Effects of cholera toxin on proliferation of cultured human keratinocytes in relation to intracellular cyclic AMP levels.

In the culture of epidermal keratinocytes, the cellular growth rate is reported to be accelerated by cholera toxin. The mechanism by which cholera toxin exerts biological effects is thought to result from changes in intracellular cyclic AMP concentrations. But in many reports cyclic AMP elevating agents appeared to inhibit growth of keratinocytes in culture. This study was done to clarify the discrepancy of this problem. Determination of cyclic AMP revealed that cholera toxin over a range of 10-14-10-8 M increased the intracellular concentration of cyclic AMP of cultured keratinocytes about 100-fold over the controls after incubation for 6 hr. When a small number (10(5)) of cells were inoculated in a 60 X 15 mm culture dish, cholera toxin strongly stimulated colony growth. When a relatively larger number (8 X 10(5)) of cells were inoculated in a dish, cholera toxin moderately accelerated cell division, and increased DNA and protein levels of the culture during early days of cultivation. But after about 20 days, of cultivation when the culture reached confluence, cholera toxin decreased both DNA and protein content in a culture dish. The cultures were pulse labeled with 3H-thyrmidine at 12 and 24 hr after the addition of 10-10 M cholera toxin, and its uptake into DNA was determined. In the early days of cultivation the uptake of 3H-thymidine increased after treatment with cholera toxin. But in the late days of cultivation, cholera toxin decreased the rate of 3H-thymidine incorporation into DNA. These results indicated that cholera toxin-cyclic AMP has effects on the proliferation of keratinocytes in culture biphasically according to cellular concentrations in culture.