INHIBITION OF MITOGEN-INDUCED DNA-SYNTHESIS BY BAFILOMYCIN A(1) IN SWISS 3T3 FIBROBLASTS

Quiescent cells (in G(0)) can be stimulated to enter the cell cycle an d proceed to DNA synthesis in S-phase by a wide range of growth factor s and mitogens. Activation of cell-surface growth factor receptors wit h intrinsic protein tyrosine kinase activity initiates autophosphoryla tion of the receptors and subsequent activation of signal transduction cascades. After activation the receptors undergo ligand-induced inter nalization to endosomes, which become acidified by the action of a vac uolar H+-ATPase (V-ATPase). The extent to which vesicular acidificatio n plays a role in mitogenic signalling by receptors with intrinsic tyr osine kinase activity remains unknown. Here we have shown that bafilom ycin A(1), a specific inhibitor of V-ATPase, inhibits endosome acidifi cation and mitogen-induced DNA synthesis in Swiss 3T3 fibroblasts. Add ition of bafilomycin A(1) at successively later times during G(1) prog ressively decreased the inhibition of DNA synthesis such that no inhib ition was observed when bafilomycin A(1) was added at the onset of S-p hase. Bafilomycin A(1) also induced a dramatic but reversible change i n the morphology of Swiss 3T3 cells. However, the rapid activation of c-fos mRNA accumulation by epidermal growth factor and insulin was una ffected by bafilomycin A(1). Together, the results suggest that activa tion of the V-ATPase plays an important role in the mitogenic signalli ng pathways that occur during the G(1) phase of the cell cycle but is not required for the initial epidermal growth factor and insulin-evoke d signalling events that lead to c-fos mRNA expression.