The interferons (IFN) are a family of naturally occurring glycoproteins which can elicit both an antiviral and an antiproliferative response in cells. The molecular basis for these manifestations of the cellular responses to IFN has been a subject of intense research for some time. An alternative consequence of IFN treatment is the restoration of shape-dependent, contact-inhibited proliferation to malignant cells lacking these properties. Research involving cell proliferation suggest that shape- dependent control may be linked to cell spreading which many, in turn, be due to alteration in either the nucleoskeleton or cytoskeleton. The intermediate filament (IF) network is an exceptionally stable, integral component of cellular skeleton. Moreover, despite a wealth of knowledge concerning the structure of both individual IF protein subunits and their polymerization into filaments, their function remains largely unclear. Within one class of the intermediate filaments, the cytokeratins, are found the developmentally regulated partner keratins, kertain 8 (K8) and keratin 18 (K18). They are initially located within the trophoblast layer of the blastocyst embryo and ultimately found in a limited number of normal adult differentiated tissues. The genes for K18, and less frquently K8, have recently been shnown to become activated in a diverse number of nonepithelial and the transformed cells. The molecular mechanisms for this inappropriate expression of keratin genes are unknown. This application proposes to use interferon as a tool for investigating the cellular and molecular basis of acquired contact-inhibited proliferation control. This will be accomplished by studying the effect of IFN on the expression of intermediate filament proteins and genes, specifically, K8 and K18 in normal and transformed human cells. Cell lines that inappropriately express K8 and K18 while remaining sensitive to the antiproliferative action of IFN will be utilized. Cells resistant to the antiproliferative activity of IFN yet which remain sensitive to the other effects of IFN will also be included in all studies so as to begin to evaluate the nature of the IFN resistance. Also proposed is the isolation of their own K8 genomic clone so as to commence an analogous dissection to that of the K18 gene with respect to potential regulatory elements. The methods utilized in this study will employ both standard biochemical methods and recombinant DNA technology.Inclusive in this analysis, but not limited to, will be keratin immunofluorescent staining of +/-IFN treated cells, immunoprecipitation of K8 and K18 from +/-IFN treated cells, Northern and S1 analysis of RNA from +/-IFN treated cells and molecular cloning techniques (including PCR) for the isolation of a K8 genomic clone. Once the fundamental principles of K8 and K18 gene regulation are ascertained in normal cells, a study of their altered genetic expression in transformed cells can be undertaken. This analysis may lead to an understanding of why cancer cells which inappropriately express K8 and K18 seem to increase their migratory and invasive ability. The goal of the present proposal is, therefore, directed towards providing not only a better understanding of the relationship between the actions of the interferons and intermediate filament protein and gene expression but also contributing information towards a broader biological understanding of cell proliferation control.
