“Using the HypOxystation is critical to our work because… unlike an incubator, it maintains the O2 levels when cells are taken in and out, avoiding spikes of normoxia.” Timothy Audas, University of Miami.

Responses to physiological cues are governed on a cellular level by changes in protein levels, and HypOxystation users Ho et al. describe their research on mechanisms mediating these responses in their newest paper "Systemic Reprogramming of Translation Efficiencies on Oxygen Stimulus”  (Cell Reports 14, 1293-1300, 2016).  Contrary to the widely held belief that transcription and mRNA levels are the main regulators of protein expression, Dr. Ho and Dr. Timothy Audas assert that translation efficiency Te of mRNA’s that are already present in the cell exerts a much larger influence than previously thought, especially in response to a stimulus such as hypoxia.

Glioblastoma and renal clear cell carcinoma cells were maintained at hypoxia (1%) in the H35 HypOxystation and at normoxia, and mRNA levels and protein expression were compared. The correlation between mRNA and protein output was quite weak, leading the authors to postulate that cellular response to oxygen stimulus occurs through a switch in Te rather than in transcription levels. Gene silencing of hypoxic and normoxic cell cultures via transient transfection with siRNA confirmed the existence of an alternative translation initiation complex binding to the 5’ cap of the mRNA, termed hypoxic eIF4F (eIF4FH), that is not in use at normoxia. The HypOxystation reliably creates physiological conditions for cells habituated to much lower oxygen than the ambient 21%, while also controlling CO2, temperature and humidity. Working inside the HypOxystation via gloveless sleeves allows users to culture and manipulate cells for extended periods of time, without ever exposing them to the “oxygen shock” of the lab atmosphere. In an interview with the University of Guelph, co-author and HypOxystation user Jim Uniacke describes using the HypOxystation to design a suicide mRNA strategy targeting hypoxic tumors.

Three classes of mRNA’s were identified, of which approximately 40% are specifically targeted for altered translation at low oxygen. Deletion of elements of the eIF4FH or eIF4F complexes significantly impacted protein levels of cells growing in hypoxic or normoxic conditions, confirming that hypoxia-specific protein expression is regulated by translation efficiency and not mRNA levels. The majority of HIF pathway targets, instead of up-regulating transcription to produce more mRNA, actually increased the Te of the mRNA’s already present, through selective recruitment by the eIF4FH complex at hypoxia. One possible determinant enabling this recruitment may be the presence of RNA hypoxia response elements, which binds RBM4 and enables eIF4FH-mediated translation. Based on these regulatory mechanisms, Ho et al. postulate the likelihood of other, stimulus-specific programs that have allowed organisms to adapt to their dynamic environments.

As co-author Dr. Uniacke recently stated in his JBC paper “Human Cells Cultured Under Physiological Oxygen Utilize Two Cap-binding Proteins to Recruit Distinct mRNAs for Translation”, “Culturing cells in ambient air could be far from physiological with respect to oxygen. Oxygen is a surprisingly neglected factor [in cell culture].” Don’t neglect your cells - Define your Environment!