HA-RAS P21-CTP LEVELS REMAIN CONSTANT DURING PRIMARY KERATINOCYTE DIFFERENTIATION

Several lines of evidence that indicate that mutation of the Ha-ras on cogene is the initiating event in mouse skin carcinogenesis. Keratinoc ytes known to possess a mutated Ha-ras have been shown to be resistant to differentiation. Thus, overstimulation of the Ha-ras signaling pat hway appears to block normal keratinocyte differentiation, and we hypo thesized that for normal keratinocytes to terminally differentiate, th e Ha-ras signaling cascade must be turned off. In the present studies, we measured the level and activity state of Ha-ras p21 protein in cul tured keratinocytes undergoing calcium-induced differentiation. We hav e employed Western blot analysis to demonstrate that Ha-ras p21 protei n levels remain constant during primary newborn and adult keratinocyte differentiation. The overall level of Ha-ras p21 was higher in immort alized, benign, and malignant mouse keratinocyte cell lines than in no rmal keratinocytes but did not change within each cell type when subje cted to differentiating conditions. The percentage of Ha-ras p21 prote in in its active, CTP-bound form also remained unchanged during primar y adult keratinocyte differentiation and in immortalized, benign, and malignant keratinocytes subjected to differentiating conditions. Our r esults indicate that terminal differentiation of primary adult mouse k eratinocytes occurred in the presence of constant levels of Ha-ras p21 -GTP, suggesting that the Ha-ras signaling pathway may be blocked at a point distal to a step involving the Ha-ras p21 protein itself. (C) 1 995 Wiley-Liss, Inc.