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 and proceed to DNA synthesis in S-phase by a wide range of growth factors and mitogens. Activation of cell-surface growth factor receptors with intrinsic protein tyrosine kinase activity initiates autophosphorylation of the receptors and subsequent activation of signal transduction cascades. After activation the receptors undergo ligand-induced internalization to endosomes, which become acidified by the action of a vacuolar H+-ATPase (V-ATPase). The extent to which vesicular acidification plays a role in mitogenic signalling by receptors with intrinsic tyrosine kinase activity remains unknown. Here we have shown that bafilomycin A(1), a specific inhibitor of V-ATPase, inhibits endosome acidification and mitogen-induced DNA synthesis in Swiss 3T3 fibroblasts. Addition of bafilomycin A(1) at successively later times during G(1) progressively decreased the inhibition of DNA synthesis such that no inhibition was observed when bafilomycin A(1) was added at the onset of S-phase. Bafilomycin A(1) also induced a dramatic but reversible change in the morphology of Swiss 3T3 cells. However, the rapid activation of c-fos mRNA accumulation by epidermal growth factor and insulin was unaffected by bafilomycin A(1). Together, the results suggest that activation of the V-ATPase plays an important role in the mitogenic signalling pathways that occur during the G(1) phase of the cell cycle but is not required for the initial epidermal growth factor and insulin-evoked signalling events that lead to c-fos mRNA expression.