Cellular and transcriptomic analysis of NS0 cell response during exposure to hypoxia

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Abstract

Mammalian cells have the ability to alter their gene expression in order to survive or adapt to a variety of environment stresses including hypoxic stress. Maintaining oxygen supply has been accepted as essential for cell survival and growth. To determine the cellular and molecular changes which take place under oxygen deprivation, an NS0 cell line producing a human-mouse chimeric antibody was cultured under hypoxic conditions (<1%). Various cellular parameters such as viability, productivity, metabolism, apoptosis and cell cycle were studied and notable changes were shown to be accompanied by changes in metabolic rates. When the cells where exposed to hypoxia for 48 h, cell growth was suppressed and cell death was detected. To better understand and explore the mechanisms underpinning these biological alterations and to identify the genes involved in the genetic reprogramming, genome-wide analyses were performed using GeneChip Mouse Genome arrays. The gene expression profiling generated by the microarray technique revealed that hypoxia, even in the early stages (12 h), induces significant changes in gene expression in NS0 cells. The primary responses to hypoxia within the cells were: (1) the up-regulation of pathways such as glycolysis that ultimately lead to alternative routes of ATP generation and increased oxygen availability; and (2) the down-regulation of genes involved in purine/pyrimidine and one carbon pool metabolisms required for DNA and RNA synthesis. By combining gene expression and physiological changes under hypoxia, it was possible to explore the mechanisms of hypoxia-induced alterations in more depth.

Introduction

Exposure to low oxygen tension can lead to a complex scenario of both metabolic and physiological changes which allow the cells to survive and even proliferate in a hypoxic environment. For example, when oxygen is limited, individual cells decrease oxidative phosphorylation and rely on glycolysis as the primary means of ATP production (Webster, 2003). Since the energy obtained from glucose is markedly reduced, enzymes involved in glycolysis are elevated in order to maintain an adequate energy level (Kim and Dang, 2006). More evidently, reduced oxygen availability in the cells can trigger a variety of cellular mechanisms, including cell cycle arrest (Alarcon et al., 2004, Yeo et al., 2006) and apoptosis (Brunelle and Chandel, 2002). The severity of hypoxia determines whether cells become apoptotic or adapt to hypoxia and survive. Under severe hypoxia (<0.01% O2) cells do not survive but initiate the apoptosis programme. However, under mild hypoxic conditions (0.01–2% O2), cells are prevented from going into apoptosis (Greijer and van der Wall, 2004). Severe hypoxia causes a high mutation rate, resulting in point mutations, followed by the initiation of a cascade of events that leads to apoptotic cell death, thereby preventing the accumulation of cells with hypoxia-induced mutations (Reynolds et al., 1996). The regulation of signaling pathways leading to cell survival or death is managed by hypoxia-inducible factor 1 (HIF-1), which can initiate apoptosis by inducing high concentrations of pro-apoptotic proteins such as BNIP3 or counteract apoptosis by inducing anti-apoptotic proteins for example IAP-2 (Greijer and van der Wall, 2004). Another major consequence of cellular hypoxia is a proliferation arrest of cells. Cells subject to severe hypoxia arrest in G1 or early S phase, but if hypoxia is not severe cells in late S, G2, or M phase complete cell division and arrest in the subsequent G1 phase (Amellem and Pettersen, 1991).

The supply of oxygen is a crucial parameter when cultivating animal cells in bioreactors, especially with high cell-density cultures. The main limitation for proper cell oxygenation is the lack of oxygen inside the carrier, mainly due to the low solubility of oxygen in the culture medium and dissolved oxygen homogeneity in the medium. The ability of various methods of oxygenation to meet the demands of high cell-density culture has been investigated using a spin filter perfusion system in a stir tank bioreactor (Emery et al., 1995) and fixed-bed bioreactor (Fassnacht and Pörtner, 1999). Exposure of hybridoma batch culture to anoxia has induced cell death and decreased monoclonal antibody (MAb) production rate (Mercille and Massie, 1994). Simpson et al. (1997) found that hybridomas died after 32 h when subjected to oxygen deprivation and that the over-expression of bcl-2 resulted in retention of cell viability above 90% under similar conditions of hypoxia. Therefore, the question arises of whether cells can survive under hypoxic conditions, which can be manifested in high cell-density culture.

The aim of this study was to characterize global changes at the genomic level related to the physiological alterations that occur during hypoxia to clarify the mechanisms of hypoxia-induced alterations, and to find out useful hypoxia-related markers of NS0 cells especially during early stages of hypoxia. This work documents a set of genes which were induced after 12 h of hypoxia; one practical application of these genes would be as hypoxia-based prognostic markers.

Section snippets

Cell line and culture conditions

An NS0 cell line transfected with the glutamine synthetase (GS) expression system and producing a human-mouse chimeric cB72.3 IgG4 antibody (Bebbington et al., 1992) was kindly provided by Lonza Biologics. Cells were maintained in glutamine-free DMEM/F12 media supplemented with 10% fetal bovine serum (Cambrex, Verviers, Belgium), 2% GS supplement (GSEM; Sigma–Aldrich, St Louis, MO), 25 μM l-methionine sulphoximine (MSX; Sigma–Aldrich) and 0.05% Pluronic F-68 (Sigma–Aldrich). NS0 6A1 cells were

Cell growth and viability

To characterize the effect of hypoxia on cell growth, NS0 6A1 cells were cultivated in two bioreactors (normoxic and hypoxic) with a volume of 150 ml and an initial cell concentration of about 3.5 × 105/ml. After 48 h of incubation, cells cultured under optimal conditions reached the maximal viable cells density of 14.8 × 105/ml (Fig. 1). The growth of NS0 cells under hypoxic condition was significantly affected, reaching the highest cell-density of 5.9 × 105/ml after 30 h of incubation. Although

Conclusions

Several notable physiological changes were observed during cell cultivation under severe hypoxia. Cultivation of NS0 6A1 cells producing a human-mouse chimeric cB72.3 IgG4 antibody led to suppressed cell growth under oxygen deprivation and to significant loss in viability after 12 h of incubation. The cells exposed to hypoxia exhibited a transient arrest in the G2 phase during the first 9 h of incubation, followed by G1 and S phase arrest. The antibody production machinery had completely ceased

Acknowledgments

We would like to thank Dr. Peadar O’Gaora (Conway Institute of Biomolecular and Biomedical Research, UCD, Ireland) for help with the analysis of the microarray results. This study was supported by a grant from the Science Foundation Ireland (SFI).

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