Effect of Pharmacological Agents on Human Keratinocyte Mitosis in Vitro I. Inhibition by Adenine Nucleotides

In the last few years many studies have suggested that cyclic adenosine 3': 5' monophosphate (cyclic AMP) may be specifically involved in regulation of proliferation and differentiation of many types of mammalian cells in vitro (1-4). In organ cultures of mouse skin, the reduction of epidermal mitosis by epinephrine is associated with an increase in adenyl cyclase activity (5, 6). This finding supports results in other in vitro systems showing that increased cyclic AMP levels are inhibitory to cellular proliferation (1, 4). Recently, Voorhees et al. (7), showed that dibutyryl cyclic AMP, but not 5'-AMP, inhibited epidermal cell division in mouse skin in organ culture. Using HeLa and L cells, Ryan and Heidrick (1) reported inhibition of cell growth by cyclic AMP but not by 5'-AMP or adenosine. Others who have investigated the role of cyclic AMP in cellular function either found no effect of 5'-AMP, ADP and ATP, or found that these nucleotides are toxic to cells (1, 2, 8). Thus, specificity of cyclic AMP has been implied. However, we report here a study, using primary cell cultures of normal human epidermal keratinocytes, which indicates that nucleotides other than cyclic AMP may affect a specific physiological function, namely mitosis, in the absence of observable toxic effects.

Materials and Methods. Normal human abdominal skin was obtained during surgery and cell cultures of epidermal keratinocytes were made from skin explants on glass coverslips according to a method described elsewhere (9). Each explant gives rise to an outgrowing sheet of epithelial cells whose origin from epidermal keratinocytes has previously been determined (9, 10). Four to five explants were placed on each coverslip. Cultures were immersed in Eagle's minimum essential medium supplemented with 10% fetal calf serum and 100 U/ml each of penicillin, streptomycin and mycostatin.