Microtubules: are they involved in the initiation of lymphocyte activation?

Purified human blood lymphocytes were stimulated with concanavalin A or phytohemagglutinin. DNA synthesis was measured with 2-h pulses of [3H]thymidine between 48 h and 73 h after stimulation. Colchicine, at concentrations between 0.1 muM and 10 muM, suppressed consequent DNA synthesis without affecting viability of the cells when added at any time up to 18 h before incorporation of [3H]thymidine was assessed. In concanavalin-A-stimulated lymphocytes, removal of the mitogen by methyl alpha-mannoside only prevented proliferation when added initially, but was without any effect when added after 20 h of stimulation, regardless of when DNA synthesis was measured. Thus, there was a period after 20 h of concanavalin A stimulation, when DNA synthesis was still sensitive to colchicine, but no longer required the presence of the mitogen. Colchicine also suppressed incorporation of [3H]leucine into protein, in resting as well as mitogen-stimulated lymphocytes. Similarly, colchicine decreased amino acid transport, as determined by uptake of alpha-amino-isobutyrate, which appeared to be the rate-limiting step in the incorporation of amino acids into protein in colchicine-treated cells. When the rate of protein synthesis was followed by the relative distribution of ribosomal particles, especially the increase of polysomes in activated lymphocytes, colchicine was without any detectable effect. The early increase in the incorporation of [14C]oleate into phospholipids was identical in the presence or absence of the microtubule-active drug. The data strongly suggest that microtubules are not involved in the initiation of lymphocyte growth or mitogenesis.