Regulation of cell proliferation under extreme and moderate hypoxia: the role of pyrimidine (deoxy)nucleotides

Abstract

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.

Review: Implications of In Vitro Research on the Effect of Radiotherapy and Chemotherapy Under Hypoxic Conditions

“…In agreement with the observations of Zö lzer and Streffer [28], Amellem et al [45] noticed that cells are able to enter the S phase during anoxia (0.01-0.13% O 2 ), although they cannot complete DNA synthesis.…”

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein

“…In a previous study, we showed that addition of deoxycytidine during moderate hypoxia in NHIK 3025 cells reversed the cell cycle-inhibitory effect of hypoxia (Amellem et al, 1994). In the present study, we therefore added 0.1 mm deoxycytidine to see if this treatment could reverse the hypoxia-induced arrest that was related to the rebinding (dephosphorylation) of pRB to nuclear structures in cells in S-phase.…”

“…NHIK 3025 cells contain integrated HPV1 8 and express the HPV1 8 E7 oncoprotein (Figure 4). The median cell cycle duration for NHIK 3025 cells was 16.5 h (Amellem et al, 1994). All cell lines were kept in exponential growth by reculturing three times a week.…”

“…It appears that reduced progression through S-phase is halted or inhibited under moderate hypoxia (greater than approximately 100 p.p.m. oxygen) due to reduced de novo synthesis of pyrimidine deoxynucleotides (Loffler, 1992;Amellem et al, 1994). Suppression of the enzymatic activity of two enzymes, which both depend on the presence of molecular oxygen, dihydroorotate dehydrogenase and ribonucleotide reductase, is presumably responsible for the reduced pyrimidine deoxynucleotide pools observed during hypoxia (Loffler et al, 1983).…”

“…Suppression of the enzymatic activity of two enzymes, which both depend on the presence of molecular oxygen, dihydroorotate dehydrogenase and ribonucleotide reductase, is presumably responsible for the reduced pyrimidine deoxynucleotide pools observed during hypoxia (Loffler et al, 1983). In some cells, addition of pyrimidine deoxynucleosides (such as deoxycytidine) under moderate hypoxia is used by the salvage pathway to restore the dCTP/dTTP pools and thus cell cycle progression (Amellem et al, 1994).…”