Cell-cycle dependence of X-ray oxygen effect: role of endogenous glutathione.

The oxygen effect was measured in human T-1 cell populations synchronized by mitotic selection and x-irradiated in vitro after they were allowed to progress to six different ages during the division cycle. Survival curves and dose-ratio calculations with 95% confidence intervals were obtained from computer fits of the data to the linear-quadratic model. The oxygen enhancement ratio (OER) values at the 1% survival increased level were 2.6 +/- 0.08 in G1/early S phase and increased to 3.0 +/- 0.15 in late S/G2 phase. The OER values at 10% survival increased linearly from 2.6 +/- 0.2 for G1-phase cells to 3.2 +/- 0.2 for late S/G2-phase cells. The increased OER in S-phase cells was the result of a greater hypoxic radioresistance compared with that measured with G1-phase cells. In parallel experiments with synchronized cell populations, glutathione (GSH) and glutathione disulfide levels were measured by the Tietze assay and also were found to increase over the same period. The molecular mechanisms responsible for the radiation response involve a number of factors, one of which in this cell line may be GSH levels, especially under conditions of hypoxic exposure. Our data are consistent with the hypothesis that G1- to late S-phase, age-dependent fluctuations in GSH content may be correlated with changes in OER during the human T-1 cell cycle. Changes in GSH content relative to its constitutive levels in the cell and alternative reductive factors (i.e., protein thiols), as well as their cellular location, may be important factors in the comparison of these findings to other cell lines.

Cell kinetics of GM-CFC in the steady state.

The kinetics of cell turnover for myeloid/monocyte cells that form colonies in agar (GM-CFC) were measured through the progressive increase in their sensitivity to 313-nm light during a period of cell labeling with BrdCyd. Two components of cell killing with distinctly separate labeling kinetics revealed both the presence of two generations within the GM-CFC compartment and the properties of the kinetics of the precursors of the GM-CFC. These precursors of the GM-CFC were not assayable in a routine GM-CFC assay when pregnant mouse uterus extract and mouse L-cell-conditioned medium were used to stimulate colony formation but were revealed by the labeling kinetics of the assayable GM-CFC. Further, these precursor cells appeared to enter the assayable GM-CFC population from a noncycling state. This was evidenced by the failure of the majority of these cells to incorporate BrdCyd during five days of infusion. The half-time for cell turnover within this precursor compartment was measured to be approximately 5.5 days. Further, these normally noncycling cells proliferated rapidly in response to endotoxin. High-proliferative-potential colony-forming cells (HPP-CFC) were tested as a candidate for this precursor population. The results of the determination of the kinetics for these cells showed that the HPP-CFC exist largely in a Go state, existing at an average rate of once every four days. The slow turnover time for these cells and their response to endotoxin challenge are consistent with a close relationship between the HPP-CFC and the Go pool of cells that is the direct precursor of the GM-CFC.

Use of 5-BrdUrd/313 nm light technique for identification of cells in initial S-phase within GM-CFCc compartment.

Long-term, low-level, BrdUrd infusion identifies two subpopulations of GM-CFCc with quite dissimilar sensitivities to 313 nm light. The responses of these two GM-CFCc subpopulations to hydroxyurea indicate that both are rapidly proliferating at the time of the assay. However, the absolute UV-light sensitivity of the S-phase components and the effects of increasing BrdUrd concentration indicate that the two GM-CFCc subpopulations passed through the previous cell cycle at widely disparate rates. Further, those GM-CFCc originating from a parental cell with a slow turnover are associated with a lower buoyant density than those GM-CFCc that have been in rapid cycle for at least two generations. These results indicate that the resistance to 313 nm-light irradiation, shown by S-phase cells in the first cell cycle of the BrdUrd labeling, may provide evidence of the proliferative history of the cell being assayed.