Role of ribonucleotide reductase in regulation of cell cycle progression during and after exposure to moderate hypoxia.

Earlier studies have shown that the retinoblastoma protein (pRb) is involved in cell-cycle regulation under conditions of moderate hypoxia, which is the term we use to denote oxygen concentrations just above the lower level giving full respiration, ie. 1300 ppm O2. We have studied the cell cycle regulatory influence of varying levels of ribonucleotide reductase under moderate hypoxia in human cancer cells with either functional (T47D and T47DHU-res) or non-functional pRb due to expression of HPV18 E7 (HeLa S3). In this study we adapted a cell-line to hydroxyurea (T47DHU-res) resulting in an increased level of ribonucleotide reductase tyrosyl-radical that is not cell-cycle dependent. Under moderate hypoxic stress, pRb becomes dephosphorylated and rebound in the cell nucleus in all phases of the cell cycle, in contrast to its function under aerobic conditions where it binds only during early G1. We have shown in this paper that, upon reoxygenation, dephosphorylation and binding to the nucleus of pRb is reversible in T47D cells, although phosphorylation is delayed by more than 12 hours. As a result of the dephosphorylation of pRb, T47D cells are between 12 and 24 hours slower in reinitiating their S-phase progression than HeLa S3 cells (not containing functional pRb) following reoxygenation. However, with an increased level of ribonucleotide reductase tyrosyl-radical in the T47DHU-res cells, the S-phase progression after reoxygenation is reinitiated earlier, although the pRb status of these cells is the same as in T47D cells.