Keratinization of human keratinocytes in cell culture: the effect of cyclic AMP

Human keratinocytes in culture were treated with dibutyryl cyclic AMP (DBcAMP) at concentrations of 10(-3) and 10(-4) M, and labeled with [3H]histidine for 48 hours. The proteins of the keratinocytes were serially extracted in 0.25 M sucrose, 0.1 M sodium citrate-citric acid (pH 2.6), 6 M urea-0.1 M Tris-HCl(pH 9.0), and 6 M urea-0.1 M Tris-HCl(pH 9.0) with 1% mercaptoethanol. The incorporation of [3H]histidine into the extract as assayed by DPM of 3H/microgram protein was increased in 0.25 M sucrose extract and Tris-urea extract of the cultures treated with 10(-3) M and 10(-4) M DBcAMP as compared with the control culture. There was no difference between the citrate-buffer extract of the control and that of the DBcAMP-treated cultures. The incorporation of [3H]histidine into the Tris-urea-mercaptoethanol extract was small, and this fraction was thought to be inactive metabolically. It was concluded that dibutyryl cycli AMP stimulated the protein synthesis of the cultured keratinocytes and induced a qualitative change toward accelerating the synthesis of keratinous protein.