PROGRESSIVE ABROGATION OF TGF-BETA-1 AND EGF GROWTH-CONTROL IS ASSOCIATED WITH TUMOR PROGRESSION IN RAS-TRANSFECTED HUMAN KERATINOCYTES

This study examined the response of human keratinocytes in different stages of transformation to exogenous TGF-beta1 and EGF as well as their receptor and growth-factor expression. Cells of the spontaneously immortalized HaCaT cell line and c-Ha-ras transfected clones (1-6, 1-7, 11-3, 11-4) exhibited different tumorigenic potentials when transplanted to athymic mice. HaCaT- and 1-6 cells were non-tumorigenic, 1-7 cells formed persisting epidermal cysts (benign tumours) and 11-3 and 11-4 cells developed into invasive squamous-cell carcinomas. TGF-beta1 inhibited thymidine uptake in a dose-dependent manner, a progressive decrease in response being associated with an increasing malignant potential (HaCaT > 1-6 > 1-7 = 11-4). HaCaT-cells and ras-clones expressed TGF-beta1 mRNA at similar levels, but cells of increasing malignant potential secreted markedly less receptor-binding TGF-beta (HaCaT > 1-6 = 1-7 > 11-3 > 11-4) into the culture medium. Whilst ras-transfected cells expressed fewer TGF-beta receptors than HaCaT cells, there was little difference between TGF-beta receptor number or affinity between the 4 transfected cell clones. The same was true for the TGF-beta receptor types, but Type-II receptors were expressed at lower levels by the malignant clones 11-3 and 11-4. When HaCaT and ras-transfected cells were investigated for their response to exogenous EGF, cells were refractory (1-7,11-4), partially stimulated (1-6) or fully stimulated (HaCaT). Cells with increasing malignant potential produced increasing amounts of endogenous TGF-alpha (11-4 = 11-3 > 1-7 = 1-6 > HaCaT). All tumorigenic ras clones expressed higher mRNA levels than HaCaT-cells. Ras-transfected clones expressed fewer high- and low-affinity EGF receptors than HaCaT cells with a tendency toward increased numbers of high-affinity EGF receptors associated with increasing malignant potential (11-4 = 11-3 > 1-7 > 1-6) but these changes were associated with a progressive decrease in receptor affinity. The results indicate that tumour progression in human epidermal keratinocytes transfected with c-Ha-ras is associated with a progressive abrogation of TGF-beta1 and EGF growth control. They suggest that the increased autonomous growth potential associated with advanced stages of epithelial tumour progression can be defined more closely using a cellular profile of TGF-beta and EGF.