REGULATION OF CELL-PROLIFERATION UNDER EXTREME AND MODERATE HYPOXIA -THE ROLE OF PYRIMIDINE (DEOXY)NUCLEOTIDES

In the present study we have used flow cytometric DNA measurements on synchronised human NHIK 3025 cells to measure cell cycle progression u nder various conditions of reduced oxygenation. Our data indicate that addition of 0.1 mM deoxycytidine or uridine has no effect on the oxyg en-dependent arrest in late G(1) or on the inhibition of cell prolifer ation through S-phase under extremely hypoxic conditions. Following re oxygenation of cells exposed to extremely hypoxic conditions in G(2) i nitiation of DNA synthesis in the subsequent cell cycle is delayed by several hours. This G(2)-induced delay is completely abolished for app roximately 60% of the cell population by addition of deoxycytidine to hypoxic G(2) cells. This finding supports our previous proposal that i mportant steps in the preparation for DNA synthesis occur during G(2) of the previous cell cycle, and it indicates that this preparation is connected to the de novo synthesis of pyrimidine deoxynucleotide precu rsors. The results show that cells are able to enter S-phase in the pr esence 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 de oxycytidine and uridine, while no such reversal is seen in the presenc e of purine deoxynucleosides (deoxyadenosine and deoxyguanosine). Thus , both deoxycytidine and uridine could replace reoxygenation under the se conditions. These results indicate that the reduction of CDP to dCT P by ribnonucleotide reductase, an enzyme which requires oxygen as an essential factor for the formation of tyrosyl radicals for its catalyt ic activity, does not seem to be the limiting step responsible for the reduced dCTP pool observed under moderate hypoxia. We conclude that t he oxygen-dependent catalytic activity of the M2 subunit of ribonucleo tide 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 dehydroge nase.