All colonies of CHO-K1 cells surviving γ-irradiation contain non-viable cells

This paper addresses the problem of the production of defective cells within clones arising from irradiated progenitor cells and is specifically aimed at answering the question of whether lethal mutations result from a generalised effect which lowers the ability of all the progeny to divide successfully or whether it represents a late expressed but unique lethal defect induced by radiation which occurs in some cells only and which causes those cells only to cease dividing. The results obtained from autoradiographic analysis of cells within individual surviving colonies (i.e. containing more than 150 cells) suggests that some cells in all clones are not synthesizing DNA over a 9-h period and that the proportion of non-synthesising cells rises with increasing dose of radiation from less than 3% in the controls to 80-85% after a progenitor dose of 12.5 Gy. Because of the possibility that cells had longer division times post irradiation, these results were repeated using Ki67 antibody labelling, a technique which identifies cells which are in cycle. The results were similar. This suggests the non-labelled cells were not reproducing. Both techniques were also used to look at the % labelling of morphologically abnormal cells in the colonies. The results suggested that up to 35% of these abnormal cells were actively cycling and about 20% were synthesising DNA. Abnormal cells did not appear in subcultures of survivor progeny suggesting that they may have failed to replate successfully and may contribute to the lethally mutated population. The idea that radiation induces a general instability in the cell population was supported by experiments where growth and the plating efficiency of irradiated progeny was measured daily. This revealed that the growth curves deviated from the control by a constant factor suggesting a division probability of about 70% of the control level after a progenitor dose of 10 Gy. The results are discussed in the context of their significance for survival curve analysis and for radiotherapy and radiation protection results.