Andrzej A. Dlugosz

Definition by specific antisense oligonucleotides of a role for protein kinase Cα in expression of differentiation markers in normal and neoplastic mouse epidermal keratinocytes

Epidermal keratinocyte differentiation is a tightly regulated, stepwise process that requires protein kinase C (PKC) activation. Studies using cultured mouse keratinocytes induced to differentiate with Ca2+ have indirectly implicated the α isoform of PKC in upregulation of “late” (granular cell) epidermal differentiation markers. Activation of this isoform is also implicated in the suppression of “early” differentiation markers keratin (K) 1 and 10 that characterizes the neoplastic phenotype produced by the v‐Ha‐ras oncogene. We used antisense oligonucleotides (AS) to directly address the role of PKCα in regulating expression of these markers in normal and v‐Ha‐ras‐transduced primary keratinocytes and a keratinocyte cell line (SP‐1) containing an activating mutation of the c‐Ha‐ras gene. Transfection of PKCα AS reduced the PKCα protein level in a dose‐dependent manner, with a maximum effect at doses of 100 nM or higher. Immunoblot analysis with antibodies against PKCα, PKCδ, PKCϵ, and PKCν confirmed that PKCα AS selectively reduced the level of PKCα but not the other isoforms. In vitro kinase assays also revealed suppression of Ca2+‐dependent PKC activity, which is the PKCα activity in this cell type, after transfection of PKCα AS. When PKCα AS‐treated normal keratinocytes were stimulated to terminally differentiate with Ca2+, induction of the late differentiation markers loricrin, filaggrin, and SPR‐1, as well as transglutaminase K mRNA, was suppressed when compared with their induction in scrambled AS‐treated controls. In neoplastic v‐Ha‐ras‐transduced keratinocytes and SP‐1 cells, transfection of PKCα AS, but not the scrambled AS control, selectively downregulated PKCα and restored differentiation specific expression of K1. These findings directly confirm that PKCα is an important component of the signaling pathway regulating terminal differentiation of normal keratinocytes and that activation of PKCα contributes to the altered differentiation program of neoplastic murine keratinocytes. Mol. Carcinog. 18:44–53, 1997.

The In Vitro Analysis of Biochemical Changes Relevant to Skin Carcinogenesis

The phenotypic alterations produced in mouse skin cells during the multistage development of squamous cancer have been well documented. In normal skin, all proliferating cells are confined to the basal cell compartment where less than 10% of the cells are in S phase when pulse-labeled with DNA precursors. Two keratins, K5 (M r 60 000) and K14 (M r 55 000), are transcribed largely in basal cells, although the proteins persist in the upper layers (Roop et al. 1988). The commitment to differentiate is associated with the loss of proliferative potential, the commencement of suprabasal migration, and the expression of two suprabasal keratins, K1 (M r 67 000) and K10 (M r 59 000) in the first spinous cell layer (Roop et al. 1988). Proliferating cells do not express K1 or K10 in normal epidermis. As cells migrate into the granular cell layer, K1 and K10 transcripts diminish and the genes for filaggrin, a M r 27 000 interfilamentous matrix protein, and loricrin, a major component of the cornified envelope, are activated and the proteins synthesized (Mehrel et al. 1990; Roop et al. 1989).