The effect of baseline O2 conditions on the response of prostate cancer cells to hypoxia.

The transcriptional response to hypoxia is largely regulated by the hypoxia-inducible factors (HIFs), which induce the expression of genes involved in glycolysis, angiogenesis, proliferation, and migration. Virtually all cell culture-based hypoxia experiments have used near-atmospheric (18% O2) oxygen levels as the baseline for comparison with hypoxia. However, this is hyperoxic compared with mammalian tissue microenvironments, where oxygen levels range from 2% to 9% O2 (physioxia). Thus, these experiments actually compare hyperoxia to hypoxia. To determine how the baseline O2 level affects the subsequent response to hypoxia, we cultured PC-3 prostate cancer cells in either 18% or 5% O2 for 2 wk before exposing them to hypoxia (∼1.1% pericellular O2) for 12-48 h. RNA-seq revealed that the transcriptional response to hypoxia was dependent on the baseline O2 level. Cells grown in 18% O2 before hypoxia exposure showed an enhanced induction of HIF targets, particularly genes involved in glucose metabolism, compared with cells grown in physioxia before hypoxia. Consistent with this, hypoxia significantly increased glucose consumption and metabolic activity only in cells previously cultured in 18% O2, but not in cells preadapted to 5% O2. Transcriptomic analyses also indicated effects on cell proliferation and motility, which were followed up by functional assays. Although unaffected by hypoxia, both proliferation and migration rates were greater in cells cultured in 5% O2 versus 18% O2. We conclude that an inappropriately hyperoxic starting condition affects the transcriptional and metabolic responses of PC-3 cells to hypoxia, which may compromise experiments on cancer metabolism in vitro.NEW & NOTEWORTHY Although human cell culture models have been instrumental to our understanding of the mechanisms involved in the cellular response to hypoxia, in virtually all experiments, cells are routinely cultured in near-atmospheric (∼18% O2) oxygen levels, which are hyperoxic relative to physiological conditions in vivo. Here, we show for the first time that cells cultured in physiological O2 levels (5% O2) respond differently to subsequent hypoxia than cells grown at 18%.

Revisiting reactive oxygen species production in hypoxia.

Cellular responses to hypoxia are crucial in various physiological and pathophysiological contexts and have thus been extensively studied. This has led to a comprehensive understanding of the transcriptional response to hypoxia, which is regulated by hypoxia-inducible factors (HIFs). However, the detailed molecular mechanisms of HIF regulation in hypoxia remain incompletely understood. In particular, there is controversy surrounding the production of mitochondrial reactive oxygen species (ROS) in hypoxia and how this affects the stabilization and activity of HIFs. This review examines this controversy and attempts to shed light on its origin. We discuss the role of physioxia versus normoxia as baseline conditions that can affect the subsequent cellular response to hypoxia and highlight the paucity of data on pericellular oxygen levels in most experiments, leading to variable levels of hypoxia that might progress to anoxia over time. We analyze the different outcomes reported in isolated mitochondria, versus intact cells or whole organisms, and evaluate the reliability of various ROS-detecting tools. Finally, we examine the cell-type and context specificity of oxygen's various effects. We conclude that while recent evidence suggests that the effect of hypoxia on ROS production is highly dependent on the cell type and the duration of exposure, efforts should be made to conduct experiments under carefully controlled, physiological microenvironmental conditions in order to rule out potential artifacts and improve reproducibility in research.