The kinetics of the formation of a G2 block from tritiated thymidine in phytohemagglutinin‐stimulated human lymphocytes

Abstract

Flow cytometry (FCM) was used to monitor the radiation effects promoted by incorporated tritiated thymidine (³H‐TdR) on phytohemagglutinin (PHA)‐stimulated human peripheral blood lymphocytes stained with propidium iodide (PI). Lymphocyte microcultures were continuously labeled or pulse‐labeled for various periods of time with different ³H‐TdR concentrations. Two types of DNA histogram analyses wer performed on unperturbed and ³H‐TdR perturbed lymphocytes. The data analyses consisted of statistical analyses between averaged groups of histograms (nonparametric analysis) and cell cycle analyses (parametric analysis) to determine the percentages of cells in G0 + G1, S and G2 + M. The results showed that (a) ³H‐TdR when added to proliferating lymphocytes under certain conditions (both short‐term continous and pulse‐labeling) caused a highly significant increase in the proportion of tetraploid (4C) cells by FCM, (b) the increase in the proportion of 4C cells represented a block in G2 and (c) the relative increase in the percentage of 4C cells was proportional to ³H‐TdR incorporation which was proportional to labeling time and concentration. Therefore, it was concluded that short labeling times be used to minimize adverse radiation effects when ³H‐TdR is used to assay substances affecting lymphocyte proliferation or in the estimation of cell cycle time.