The Choline Kinase Inhibitor Hemicholinium-3 Can Inhibit Mitogen-Induced DNA Synthesis Independent of Its Effect on Phosphocholine Formation

In NIH 3T3 cells, phosphocholine (PCho) stimulates mitogenesis in synergism with insulin, ATP, and sphingosine-1-phosphate (S1P) via an extracellular target. Intracellular PCho also has been suggested to mediate the mitogenic effects of fibroblast growth factor (FGF) and several other growth factors based, in part, on the observed inhibition of growth factor-induced mitogenesis by the choline kinase inhibitor hemicholinium-3 (HC-3). Here we examined the specificity of HC-3 effects on mitogenesis in serum-starved NIH 3T3 and Swiss 3T3 cells. In both cell lines, FGF greatly enhanced DNA synthesis in a medium containing 28 μM choline, and it also stimulated the formation of [14C]PCho from both 50 μM and 5 mM [14C]choline. HC-3 (2 mM) inhibited basal or FGF-induced formation of [14C]PCho and [14C]phosphatidylcholine as well as the uptake of [14C]choline only at the 50 μM, but not the 5 mM, concentration of [14C]choline. In addition, HC-3 (1 mM) from three different sources (95–99.9% purity) inhibited FGF-stimulated DNA synthesis by 53–58% which was not reversed by 5 mM choline. The choline analogue dimethylethanolamine (1 mM) also inhibited FGF-stimulated formation of [14C]PCho from 50 μM [14C]choline, but it had no effect on FGF-induced DNA synthesis. Of the other growth regulators examined, synergistic stimulation of DNA synthesis by extracellular PCho and S1P or PCho and ATP via choline kinase-independent mechanisms was inhibited by 2 mM HC-3. However, HC-3 failed to inhibit the synergistic mitogenic effects of PCho and insulin or S1P and insulin. The results suggest that FGF-induced mitogenesis does not require PCho formation and that HC-3 can inhibit DNA synthesis independent of its inhibitory effects on choline metabolism.