Characterization of radiation resistant hypoxic cell subpopulations in KHT sarcomas. (I). Centrifugal elutriation

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Abstract
Studies were performed to determine the location, with respect to cell cycle phase, of the radiobiologically hypoxic cells in KHT sarcomas. Cells dispersed from solid KHT tumours were separated into subpopulations at different stages of the cell cycle by centrifugal elutriation. Flow cytometric analysis of the DNA content of these subpopulations indicated that the degree of synchrony that could be achieved was greater than or equal to 95% for G1 cells, 70-75% for S cells and 70-75% for G2M cells. The approach to locate and characterize hypoxic cells was based on the premise that due to their lack of oxygen such cells would preferentially survive radiation treatment. Consequently KHT sarcomas were irradiated in situ either in dead animals or in animals breathing air. Following treatment, the tumours were dissociated, the cells elutriated into the various phases of the cell cycle and clonogenic cell survival was determined. Complete dose-response curves were determined for cells in the G1, S and G2M cell cycle phases. The various cell cycle subpopulations obtained from tumours irradiated while mice breathed air, all demonstrated survival curves with a break and final slope which paralleled that of the corresponding anoxic cell survival curve. From these curves the proportion of hypoxic tumour cells in the G1 phase was calculated to be 10.1 +/- 1.7%. Because the elutriated S and G2M enriched cell fractions were to some extent contaminated by cells from other phases of the cell cycle, the percentage of hypoxic S and G2M tumour cells was estimated to range from 0-7% and 0-5% respectively. However, since G1 cells comprised the majority of all the neoplastic cells in these tumours, the data suggest that hypoxic cells in KHT sarcomas are found primarily in this cell cycle stage.