In vitro analysis of the control of keratinocyte proliferation in human epidermis by physiologic and pharmacologic agents

Abstract

Human keratinocytes grown in vitro as epithelial outgrowths or as organ cultures maintain many of their normal functions such as proliferation and keratinization. These in vitro systems have been used to analyze the effect of various agents on proliferation. All adenine nucleotides, including dibutyryl cyclic AMP, blocked mitosis in the G2 part of the cell cycle at concentrations of 1 x 10-4 M. Some nonadenine nucleotides also showed this effect, but only at higher concentrations, an indication that the effect was specific for adenine nucleotides. Dibutyryl cyclic AMP and theophylline both depressed the incorporation of [3H] thymidine into DNA. Catecholamines such as isoproterenol, epinephrine, and norepinephrine were also potent inhibitors of mitosis (G2 block) at concentrations of 1 x 10-8 to 1 x 10-10 M. The fact that the effect could be blocked by the beta blocking agent, propranolol, suggests the existence of specific membrane receptor sites. However, dichloroisoproterenol, another beta blocker, had distinct inhibitory properties in itself and thus indicated that the mechanism of action of catecholamines in human keratinocytes is complex and may involve more than binding to specific receptor sites. Histamine at a concentration of 2 x 10-6 M was also a strong mitotic inhibitor. This finding is directly opposed to that in rat skin where mitosis is stimulated. Imidazole acetate, a histamine breakdown product, was found to be a striking mitotic stimulator in organ culture. A water extractable protein (chalone) from human skin also caused a block in G2. Most of the substances tested occur naturally in the cell or organism and their ability to stimulate or depress proliferation in vitro suggests that they play a regulatory role in vivo.