In the present study we have used flow cytometric
DNA measurements on synchronised human NHIK 3025 cells to measure cell cycle progression under various conditions of reduced oxygenation. Our data indicate that addition of 0.1 mM
deoxycytidine or
uridine has no effect on the
oxygen-dependent arrest in late G1 or on the inhibition of cell proliferation through S-phase under extremely hypoxic conditions. Following reoxygenation of cells exposed to extremely hypoxic conditions in G2 initiation of
DNA synthesis in the subsequent cell cycle is delayed by several hours. This G2-induced delay is completely abolished for approximately 60% of the cell population by addition of
deoxycytidine to hypoxic G2 cells. This finding supports our previous proposal that important steps in the preparation for
DNA synthesis occur during G2 of the previous cell cycle, and it indicates that this preparation is connected to the de novo synthesis of
pyrimidine deoxynucleotide precursors. The results show that cells are able to enter S-phase in the presence of 100-1,300 p.p.m. (0.01-0.13%)
oxygen (here denoted 'moderate
hypoxia'), but they are not able to complete
DNA synthesis under such conditions. However, the cell cycle inhibition induced under moderate
hypoxia is partially reversed in the presence of exogenously added
deoxycytidine and
uridine, while no such reversal is seen in the presence of
purine deoxynucleosides (
deoxyadenosine and
deoxyguanosine). Thus, both
deoxycytidine and
uridine could replace reoxygenation under these conditions. These results indicate that the reduction of
CDP to
dCTP by
ribonucleotide reductase, an
enzyme which requires
oxygen as an essential factor for the formation of tyrosyl radicals for its catalytic activity, does not seem to be the limiting step responsible for the reduced
dCTP pool observed under moderate
hypoxia. We conclude that the
oxygen-dependent catalytic activity of the M2 subunit of
ribonucleotide reductase is still intact and functional in NHIK 3025 cells even at
oxygen concentration as low as 100 p.p.m. Therefore the cell cycle inhibition observed is probably due to inhibition of the respiratory chain-dependent
UMP synthesis at the stage of
dihydroorotate dehydrogenase.