Response of human lymphocytes to mitogen: At what stage is there a requirement for Ca2+?

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Abstract
In order for significant DNA synthesis to be observed in the case of human lymphocytes stimulated for 36 h in presence of phytohemagglutinin (PHA), Ca2+ must be present in the medium continuously for at least 20 h. Access to Ca2+ for 10 h periods during the first 30 h was not sufficient to permit DNA synthesis to occur. Addition of the chelator EGTA [ethylene glycol bis (.beta.-aminoethyl ether) tetraacetic acid] from 0-60 h after stimulation caused severe inhibition of incorporation of labeled thymidine when this was measured after 36-144 h of culture. Equimolar calcium reversed the inhibition caused by EGTA. Incorporation of labeled uridine and leucine showed a temporal pattern of dependence on the presence of Ca2+ in the medium similar to that of thymidine. Ca2+ appeared not to be required in the medium during the last half (i.e., 20-36 h) of the presynthetic G1 phase nor during S phase since removal of Ca2+ from the medium after 20 h did not prevent a subpopulation of lymphocytes from entering S phase 16 h later.