12-Hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) does not stimulate proliferation of human neonatal keratinocytes

We have developed an assay to study the effect of drugs on the proliferation of neonatal human skin-derived keratinocytes in vitro. Expanding populations of neonatal keratinocytes were cultured in low concentrations (0.5%) of fetal calf serum for up to 12 d. Growth of the cultures was determined by measurement of DNA using a sensitive fluorimetric assay. Addition of 10(-9)-10(-6) M 12(RS)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(RS)-HETE) neither stimulated keratinocyte proliferation nor enhanced the incorporation of [3H]thymidine. The ability of neonatal keratinocytes in low serum medium to respond to exogenous factors was demonstrated by increased growth in response to a mixture of cholera toxin, hydrocortisone, and epidermal growth factor. Confluent keratinocyte cultures in 10% human AB serum exposed to 12(S)-HETE for 72 h also showed no changes in DNA, [3H]thymidine incorporation, or labeling index. Metabolism of 12(S)-[3H]HETE was greater in cultures containing low concentrations of serum but there was no evidence for the formation of 12,20-dihydroxyeicosatetraenoic acid.