Effects of Extreme Hypoxia on the Growth and Viability of EMT6/SF Mouse Tumor Cells in Vitro¹

The purpose of this study was to characterize a model system in which to study hypoxic cell biology in vitro as a function of time under extremely hypoxic conditions. EMT6/SF cells that were maintained at 37° under hypoxic conditions showed no increase in cell number for up to 70 hr. The mitotic index of hypoxic cultures was less than 0.1%, compared to 2.3 to 3.0% in aerated cultures. The plating efficiency of hypoxic cells decreased with time to 20 to 30% of control values by 70 hr. Aerated cultures consumed glucose more rapidly than did hypoxic ones, due to increasing cell number in air. But, on a per cell basis, hypoxic and aerated cells consumed glucose at equal rates (≃1.2 × 10^-4 µg/cell/hr). Virtually 100% of the glucose consumed was converted into lactic acid in both aerated and hypoxic cultures. The labeling index and rate of incorporation of [³H]thymidine decreased exponentially with time in hypoxia. However, the percentage of cells with S-phase DNA content remained nearly constant for up to 72 hr. The rate of protein synthesis was suppressed in hypoxic cultures to between 20 and 50% of control (aerated) rates. When cultures were reaerated following 45 hr of hypoxia, ≃12 hr was required for resumption of DNA synthesis and cell division. The application of this system to further study of hypoxic cell biology is discussed.