10, FEI, Hillsboro, USA) 2.5 Quantification of MDC-labelled vacuoles Autophagic vacuoles were labelled with monodansylcadaverine (MDC, Sigma, St Louis, USA) by incubating cells with 0.05 mM MDC in PBS at 37°C for 15 min. After this A c c e p t e d M a n u s c r i p t 7 incubation, cells were washed four times in PBS and the fluorescence associated with MDC uptake by autophagic vacuoles was measured by excitation at 380 nm and emission at 450 nm with a fluorometer (Perkin Elmer LS45 fluorescence spectrometer, Zaventem, Belgium). The protein concentration was measured and fluorescent value was normalized by total amount of proteins.

A c c e p t e d M a n u s c r i p t 8

 8 The siGENOME smartpool of siRNA BNIP3 (M-004636-01) and Atg5 (M-004374-04) were obtained from Dharmacon (Lafayette, USA). siGENOME smartpool of siRNA were transfected with the DharmaFECT1 transfection reagent (Dharmacon, T-2001, Lafayette, USA) according to the manufacturer's recommendations. Cells seeded at 40,000 cells/cm2

A c c e p t e d M a n u s c r i p t 9 TGAGCTGTTTCTTCTTCCGGTAGT

 9 ; BNIP3-F : TTTGCTGGCCATCGGATT/BNIP3-R : ACCAAGTCAGACTCCAGTTCTTCA). Amplification reaction assays contained 1x SYBR

A c c e p t e d M a n u s c r i p t 11

 11

  A c c e p t e d M a n u s c r i p t 12 the conversion of LC3-I into LC3-II. The cleavage of PARP-1 protein was not detectable before 16 hours of incubation with etoposide, whereas the conversion of LC3-I into LC3-II was already observed after 8 hours of incubation. It has to be noted that LC3-II abundance then decreased. This result indicates that the activation of autophagy by etoposide occurs before the induction of apoptosis (Figure3A). To study the LC3 relocalization in membrane of autophagic vacuoles, cells transiently transfected with GFP-tagged LC3 were used (Figure3B). After 16 hours of incubation with or without etoposide under hypoxia or under normoxia, cells were fixed, nuclei were stained with TO-PRO-3 and cells were observed in a confocal fluorescent microscope to determine the localization of LC3-GFP. During autophagy, LC3-II is recruited to the autophagic vacuoles membrane giving a punctuate labelling. Such labelling was observed in cells incubated in the presence of etoposide under

  Zhang et al. showed that BNIP3-induced mitochondrial autophagy promotes cell survival under A c c e p t e d M a n u s c r i p t 19

Figure 1 :

 1 Figure 1: Hypoxia protects HepG2 cells against etoposide-induced apoptosis. HepG2 cells were incubated for 16 hours under normoxic (Nx; 21% O 2 ) or hypoxic (Hx; 1% O 2 ) conditions with (Ne-He) or without (Nx-Hx) etoposide (50 µM). (A) Nuclear fragmentation was assessed by nuclei labeling with DAPI using non-confocal fluorescent microscope. Fragmented nuclei are pointed by yellow arrows. (B) PARP-1 cleavage was detected in total cell extracts by Western blot analysis, using a specific antibody. Immunodetection of αtubulin was used to assess the equal protein loading on the gel.

Figure 2 :

 2 Figure 2: Etoposide increases the abundance of autophagic vacuoles. HepG2 cells were incubated for 16 hours under normoxic (Nx; 21% O 2 ) or hypoxic (Hx; 1% O 2 ) conditions with (Ne-He) or without (Nx-Hx) etoposide (50 µM). (A) Cells were fixed in glutaraldehyde and postfixed in osmium tetroxide. Ultramicrotome sections were poststained and imaged on transmission electron microscope. (B) Autophagic vacuoles were labeled with fluorescent marker MDC and MDC-associated fluorescent was measured. Results are normalized by total amount of protein and are presented as means ± 1 SD (n = 3). Statistical analysis was carried out with the Student's t test. ns : non significant; * : 0.05 > P > 0.01; ***: P < 0.001.

Figure 3 :

 3 Figure 3: Etoposide increased LC3-I conversion to LC3-II while hypoxia decreases total LC3 abundance. (A) HepG2 cells were incubated for increasing periods from 2 to 24 hours under normoxic (Nx; 21% O 2 ) or hypoxic (Hx; 1% O 2 ) conditions with (Ne-He; 50 μM) or without (Nx-Hx) etoposide (50 µM). LC3 and PARP-1 were detected in total cell extracts by Western blot analysis, using specific antibody. Immunodetection of α-tubulin was used to assess the equal protein loading on the gel. Thapsigargin was used as a positive control for autophagy. (B) LC3-GFP expressing cells were incubated for 16 hours under normoxic (Nx; 21% O 2 ) or hypoxic (Hx; 1% O 2 ) conditions with (Ne-He) or without (Nx-Hx) etoposide (50 µM). At the

Figure 4 :

 4 Figure 4: Etoposide-induced autophagy contributes to apoptosis and cell death under normoxia but not under hypoxia. HepG2 cells were transfected with Atg5 siRNA or negative control RISC free siRNA for 24 hours. Eight hours later, cells were incubated for 16 hours under normoxic (Nx; 21% O 2 ) or hypoxic (Hx; 1% O 2 ) conditions with (Ne-He) or without (Nx-Hx) etoposide (50 µM). (A) At the end of the incubations, Atg5 and LC3 were detected in total cell extracts by Western blot analysis, using specific antibody. Immunodetection of βactin was used to assess the equal protein loading on the gel. (B) Caspase-3 activity was assayed by measuring fluorescence intensity associated with free AFC released from the cleavage of the caspase-3 substrate Ac-DEVD-AFC. Results were normalized by fluorescence intensity of control cells and are presented as means ± 1 SD (n = 3). (C) LDH release was assessed after 40 hours of incubation. Results were normalized by the LDH release of control cells and are presented as means ± 1 SD (n = 3). Statistical analysis was carried out with the Student's t test. ns : non significant; * : 0.05 > P > 0.01; ** : 0.01 > P > 0.001; ***: P < 0.001.

Figure 5 :M a n u s c r i p t 31 (

 531 Figure 5: BNIP3 has no effect on etoposide-induced autophagy and apoptosis under normoxia but protects against etoposide-induced apoptosis under hypoxia through an autophagyindependent pathway. (A and B) HepG2 cells were incubated for 16 hours under normoxic (Nx; 21% O 2 ) or hypoxic (Hx; 1% O 2 ) conditions with (Ne-He) or without (Nx-Hx) etoposide

Figure 6 :

 6 Figure 6: Schematic representation of the involvement of autophagy and BNIP3 on the etoposide-induced apoptosis and the protective effect conferred by hypoxia against it. Etoposide induces DNA double strand breaks (DSB) and causes apoptosis and cell death.Hypoxia protects cells against etoposide-induced apoptosis. Autophagy is induced by etoposide both in normoxic and hypoxic cells. In normoxic cells, autophagy contributes to etoposide-induced apoptotic cell death. In contrast, autophagy has no effect on etoposideinduced apoptotic cell death and on the protective effect conferred by hypoxia. BNIP3 is

Figure

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Acknowledgements

J.-P. Cosse is recipient of a FRIA fellowship (Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture). This article presents results of the Belgian program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Science Policy Programming. The responsibility is assumed by its authors. We thank Prof.

Bassam Janji (LHCE CRP-Santé, Luxembourg) for providing the pEGFP-LC3m plasmid and the department of electron microscopy (FUNDP).


Philip Moore
email: philip.moore@ncl.ac.uk

Matt A King

Satellite Gravity Gradiometry: Secular Gravity Field Change over Polar Regions

Keywords: GOCE, gravity field, temporal variations, gradiometry 1

The ESA Gravity and steady state Ocean and Circulation Explorer, GOCE, mission will utilise the principle of satellite gravity gradiometry to measure the long to medium wavelengths in the static gravity field. Previous studies have demonstrated the low sensitivity of GOCE to ocean tides and to temporal gravity field variations at the seasonal scale. In this study we investigate the sensitivity of satellite gradiometry missions such as GOCE to secular signals due to ice-mass change observed in Greenland and Antarctica. We show that unaccounted ice mass change signal is likely to increase GOCE-related noise but that the expected present-day polar ice mass change is below the GOCE sensitivity for an 18 month mission. Furthermore, 2-3 orders of magnitude improvement in the gradiometry in future gradiometer missions is necessary to detect ice mass change with sufficient accuracy at the spatial resolution of interest.

Introduction

A c c e p t e d M a n u s c r i p t

Mass redistribution within and on the Earth's surface cause temporal changes to the Earth's gravity field. These temporal signatures are measurable by geodetic techniques including space-borne instrumentation. Over the last decade the scientific community has had access to data from the Gravity Recovery and Climate Experiment (GRACE) mission (Tapley et al., 2004) using precise inter-satellite measurements between a tandem pair of near polar satellites. GRACE has provided the static gravity field to degree and order 100-150 and monthly snapshots of the temporal gravity field for mass redistribution studies. Investigation of these signatures has provided a wealth of knowledge particularly in the areas of hydrology (e.g. [START_REF] Ramillien | Land water storage contribution to sea level from GRACE geoid data over 2003-2006[END_REF] and cryospheric science including insight into the changing ice mass over Greenland (e.g. [START_REF] Velicogna | Short term mass variability in Greenland, from GRACE[END_REF] and Antarctica (e.g. [START_REF] Velicogna | Measurements of time-variable gravity show mass loss in Antarctica[END_REF][START_REF] Chen | Antarctic regional ice loss rates from GRACE[END_REF].

The ESA Gravity and steady state Ocean and Circulation Explorer, GOCE, mission [START_REF] Muzi | GOCE: the first core Earth explorer of ESA's Earth observation programme[END_REF] will utilise the principle of satellite gravity gradiometry to measure the long to medium wavelengths in the static gravity field. While the GRACE mission is based on sensing the differential gravitational forces acting on two point masses orbiting some 220km apart satellite gradiometry carries an array of accelerometers on a single spacecraft to measure the differential accelerations. With GOCE the distance between the point masses, namely the accelerometers, is reduced to 0.5m with the differential accelerations providing the components of the satellite gradient tensor. GOCE was launched into a near polar orbit at an altitude of about 270km on 17 March 2009.

A c c e p t e d M a n u s c r i p t

The requirements of the GOCE gradiometry mission have advanced the technological development of satellites. Apart from the highly advanced accelerometers, which are two orders of magnitude more precise than those carried on GRACE, the satellite is a first in both the design of the satellite to ensure a highly stable flight through the Earth's atmosphere and the drag-free control system (e.g. [START_REF] Canuto | Drag-free and attitude control for the GOCE satellite[END_REF] to neareliminate the effects of the atmosphere. A feedback mechanism from the accelerometers will control firing of ion thrusters to compensate for the drag and other surface force effects. GOCE will carry six accelerometers to measure the gravity gradients along three orthogonal axes. The scientific drivers behind the GOCE mission are described in ESA (1999) for example. Given the band-width limitation of the accelerometers, gradiometry will not be able to measure gravity field signatures at the longest wavelengths. The harmonics causing these long wavelength signatures will instead be recoverable from orbital perturbations utilising precise positioning from the GPS/GLONASS receiver which also provides the necessary orbital positioning. The actual orbit and operational procedure for GOCE will be finalised once in orbit. The pre-launch scenario envisages two six-month measurement phases either side of a six-month period of hibernation during which passage through the Earth's umbra limits power from the solar cells. However, given the low level of solar activity in 2009 other scenarios may be feasible including the potential for continuous measurements over an 18 month period.

Although designed to measure the static gravity field several authors have investigated the sensitivity of the GOCE mission to the temporal field. For example [START_REF] Han | Effect of High-Frequency Temporal Aliasing on GOCE Gravity Field Solution[END_REF], [START_REF] Jarecki | Temporal gravity variations in GOCE gradiometric data[END_REF] and Han et al. (2006) investigated the effects of the atmosphere, oceans and hydrology in terms of the geoid and gravity gradients.

A c c e p t e d M a n u s c r i p t

These studies have shown GOCE to be insensitive to ocean tides and other mass redistributions at the seasonal scale. Others such as [START_REF] Vermeersen | The potential of GOCE in constraining the structure of the crust and lithosphere from post-glacial rebound[END_REF] and [START_REF] Vermeersen | High-harmonic geoid signatures related to glacial isostatic adjustment and their detectability by GOCE[END_REF] have investigated the possibility of using GOCE for glacial isostatic adjustment (GIA) studies. In particular, these have identified the possibility of using GOCE measurements to better constrain ice history. In this study we investigate the sensitivity of satellite gradiometry missions such as GOCE to secular ice-mass change observed in Greenland and Antarctica. We utilise rates estimated from the GRACE mission as well as from ERS and ENVISAT radar altimetry and elsewhere. Satellite derived results need to be corrected for GIA for true ice-mass studies but for our purposes it is the total signal over these areas that has been used as the appropriate temporal change affecting space missions. Ice mass loss over Greenland has been observed in several studies and here we use results pertinent to the whole area and to Eastern Greenland which has been shown to be the area of rapid change. Similarly, over West Antarctica the total GRACE mass change is utilised. We also investigate one of the areas of most rapid change in Antarctica namely the Pine Island Glacier with the rates taken from [START_REF] Shepherd | Inland Thinning of Pine Island Glacier, West Antarctica[END_REF].

In practice, the GOCE gradiometry will be accompanied by a non-tidal mass correction computed from an atmospheric and ocean model. In addition the correction will include large scale mass redistribution corrections for the very long wavelengths.

These corrections, to be computed from the annual variations of the GRACE spherical harmonic coefficients up to degree and order 20, will effectively account for the ice mass variation within the GOCE gradiometry at spatial scales of 1000km or larger. However, contributions at smaller spatial scales will be excluded from the correction fields. This study is thus motivated by the question to what extent is GOCE The study simulates the expected gradiometer signals due to the ice mass change and quantifies the sensitivity in terms of the magnitude of the signal and its spectral power density. In addition, we investigate sensitivity of potential recovery of localized signals over an extended period by using band-width limited windowing functions [START_REF] Wieczorek | Localized spectral analysis on the sphere[END_REF]. By utilising the distinct spatio-temporal spectra of the signal and error (e.g. [START_REF] Han | Spatiospectral localization of global geopotential fields from GRACE reveals the coseismic gravity change due to the 2004 Sumatra-Andaman earthquake[END_REF][START_REF] Han | Localized spectral analysis of global satellite gravityfields for recovering time-variable mass redistributions[END_REF][START_REF] Migliaccio | Detecting geophysical signals in gravity satellite missions[END_REF] the signal-to-noise ratio over the polar regions can be enhanced. In this study we follow the approach of [START_REF] Han | Spatiospectral localization of global geopotential fields from GRACE reveals the coseismic gravity change due to the 2004 Sumatra-Andaman earthquake[END_REF] and [START_REF] Han | Localized spectral analysis of global satellite gravityfields for recovering time-variable mass redistributions[END_REF] where spatiospectral localization around the epicentre of the 2004 Sumatra-Andaman earthquake facilitated recovery of the geophysical signal even though the signature was unobservable in the original GRACE fields. The methodology takes advantage of the distinct spatiotemporal characteristics of the signal and measurements error to enhance the signal-to-noise ratio of the locally intense signal to the more globally uniform errors in the measurements.

In the final section we consider an idealised future gradiometer mission where the gradiometry is either noise-free or affected by white noise over the band-width of the local signal to investigate potential recovery using 30 day snapshots of data as the mission overflies the Pine Island Glacier.

GOCE Gradiometry

A c c e p t e d M a n u s c r i p t

The nominal initial GOCE orbit is taken to be at an initial altitude of 270km with semi-major axis of 6648.1363km. The orbit was assumed to be circular with inclination i = 96.65475º. Positioning was computed for a 180 day period with the orbit subject to gravitational effects from the Earth and third bodies. Surface forces were set to zero to mirror the drag-free environment. The altitude of the satellite was seen to oscillate around 265 km. The orbital computations for this period provided the positioning to derive the simulated SGG data. For long-term studies over the lifetime of GOCE the orbit was assumed to repeat after the 6 month hibernation to give a total of 360 days of observations from a 540 day lifetime.

Based on the computed positioning a data set of radial gravity gradients, T rr , was derived at 1 s intervals, the GOCE sampling interval. Noise was subsequently added utilising a characterisation (e.g. [START_REF] Abrikosov | Recovery of the Earth's gravity field from GOCE satellite gravity gradiometry: A case study[END_REF] of the GOCE power spectral density (PSD) in the radial direction. The noise corresponded to a PSD of 1 mE/√Hz over the measurement band width (MBW) of 5*10 -3 -1*10 -1 Hz with a PSD proportional to the inverse of the frequency at frequencies below 5*10-3 Hz. The upper frequency is the Nyquist limit of twice the sampling rate. To illustrate the noise threshold on the sensitivity of GOCE gravity field recovery T rr was simulated for the differences between EGM08 [START_REF] Pavlis | An Earth Gravitational Model to degree 2160[END_REF] and EGM96 [START_REF] Lemoine | The Development of the Joint NASA GSFC and the NIMA Geopotential Model EGM96[END_REF] to degree and order 360. The PSD for this data set is given in Figure 1. Also plotted is the assumed PSD for the gradiometer noise, with the constant value over the MBW.

With these noise considerations and altitude near 270km satellite gradiometry cannot determine the longer wavelengths in the gravity field while some shorter-wavelengths are also below the GOCE sensitivity. Gton from the 2005 mass balance reported by [START_REF] Rignot | Changes in the velocity structure of the Greenland ice sheet[END_REF]. On taking the area of Greenland as 1,819,739 km 2 the Rignot and Kanagaratnam value equates to ≈ 0.12m/yr loss in terms of equivalent water height over the entire area. [START_REF] Luthcke | ITG-CHAMP01: a CHAMP gravity field model from short kinematic arcs over one-year observation period[END_REF] identify South-east and East Greenland (their areas 3b, 4a and 4b) to be the region of high ice loss amounting to 146 Gton per year over an area of 279166 km 2 (see Figure 2). This is equivalent to an ice loss of 0.5 m/yr over the region in equivalent water height. The largest loss of ice per unit area is identified as 75 Gton p.a. in South-East Greenland (their area 3b) of area of about 56109 km 2 equivalent to an ice mass loss of ≈ 1.3m/yr. These rates provide us, in the worst case, with an order of magnitude estimate of likely ice mass change in these regions to be used in our simulations.

To quantify the corresponding impact on the GOCE gradiometry the spatial distribution of the surface ice mass change over the selected area was converted to The observations can be considered as the temporal change to the static base model.

In this analysis it is assumed that the temporal field represents the anomalous potential to be estimated from GOCE.

Figure 3 shows the radial gravity gradient signal of a secular rate of 0.2 m/yr of ice mass over the entire Greenland land mass exhibiting a quasi-secular increase over the two observation periods. For an 18 month period the signal is less than 0.15 mE (1 mE is equivalent to 10 -12 Gal cm -1 or 10 -12 s -2 ). The PSD of the radial gradiometry is shown in Figure 4 for the first 30 days and last 30 days of the 18 month period. It

shows that the power, with maximum value 1.9 mE/√Hz, is however always below the GOCE measurement noise level of 1 mE/√Hz over the frequency range (Figure 1).

Although GOCE is insensitive to the Greenland change it is apparent that neglect of the ice mass loss may increase the PSD noise in the signal to over 1 mE/√Hz at some frequencies.

A similar analysis for an assumed ice mass change of 0.5 m/yr over the area of East and South-East Greenland in Figure 2 is summarised in Figures 5 and6. The increase in ice mass loss over the area has now yielded a larger signal but the smaller area results in a reduced PSD compared with the whole of Greenland. [START_REF] Han | Spatiospectral localization of global geopotential fields from GRACE reveals the coseismic gravity change due to the 2004 Sumatra-Andaman earthquake[END_REF][START_REF] Han | Localized spectral analysis of global satellite gravityfields for recovering time-variable mass redistributions[END_REF]. In the terminology of [START_REF] Han | Localized spectral analysis of global satellite gravityfields for recovering time-variable mass redistributions[END_REF] the signal is non-stationary while the noise is more likely to be stationary. Techniques of spatiospectral localization [START_REF] Wieczorek | Localized spectral analysis on the sphere[END_REF] can enhance the signal as the effect decays rapidly outside the region of interest while the measurement errors are relatively uniform over the Earth. Thus, the signal to noise ratio (SNR) of the localized signal is a better measure of the observability of the event. In this way, the 2004 Sumtra-Andaman earthquake was detected by localized analysis of the monthly GRACE gravity field solutions [START_REF] Han | Spatiospectral localization of global geopotential fields from GRACE reveals the coseismic gravity change due to the 2004 Sumatra-Andaman earthquake[END_REF]. Utilising the localized zonal window function h(θ) (Eq. 8 of [START_REF] Wieczorek | Localized spectral analysis on the sphere[END_REF] of spherical cap size θ 0 and expanded up to degree L h = 2π/ θ 0 -1 the SNR of the degree variances of the global gravity field solution and the localized solution are presented in Eq. 3-8 of [START_REF] Han | Localized spectral analysis of global satellite gravityfields for recovering time-variable mass redistributions[END_REF]. In this study we utilise both the SNR of the signal, where both measurement noise and signal are expanded in spherical harmonics, and the SNR on using a localized window centred on the source of the signal. The spherical harmonic expansion of the measurement noise was taken from [START_REF] Ditmar | Computation of spherical harmonic coefficients from satellite gravity gradiometry data: regularization issues[END_REF]. The localized SNR is defined for degrees

L h ≤ l ≤ L s -L h
where L s is the maximum degree of the solution field. For Greenland a spherical cap of θ 0 =40° gives L h = 8. Figure 7 shows the SNR for the signal in terms of spherical harmonics and localized by the window function for the ice mass change after 18 months for Greenland and East and South-East Greenland. After application of the East Greenland. Even for a GOCE-like gradiometer an order more accurate than GOCE the signal is only observable for 8 ≤ l ≤ 18. A gradiometer three orders more accurate than GOCE will be necessary for recovery of ice-mass change over

Greenland within an 18 month mission.

Secular ice mass change over Antarctica.

To quantify the signature on GOCE gradiometry from ice mass change over Antarctica we used the secular rates as given by the GRACE mission. Figure 8 The signal over Antarctica is dominated by a negative rate over the West Antarctic Ice Sheet (WAIS), including over Pine Island Glacier. Also evident is an apparent accumulation of mass over the Filchner Ronne Ice Shelf. To investigate the WAIS we have taken an ice mass change of 0.06 m/yr for the region 72°-78°S and 225°-270°E.

The corresponding T rr contribution is less than 0.05 mE over the 18month period while the resultant PSD is below the measurement noise by an order of magnitude with a maximum value of ≈ 0.1 mE/√Hz. Equivalently, data from a mission lifetime of a factor of 10 larger than the 18 month assumed here would still just approach the noise level in the MBW. Such a conclusion is not unexpected as the GRACE data underpinning the WAIS analysis is derived from spatial averaging to the extent that the resultant mass change is at large spatial scales whilst satellite gradiometry is more

Regional Solution

The previous analyses considered the magnitude of the radial gravity gradient and the associated PSD in a sensitivity analysis of ice mass change over Greenland and WAIS on GOCE. In this section, we now contemplate the possibility of an idealised gradiometry mission and seek to recover the observed change in a simulation based on a localized study. For this aspect we have chosen Pine Island Glacier in the WAIS.

Pine Island Glacier is one of the areas of most rapid change in Antarctica. Figure 10, replotted from [START_REF] Shepherd | Inland Thinning of Pine Island Glacier, West Antarctica[END_REF], shows the measured rate of change of ice as inferred from ERS altimetry (1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999). As an extreme characterisation of potential ice mass change the area of 23660km 2 bounded by 74.5ºS -76ºS and 261ºE -266ºE was considered to be experiencing a change of 0.5m/yr of ice. As for the whole Antarctica study a set of radial gravity gradient measurements were simulated and the PSD deduced. Although the maximum Trr signal after 18 months of 0.1 mE is a factor of two larger than for the WAIS the PSD is lower than that of the WAIS with a maximum value of 3.10 -2 mE/√Hz.. The sensitivity of gradiometry to shorter wavelengths is however evident in the less rapid reduction in the PSD amplitudes with signatures close to the maximum being measured at frequencies from 10 -3 Hz to 10 -2

A c c e p t e d M a n u s c r i p t

Hz. The SNR for Pine Island is plotted in Figure 9. The rapid ice-mass change is evident in the increased sensitivity compared to WAIS at degrees l ≥ 30.

Given the regional nature of the analysis a localized base function methodology based on that proposed by [START_REF] Ilk | Gravity Field Recovery and Validation by Analysis of Short Arcs of a Satellite-to-Satellite Tracking Experiment as CHAMP and GRACE[END_REF] and Mayer-Gürr et al. ( 2005, 2006) is adopted.

Accordingly, the anomalous potential, T, at satellite point position r, is represented as

    max 1 ) , ( I i Q i i r r T  where i Q
r is the surface point corresponding to node Q i , I max the total number of nodes and α i the coefficients to be estimated from the gradiometry. The localized base function Φ is given by

) (cos ) , ( , 1 1 max i Q i r r n n N n e n e Q P r R k R GM r r            
where G is Newton's gravitational constant, M the mass of the Earth of radius R e , P n the Legendre polynomial of degree n, to the expected signal we utilised general values for k n taken from the differences between EGM08 and EGM96. Figure 11 shows that the local base functions rapidly decay to zero with geocentric angle.

For the localized analysis the nodes were restricted to latitudes 60ºS to 90ºS. The nodes were spaced on N lat =60 circles of latitude formed using N tri = 5 equal triangles with common apex at the South Pole, each spanning 72º of longitude. Over each triangle a total of j nodes were equally distributed along the jth circle of latitude counting away from the pole. The total number of nodes was thus I max =N tri * N lat *(N lat +1)/2. Of these nodes those between 84ºS and 90ºS were removed given the polar gap due to the GOCE inclination. With Imax= 9150 this left a total of 8760 nodes and coefficients α i to be estimated. A subset of the nodes across Antarctica and Pine Island Glacier is shown as Figure 12. The simulated T rr data was taken for separate 30 day periods utililising short arcs across the pole of ≈16min duration. Each 30 day period involved 84265 from 518400 observations in the global set. In the analysis the data was assumed to be noise free with no restriction due to band-width whilst the satellite positioning was assumed exact. The application of white-noise at a level significantly below the PSD across the band-width of the measurements will lead to similar results. Thus, it is assumed that coloured noise as seen with GOCE is not significant in any future gradiometer mission. At the GOCE altitude the maximum Trr over the 18 months never exceeded 0.1 mE whist the PSD was always below 0.04 mE/√Hz. In practice, a gradiometer with reduction in noise by 2-3 orders of magnitude compared with GOCE is necessary. To illustrate the enhancement due to a decrease in altitude an alternative data set at initial altitude of 230km (mean altitude 225km) was derived. The radial gravity gradients for the two altitudes are shown in The true geoid height rates for the Pine Island Glacier are plotted as Figure 14a. There is some spatial distortion of the constant signal over the area 74.5ºS -76ºS and 261º-266ºE due to the restriction to degree and order 360 in the gravity field. The maximum rate of change is 2.89 mm over the 18 months. Figures 14b and14c plot the corresponding rates for the regional solution from gradiometry at mean altitude of 265 km (max change 2.70 mm) and 225 km (max 3.06 mm) respectively. Note that as the effective rates corresponded to the midpoint of month 17 the rates were multiplied by 18/17.5 for consistency with Figure 14a. The lower altitude is preferable in terms of gravity field recovery but the higher altitude also supports recovery to a high level.

The analysis shows that the local base function approach is capable of measuring changes over a region such as Pine Island Glacier and that gradiometry can, in theory, support solutions recovered from 30 days of data as for GRACE.

Conclusions:

Studies

  satellite gravity gradiometry (SGG) follow-on mission sensitive to ice mass change over Antarctica/Arctic over an 18 months or longer lifetime at long to medium spatial scales.

  Ice mass loss from Greenland has been confirmed from both in situ measurements and from GRACE studies. For example[START_REF] Luthcke | ITG-CHAMP01: a CHAMP gravity field model from short kinematic arcs over one-year observation period[END_REF] employed a mass concentration approach with GRACE data to deduce the rate of change for Greenland divided into six interior regions above 2000m and a further 6 coastal areas below 2000m for July 2003 to July 2005. The interior regions were inferred to have an accumulation of ice of about 40Gton p.a. whilst the coastal areas had a net loss of 140 Gton p.a. East Greenland evidenced the greater loss of ice. The total loss over Greenland of 100 Gton p.a. is considerably less than the ice mass loss of -224±41

  A c c e p t e d M a n u s c r i p t spherical harmonics rates using the surface load Love number approach of[START_REF] Wahr | Time-Variability of the Earth's Gravity Field: Hydrological and Oceanic Effects and Their Possible Detection Using GRACE[END_REF]. Changes from the rates were converted to gravity harmonics up to deg/order 360 at each epoch. These harmonics were subsequently used to generate a global data set of simulated gradiometer observations in the radial direction, T rr , for the mission.

  Consideration of the gravity gradient signal and the associated PSD gives some insight into the observability but does not necessarily indicate the sensitivity of the gravity field solution to ice mass change. A complementary measure is the degree variance of the gravity field solution in the presence of realistic noise. As the signals originate from a relatively small geographical region it is necessary to localise the signal and noise

  SNR has improved but is below unity for all degrees with a maximum SNR of less than 0.3 for all Greenland and 0.25 for East and South

  is a replot of the figure given in Moore and King (2008) which shows the rate of change of equivalent water height for Aug 2002 -Jan 2006 in mm/yr over Antarctica. This is the spatially averaged mass change and thus includes GIA as well as ice mass rates.

  Figure 7 the SNR for WAIS is plotted as Figure 9. For WAIS a spherical cap of θ 0 =40 was used. Once again the localized SNR shows an enhancement over the global equivalent but three orders of magnitude below the GOCE measurement sensitivity for degrees l ≥40.

  harmonics. Rather than risk possible aliasing due to tuning the covariances A c c e p t e d M a n u s c r i p t

  Figure 13 for the final month of the 18 month time span considered in the study. The decrease from 265 to 235 km in mean altitude effectively doubles the magnitude of the largest signal.

Figure 1 :

 1 Figure Captions

Figure 2 .

 2 Figure 2. 1° x 1° latitude/longitude block diagram of Greenland (grey), showing the East and South East regions (black).

Figure 3 .

 3 Figure 3. Radial gravity gradient signal over a 540 day period resulting from 0.2 m/yr ice mass change over Greenland.

Figure 4 .

 4 Figure 4. PSD of radial gravity gradient signal resulting from 0.2 m/yr ice mass change over Greenland. Days 0-30 (grey) and 510-540 (black).

Figure 5 .

 5 Figure 5. Radial gravity gradient signal resulting from 0.5 m/yr ice mass change over East and South-East Greenland.

Figure 6 .

 6 Figure 6. PSD of radial gravity gradient signal resulting from 0.5 m/yr ice mass change over East and South-East Greenland. Days 0-30 (grey) and 510-540 (black).

Figure 7 .

 7 Figure 7. GOCE degree variance signal-to-noise ratio (SNR). Global spherical harmonics: Greenland (circles) and SE Greenland (crosses). Localized harmonics (Lh): Greenland (solid line) and SE Greenland (stippled line).

Figure 8 .

 8 Figure 8. Mass change rates in mm/yr of equivalent water heights for the period 2002-2006 estimated from GRACE (replotted from Moore and King, 2008).

Figure 9 .

 9 Figure 9. GOCE degree variance signal-to-noise ratio (SNR). Global spherical harmonics: Western Antarctic Ice shelf (WAIS) (circles) and Pine Island (PI) (crosses). Localized harmonics (Lh): WAIS (solid line) and PI (stippled line).

Figure 10 .

 10 Figure 10. The rate of elevation change of the lower 200 km of Pine Island Glacier between 1992 and 1999 (coloured scale) registered with a map of the ice surface

Figure 11 .

 11 Figure 11. Normalised local base function Figure 12. Distribution of nodes across Antarctica with Pine Island Glacier shaded grey.

Figure 13 .

 13 Figure 13. Simulated Pine Island Glacier radial gravity gradiometry signal from mission at 230 km (black) and 270km (red).

Figure 14 .

 14 Figure 14. Change in geoid height (mm) over 18 months. Top: (a) true signal (max 2.89 mm); middle: (b) regional solution from gradiometry at 265 km (max 2.70 mm); lower: (c) regional solution from gradiometry at 225 km ( max 3.06 mm).

Figure 1 :

 1 Figure 1: Power spectral density of the differences between EGM08 and EGM96 to

Figure 2 .

 2 Figure 2. 1° x 1° latitude/longitude block diagram of Greenland (grey), showing the

Figure 3 .Figure 5 .

 35 Figure 3. Radial gravity gradient signal resulting from 0.2 m/yr ice mass change over

Figure 11 .

 11 Figure 11. Normalised local base function.

Figure 12 .Figure 13 .

 1213 Figure 12. Distribution of nodes across Antarctica with Pine Island Glacier shaded

Acknowledgments