Resistance of adult keratinocytes to differentiation-induced decrease in Ha-ras mRNA levels observed in newborn...

During two-stage mouse skin tumorigenesis, the mouse c-Ha-ras oncogene undergoes activation by point mutation after initiation with polycyclic aromatic hydrocarbons. Furthermore, initiated epidermal cells containing an activated Ha-ras oncogene have been shown to be resistant to calcium-induced terminal differentiation. However, the relationship between Ha-ras expression and the differentiation process is not well understood in either normal or initiated cells. Before attempting to explore the role of Ha-ras expression in epidermal differentiation during tumorigenesis, we felt that investigation of Ha-ras gene expression in normal primary epidermal cells undergoing differentiation was warranted, since primary cultures of normal newborn and adult keratinocytes presumably contain the stem cells from which skin tumors arise. In the present studies, northern blot analysis was used to compare Ha-ras expression in normal newborn and adult epidermal cells undergoing differentiation. Steady-state levels of Ha-ras mRNA remained unchanged in primary cultures of normal adult epidermal cells during calcium-induced differentiation, whereas steady-state levels of Ha-ras transcripts decreased during calcium-induced differentiation in primary newborn epidermal cells. Differentiation was induced by switching the adult and newborn keratinocytes from medium containing 0.05 mM Ca2+ to medium containing one of three different calcium concentrations (0.15, 0.5, or 1.2 mM Ca2+). The decrease in Ha-ras mRNA levels observed during differentiation in newborn keratinocytes occurred as an intermediate event in the differentiation process, was specific for the Ha-ras gene, and was not due to a general decrease in transcriptional activity during differentiation. Characteristic patterns of keratin 14 gene expression and cornified envelope formation were observed, verifying that the differentiation process had been induced in both the primary adult and newborn epidermal cells. That adult keratinocytes are resistant to the differentiation-induced reduction in Ha-ras mRNA expression observed in newborn keratinocytes may explain the difference in in vivo tumorigenic potentials of newborn and adult skin.