Discrimination of slow growth from non-survival among small colonies of diploid Syrian hamster cells after chromosome damage induced by a range of x-ray doses.

Synchronous samples of cultured diploid Syrian hamster cells (BHK 21 C13/A3) were obtained by mitotic selection, transferred to an observation chamber and given a single X-ray dose in the range 0.2 to 3.8 Gy. Each cell's radiation response was followed by visual observations of its progress alive through post-radiation mitosis (M1), and subsequently of its clonogenicity, by methods already published (Grote et al. 1981 a, b; Joshi et al. 1982a). Our recent paper about the same cell samples (Joshi et al. 1982b) showed that the probability of reaching M1 is nearly unity in controls and over the whole dose range (mean greater than 0.99). The present paper describes the clonogenicity of each sample, based on five daily cell counts at the site of each initial cell. The frequency of viable colonies falls from 98 per cent for unirradiated cells to 8 per cent in the 3.8 Gy sample, but the proportion of these which grow slowly rises from 3 to more than 70 per cent. There was substantial overlap in cell numbers reached by larger abortive colonies and smaller slow-growth colonies, and many of the later had not reached 50 cells at the last count at 5 1/2 days. Impaired colony growth (slow growth or stop growth) was strongly correlated with chromosome fragment loss detected as micronuclei in the daughter cells of M1.

X-ray-induced chromosome damage in live mammalian cells, and improved measurements of its effects on their colony-forming ability.

We have improved the precision of the technique described by Grote et al. (1981 a,b) for the observation of the radiation responses of live cultured mammalian cells with an incubated phase-contrast microscope: the colony-forming abilities of single cells obtained by selective detachment of mitoses (instead of cell pairs as previously) may now be followed individually and may be directly compared with chromosome damage detected after post-radiation mitosis (M1). An X-ray dose of 1.4 Gy to diploid Syrian hamster cells (BHK 21 C13) in G1 had no effect on cell ability to reach M1. If chromosome fragment loss was then detected (as micronuclei) in the daughter-cell pair then colony-forming ability nearly always deteriorated, and either a stop-growth (79 per cent) or a slow-growth (21 per cent) colony resulted; but chromosomal bridges which persisted beyond M1 broke during interphase 1 and themselves caused no detectable cell damage additional to that attributable to the micronuclei which accompanied them.

A method for the scrutiny of live mammalian cells in culture and for the measurement of their proliferative ability after x-irradiation.

A method is described for the observation of live mammalian cells in culture with an incubated phase-contrast microscope. A sample of plated cells may be watched and their respective capacities to form a colony measured by daily cell counts. The method has first been used to make direct estimations of the plating efficiency of the diploid line of Syrian hamster fibroblasts, BHK 21 C13, and then to observe the response of synchronous samples of these cells to 220 kV X-rays. A dose of 1.4 Gy given in Gl has no immediate detectable effect on cell or unclear morphology, and cell capacity to reach post-irradiation mitosis in unimpaired apart from delay. In contrast, after this mitosis is completed, descendant cells from some mitoses retain a normal form and clonogenic capacity, whereas the cells from other mitoses show varying degrees of abnormality and produce either slow-growth or stop-growth (micro-) colonies.

Observations of radiation-induced chromosome fragment loss in live mammalian cells in culture, and its effect on colony-forming ability.

Our preceding paper (Crote, Joshi, Revell and Shaw 1981) described a method for the direct scrutiny of live cultured mammalian cells with a microscope, and reported that all diploid Syrian hamster cells (BHK 21 C13) of a sample given 1.4 Gy of 220 kV X-rays in Gl reached post-radiation mitosis without discernible abnormality, but then diverged in observed behaviour: descendent cells from some first mitoses continued to proliferate normally while cells from other first mitoses behaved abnormally and produced either slow-growth or stop-growth colonies. This paper completes our study of the same irradiated cell sample, and shows that these post-mitotic differences in clonogenic ability were related to acentric chromosome fragment losses at post-radiation mitosis, which were detected in live daughter-cell pairs as micronuclei. The proportion of live daughter-cell pairs scored as deficient was at least 80 per cent of the proportion of comparable fixed-and-stained mitoses with detected acentric fragments.