DNA synthesis inhibitors block protein synthesis in the human lymphocyte

We examined the requirement for early DNA synthetic events in the protein synthetic response of PHA-stimulated human lymphocytes using specific DNA synthesis inhibitors. The incorporation of H3-thymidine into DNA and H3-leucine into protein was assessed in PHA-stimulated lymphocytes cultured with cytosine arabinoside (8.5 X 10(-6)M), hydroxyurea (1.0 X 10(-2)M) or mitomycin-C (1.5 X 10(-2)M), each of which reduced DNA synthesis to the level of unstimulated lymphocytes. Under those conditions, protein synthesis was suppressed to 2, 14 and 1% of uninhibited cells at 48 h, respectively. Delaying addition of these inhibitors to 6, 12 and 24 h after initiation of PHA stimulation resulted in a parallel loss of inhibition of both RNA and protein synthesis. However, during the first 24 h of uninhibited mitogen stimulation, there is no DNA synthesis as measured by either H3-thymidine uptake or by flow cytometry. In contrast to the inhibition of protein synthesis when measured after 48 h, PHA-stimulated H3-leucine incorporation during the first 24 h was not affected by DNA synthesis inhibition. These results suggest that early events related to DNA synthesis are required for optimum mitogen-induced protein and RNA synthesis, but that new DNA synthesis does not occur until after 24 h of mitogen stimulation.