Impact of bis-(beta-chloroethyl) sulfide on keratin protein and intermediate filaments in cultured keratinocytes as indicated by monoclonal antibody binding.

Abstract

BCES is a highly toxic alkylating vesicant for human skin. The blister formed at the dermal-epidermal junction, is associated with the necrosis of the basal layer, which appears to be selectively susceptible to BCES. Rat keratinocytes grown in low calcium medium (0.11 mM) form a monolayer culture consisting primarily of a proliferative population, but including cells which exhibit characteristics of differentiated cells. Keratin polypeptides, the major constituent of the intermediate filament systems of epithelial cells, change as the cell differentiates. Anti-keratin monoclonal antibodies can act as specific and sensitive probes for normal changes and abnormalities resulting from toxic chemical exposure. 2D6, an anti-rat keratin monclonal antibody, developed during this project, binds only the basal layer in tissue cross sections. Immunoblot analysis of tissue extracts reveals binding to a 55K Mr protein extracted from basal cells and a 54.5K Mr protein extracted from the differentiated layers. BCES induces numerous changes in this rat keratinocyte culture system that can be in part characterized by the binding of 2D6. Exposure to BCES between 1 $\mu$M and 10 $\mu$M induces what appears to be a dose-related increase in 2D6 expression in the monolayer population from 10% to 40% followed by a reduction at higher doses. Further characterization of cellular changes that occur after exposure to 10 $\mu$M BCES revealed that BCES can cause an increase in the average amount of 2D6 binding antigen per cell, and that exposure can result in the generation of a new 2D6 binding protein in a specific subpopulation of the monolayer. Exposure to BCES, then, can change the regulation of the production of keratin proteins and therefore the condition of the intermediate filament system in certain epidermal cells. Keratin expression is a highly orchestrated part of the differentiative process and the keratin present in the cell in part defines the cell condition at any given stage. BCES, then, appears to be able to disrupt or change the culture differentiative process and redefine the cell condition by the generation of a new keratin protein.