Cycling mammalian cells that are rendered extremely hypoxic (less than 4 ppm O2) tend to accumulate in a pre-DNA-synthesis stage. It is not clear whether or not this is the result of an active regulation by the cells. In the present study we have rendered cells, synchronized by mitotic selection, extremely hypoxic over a relatively long period of time (up to 48 h). We have recorded cell cycle progression during hypoxia as well as cell inactivation depending on where in the cell cycle the cells were located when the hypoxic treatment was started. Three main conclusions are drawn: 1 the cell cycle arrest in late-G1 is complete even during a long-lasting (24 h) hypoxic treatment: 2 while cells in early- and mid-S are completely arrested and quickly inactivated under hypoxic conditions, cells in late-S, G2 and mitosis are able to continue cell cycle progression and divide; 3 whether the cells are located in G2, mitosis or early-G1 at the onset of hypoxia, they were able to survive relatively long-lasting hypoxic treatment. The present results are in favour of the view that the pre-DNA-synthetic arrest induced by extreme hypoxia may function to rescue the cells from severely damaging effects that would appear if the cells were able to initiate DNA synthesis.

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Summary We have studied the role of the oxygen-dependent pyrimidine metabolism in the regulation of cell cycle progression under moderate hypoxia in human cell lines containing functional (T-47D) or non-functional (NHIK 3025, SAOS-2) retinoblastoma gene product (pRB). Under aerobic conditions, pRB exerts its growth-regulatory effects during early G, phase of the cell cycle, when all pRB present has been assumed to be in the underphosphorylated form and bound in the nucleus. We demonstrate that pRB is dephosphorylated and re-bound in the nucleus in approximately 90% of T-47D cells located in S and G2 phases under moderately hypoxic conditions. Under these conditions, no T-47D cells entered S-phase, and no progression through S-phase was observed. Progression of cells through G2 and mitosis seems independent of their functional pRB status. The p21 WAF1/CIP1 protein level was significantly reduced by moderate hypoxia in p53-deficient T-47D cells, whereas p1 6INK4a was not expressed in these cells, suggesting that the hypoxia-induced cell cycle arrest is independent of these cyclindependent kinase inhibitors. The addition of pyrimidine deoxynucleosides did not release T-47D cells, containing mainly underphosphorylated pRB, from the cell cycle arrest induced by moderate hypoxia. However, NHIK 3025 cells, in which pRB is abrogated by expression of the HPV18 E7 oncoprotein, and SAOS-2 cells, which lack pRB expression, continued cell cycle progression under moderate hypoxia provided that excess pyrimidine deoxynucleosides were present. NHIK 3025 cells express high levels of p16INK4a under both aerobic and moderately hypoxic conditions, suggesting that the inhibitory function of p1 6INK4a would not be manifested in such pRB-deficient cells. Thus, pRB, a key member of the cell cycle checkpoint network, seems to play a major role by inducing growth arrest under moderate hypoxia, and it gradually overrides hypoxia-induced suppression of pyrimidine metabolism in the regulation of progression through S-phase under such conditions.

Sary 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 GI or on the inhibition of cell prolferation 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 aboLshed 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 nbnonucleotide 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.

The work demonstrates that prolonged hypoxia followed by re-oxygenation results in a G2 delay similar to that observed after DNA damage. Furthermore, chronic hypoxia results in decreased radiation survival for at least 50h following the reintroduction of oxygen. The hypoxia-induced radiosensitization following 7 h of re-oxygenation could in large part be explained by the synchronous cell cycle progression that occurred.