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.
Supported by National Cancer Institute Research Grants CA 20529 and CA 13525.