Keratinization involves the expression of numerous differentiated traits. Variations in the pattern of keratinization in oral mucosa and epidermis reflect varying degrees of expression of these traits. To determine how keratinization is controlled, several markers of epithelial differentiation will be examined in cultured human keratinocytes. I. Keratin Production: Kinetic studies of keratin synthesis in cultured cells suggest that keratin production is controlled differently in the lower and upper strata of keratinizing epithelia. To test this hypothesis, aspects of keratin synthesis will be characterized in keratinocytes stimulated to undergo enhanced keratinization in culture. Keratin is heavily phosphorylated. Experiments will define the kinetics of this reaction during maturation, and the role of these charged groups in filament assembly. II. Terminal Differentiatio (TD): An assay developed to measure the rate of TD will be used to explore two central questions in keratinization...does TD follow or precede transit out of the basal layer? and, is the probability of TD following cell replication a fixed property of the keratinocyte. III. Productive Viral Replication: Replication of Adenovirus type 2 (Ad-2) is a newly discovered marker of keratinocyte differentiation. To determine how viral replication is blocked in infected basal cells, the transcriptional activity and physical state of Ad-2 DNA will be examined in these cells. Human papilloma virus (HPV) has been shown to persist in keratinocytes as stable episome and to synthesize viral antigens after repeated passage of infected cells. To explore this model of viral latency, HPV RNA will be isolated from early and late passage cells. Transcripts will be mapped and where possible used to identify viral specific proteins. IV. Somatic Cell Genetics of Keratinocytes: To elucidate the genetic controls in keratinization, expression of keratinocyte markers will be studied in human keratinocyte X mouse fibroblast hybrids. Where possible, chromosomal mapping will be performed. These experiments draw upon disciplines and techniques not traditionally used in the study of keratinization. It is hoped that concepts and mechanisms heretofore unknown, will be discovered and that a useful body of knowledge will be generated concerning disorders of keratinization.
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