Studies on in vitro lymphocyte proliferation in cultures synchronized by the inhibition of DNA synthesis: I. Variability of S plus G2 periods of first generation cells

Abstract

The addition of amethopterine plus adenosine, fluorodeoxyuridine or thymidine in high concentrations to PHA-stimulated human peripheral lymphocytes in vitro resulted in reversible accumulation of the great majority of stimulated cells at the first G1/S point of the cell cycle or early in the S phase—provided that conditions preventing DNA precursors reutilization were maintained. The kinetics of cell proliferation was studied in such cultures up to 24 h following release of the block. A diffuse, asymmetric and apparently bimodal time-distribution of mitotic activity was found. The variability of the S plus G2 periods in first generation cells was estimated from these results: Minimal S plus G2 was in the 6–9 h range; the maximal time was over 24 h. Median S plus G2 duration thus calculated was in the 16–18 h range, as compared with 17.7 h previously found in a random population of second and subsequent generation cells. Hence, it was concluded, that the duration of S plus G2 does not depend upon number of generations in vitro. Indirect evidence was obtained, indicating that PHA-stimulated lymphocytes may enter the first in vitro DNA synthesis period after more than 72 h in culture. Thus it was inferred that the duration of the “growth induction period” of stimulated lymphocytes is also subject to considerable variability. The advantages of procedures based on reversible inhibition of DNA synthesis, in quantitative studies on lymphocyte stimulation and proliferation, are discussed.