Inhibition of mitogen-induced DNA synthesis by bafilomycin A1 in Swiss 3T3 fibroblasts Available to Purchase

Quiescent cells (in G₀) 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 A₁, a specific inhibitor of V-ATPase, inhibits endosome acidification and mitogen-induced DNA synthesis in Swiss 3T3 fibroblasts. Addition of bafilomycin A₁ at successively later times during G₁ progressively decreased the inhibition of DNA synthesis such that no inhibition was observed when bafilomycin A₁ was added at the onset of S-phase. Bafilomycin A₁ 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 A₁. Together, the results suggest that activation of the V-ATPase plays an important role in the mitogenic signalling pathways that occur during the G₁ 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.