The effect of donor age on the proliferative response of human and mouse keratinocytes to phorbol, 12-myristate, 13-acetate

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Epidermal keratinocytes grow clonally when provided with a 3T3 feeder layer and medium supplemented with 20% foetal bovine serum, hydrocortisone and cholera toxin. In this culture system the proliferative response of freshly isolated human and mouse keratinocytes to a short exposure (24 h) of the tumour promoter phorbol, 12-myristate, 13-acetate (PMA) was dependent on the donor age but was independent of the species or the biopsy site. Human keratinocytes from early (16-18 week) foetal donors displayed a strong proliferative response to PMA as determined by a 5- to 7-fold increase in colony number and an increase in the average colony size. In contrast, adult and juvenile keratinocytes of all ages from both mice and humans displayed an inhibition of colony forming efficiency and a reduction in colony size. When continuously passaged in culture human foetal keratinocytes gradually changed the pattern of their response to PMA so that they were inhibited from growing rather than being stimulated and after 21 days (three passages) their response was quantitatively similar to juvenile keratinocytes. Co-culture of juvenile keratinocytes with irradiated foetal keratinocytes did not alter their response to PMA so that the observed proliferative response of foetal keratinocytes to PMA is not readily explained by the autosecretion of mitogens or other regulatory molecules by these cells. Late foetal (17 days gestation), neonatal and post-neonatal (5-10 days old) mouse keratinocytes were also inhibited from growing by PMA but the magnitude of the effect was directly related to the age of the mouse and was in all cases less than that observed with adult mice. The relationship of these results to the mechanism of action of phorbol esters in epidermis is discussed.