Effect of minoxidil on pre- and postconfluent keratinocytes

Studies in our laboratory have shown that minoxidil prolongs the life of keratinocytes in culture and extends the time after confluence that cells can be subcultured. These data suggest that the drug reduces the rate at which cells are lost from the germinative pool and hence slows senescence. In a dose-response study with minoxidil, the maximal effect of the drug was seen at doses from 6 to 12 micrograms/ml; however, activity could be detected at doses below 1 microgram/ml. Cells subcultured during log growth failed to demonstrate that minoxidil increased the total number of generations attainable under these conditions, although as expected, epidermal growth factor extended the life span of cells. When the experiments were repeated in a keratinocyte cell line that does not require a fibroblast feeder layer, the same results were obtained, indicating that the difference observed between log phase and postconfluence growth cannot be explained by the presence of fibroblasts. Minoxidil's effect on postconfluent cells was blunted by the addition of cholera toxin to the medium, suggesting that elevation of cyclic adenosine monophosphate cannot be a mechanism, although reduction of cyclic adenosine monophosphate is a possibility. Finally, maintaining keratinocytes at a 20 to 40 mM calcium concentration greatly reduced the ability of postconfluent cells to be subcultured, in comparison with the normal calcium concentration of 2 mM. That minoxidil almost completely reversed this inhibitory effect suggests it may work by preventing cross-linking by transglutaminase, which is activated by elevated calcium concentrations.