Responses of human cervical keratinocytes in vitro to tumour promoters and diethylstilboestrol.

Abstract

We have compared the responses of normal human cervical keratinocytes (HCE) to diethylstilboestrol (DES), and the promoting agents, phorbol-12-myristate-13-acetate (PMA) and mezerein using the loss of cloning efficiency as a measure of terminal differentiation in vitro. Dose-response studies showed that normal HCE are growth inhibited by chronic exposure to DES at concentrations greater than or equal to 2.5 X 10(-5) M, to PMA at concentrations greater than 10(-8) M and mezerein at concentrations greater than 10(-9) M. Compared to acetone controls, promoter or DES-treated cells exhibited a 10- to 12-fold increase in cornified-envelope formation. Normal HCE exhibit a heterogeneous response to PMA in that 85-90% of colony-forming cells lose their colony-forming ability after a 24-h exposure to 10(-6) M PMA. The PMA-resistant subpopulation, PMAR, remains constant and is not reduced even after 96 h chronic exposure to PMA. In contrast, the colony-forming ability of normal HCE is almost totally suppressed after 24 h exposure to 10(-6) M mezerein. After 24 h incubation with 5 X 10(-5) M DES, 20% of normal HCE are capable of colony formation but this resistant fraction is eliminated after 96 h chronic exposure. Cornified-envelope formation was negligible in malignant cervical keratinocytes grown in the presence of DES or promotors and these cells were characterised by a very large PMAR fraction - 85 - 90% of cells retained colony-forming ability after exposure to 10(-6) M PMA for 24 h. Furthermore, 90-100% of malignant cervical keratinocytes retained their colony-forming capacity after exposure to 10(-6) M mezerein. However, colony-forming ability declined steadily in the presence of 5 X 10(-5) M DES and after 96 h only a tiny fraction, 1% of malignant cervical keratinocytes could form colonies on replating. The mechanisms by which DES inhibits growth and induces cornified-envelope formation in HCE would appear to be distinct from those activated by PMA and mezerein.