Induction of keratinocyte proliferation by a short treatment with keratinocyte-conditioned medium

The effect of a short treatment with keratinocyte-conditioned medium (KCM) on the growth of normal human epidermal keratinocytes was investigated. Serum-free MCDB153 medium was conditioned by keratinocytes for 24 h after plating. Following attachment to uncoated plastic surfaces (4 h after plating), cells were exposed for 20 h to KCM. After 10 days of culture in MCDB153 medium, an increase of about six-fold in cell number was observed in KCM-treated plates over controls, indicating that a short treatment with KCM is sufficient to induce cell proliferation. The effect of addition of KCM at different times after plating was also evaluated: KCM treatment resulted to exert its maximum effect on cell proliferation, when performed immediately after the completion of attachment of cells to the surface of the dish. Mitogenic activity present in KCM is not inhibited by heparin sulphate. The kinetics of accumulation of this early secreted growth-stimulating activity showed that a plateau is reached within 24 h of conditioning. These data suggest that this mitogenic activity should not be amphiregulin. The observation that, following KCM treatment, the majority of cells is able to incorporate [3H]-thymidine as compared to controls suggests that the observed final increase in cell number is due to an increase in the number of cycling cells rather than to a shortening of the cell cycle.