Effects of ambient oxygen concentration on the proliferation and viability of cultured human corneal epithelial cells

Abstract

Ambient oxygen (O2) affects the metabolism and other functions of corneal epithelial cells. The effects of O2 concentration on the proliferation and viability of corneal epithelial cells in culture were investigated. Simian virus 40-transformed human corneal epithelial (HCE) cells were maintained at 37°C in a humidified incubator containing 5% CO2 and 95% air. The cells were subsequently transferred to a multigas incubator and exposed to 5% CO2 and either 1, 21, or 60% O2 plus 94, 74, or 35% N2, respectively. Cell proliferation was evaluated by determination of cell number and measurement of the incorporation of bromodeoxyuridine. Cell lysis was quantified by measurement of the release of lactate dehydrogenase. Apoptosis was evaluated by flow cytometric analysis of cells stained with annexin V and propidium iodide as well as by immunoblot analysis of cleavage of caspase-7. The phosphorylation (activation) of Akt was also detected by immunoblot analysis. Hyperoxia (60% O2) inhibited the increase in cell number and the incorporation of bromodeoxyuridine apparent in HCE cells exposed to normoxia (21% O2). It also induced the release of lactate dehydrogenase, an increase in the proportion of apoptotic (annexin V+, propidium iodide−) cells, the cleavage of caspase-7, and the phosphorylation of Akt. None of these effects was observed in cells exposed to hypoxia (1% O2). The amounts of the cleaved forms of caspase-3, 6, and 9 did not differ among HCE cells cultured under 1, 21, or 60% O2. These results indicate that hyperoxia inhibited the proliferation of, and induced death by apoptosis in, cultured human corneal epithelial cells. The antiapoptotic protein Akt was also activated in cells exposed to hyperoxia, possibly reflecting a protective response to oxygen toxicity.