Studies on in vitro lymphocyte proliferation in cultures synchronized by the inhibition of DNA synthesis: II. Kinetics of the initiation of the proliferative response

Reversible inhibition of DNA synthesis can be produced by adding amethopterine plus adenosine to a culture of cells. This results in accumulation of cells at the G1/S point. This blocking procedure was used in PHA-stimulated lymphocyte cultures to find the proportion of cells which respond to the mitogen. It was also used to study the different rates at which stimulated cells reached the G1/S point. The induction period between addition of PHA and the onset of DNA synthesis was estimated by scoring labelled cells in PHA-stimulated human lymphocyte cultures after different periods of continuous incubation with the DNA synthesis inhibitors, followed by release of the block with 3H-thymidine. The induction period varied markedly from cell to cell, the extreme values being separated by more than 50 h. It was shown that this variability did not depend upon the time of exposure to, or availability of, PHA in the culture medium. The curve showing the rate of accumulation of cells at the G1/S point was found to be biphasic in several experiments and may reflect the presence of two PHA-reactive lymphocyte subpopulations. A biphasic curve was seen particularly in cultures stimulated with lower than optimal concentrations of PHA. The arrival of cells at the G1/S point was delayed in cultures stimulated with still lower concentrations of PHA. The ability of cells to enter the S phase following prolonged G1/S block seemed to depend upon the continuous presence of PHA in the culture medium.