The research proposed in this grant application is designed to elucidate the basic regulatory events that control the differentiation of mouse keratinocytes. The overall goal of this project is to test the hypothesis that Ca++ induced terminal differentiation of keratinocytes is brought about by changes in gene expression. Balb/MK keratinocytes will be used as the experimental cell system. cDNA sequences that represent the Ca++ induced differentiation gene will be cloned, isolated, and characterized. Functional analysis will seek to prove that specific mRNA species and/or its protein product is directly involved in the initiation of differentiation. Characterization of the expression of the putative genes that bring about differentiation will include: the kinetics and biology of expression, complete sequence, and the function of gene products (proteins) that control differentiation. A major goal is to demonstrate whether expression of the keratinocyte phenotype can be separated from terminal growth inhibition. The effects of transforming oncogenes on the induction of differentiation will be determined. Since the MK cell line is committed to keratinocyte lineage, the DNA from keratinocytes will be transfected into embryonic epithelial cells that are not committed to a developmental lineage in order to isolate the gene responsible for commitment to keratinocyte differentiation. A final goal of this application will be to investigate the cellular regulation of the gene(s) that control differentiation induced growth inhibition. Genomic clones of genes that regulate differentiation will be used to identify cis acting sequences and trans acting factors. It is proposed that an experimental system can be designed to analyze the Ca++ modulated switch leading to growth inhibition and differentiation. A possible two stage model for the differentiation of keratinocyte cells will be examined. The understanding of this control system -- stimulation/inhibition of proliferation and differentiation -- will allow a better understanding of the processes leading to neoplastic growth.
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