Verma, Alka; (1996) The development of a human cell culture assay for skin tumour promoters. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A valid model of initiated epidermis requires that normal keratinocytes should suppress the growth of initiated keratinocytes and that this suppression should be overcome on exposure to tumour promoters. Subgenomic SV40 transformed human keratinocytes (HK-4), which display the initiated phenotype, were cultured within a confluent monolayer of normal human keratinocytes and exposed to various tumour promoters and anti-promoters. This system appeared to be successful in identifying both phorbol ester and non-phorbol ester type tumour promoters in a dose-dependent manner as determined by the size of the colonies of the HK-4 cells. The ranking of three phorbol ester-type tumour promoters matched the order of potency of these compounds as tumour promoters in mouse skin. In this model tumour promoters appear to induce the clonal expansion of HK-4 cells indirectly by selectively stimulating the differentiation of the normal cells. The anti-promoters retinoic acid and fluocinolone acetonide were unsuccessful in inhibiting TPA induced colonies in this human model. A major reason for the absence of anti-promoter activity in the co-culture has been attributed to the abnormal conditions of the cells (i.e grown on plastic and submerged in medium). The presence of a dermis and an air-liquid interface is important in cellular functions such as differentiation and proliferation. Cells grown as organotypic cultures better represent the in vivo situation compared with those in submerged cultures. Therefore the co-culture of HK-4 and normal human keratinocytes was incorporated within a skin equivalent at the air-liquid interface to produce a three dimensional model of initiated human epidermis. Mimicking the situation in vivo may give rise to a more accurate interpretation of tumour promotion in human skin. In addition, this system allows test samples to be added either topically (placed on the surface of the skin equivalent) or systemically (placed in the medium below the dermis).

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