The overall objective of this renewal application is to extend our investigations into the role that Ha-ras gene expression plays during skin tumorigenesis in vivo, and to delineate the various factors involved in regulating the expression of this proto- oncogene in epidermal cells. Experiments are proposed to investigate regulation of Ha-ras gene expression at both the transcriptional and post-transcriptional level. We first propose to employ the heteroduplex mismatching method to characterize the Ha-ras mRNA transcripts previously observed by us to be present at elevated levels in developing papillomas, to determine whether Ha- ras transcription is originating from the normal or point-mutated allele, or both. We will also investigate transcriptional regulation in cis of the mouse c-Ha-ras gene in vitro, by identifying and characterizing cis- regulatory elements of the Ha- ras oncogene, using the transient gene expression assay for chloramphenicol acetyltransferase. In addition to examining factors which regulate expression in cis, we will investigate elements that may regulate expression of the Ha-ras gene in trans: A series of experiments is proposed which will manipulate trans- regulatory elements involved in expression of the mouse Ha-ras promoter in vitro. The glucocorticoids (which have long been known to inhibit skin tumor development) and tumor promoters (which encourage skin tumor development) will be used to characterize trans-acting regulatory factors in vitro. In addition transcriptional studies, we propose to carry out experimental cis- and trans-transcriptional regulation of the mouse c-Ha-ras gene in vivo. Mouse skin cells will be transfected with the normal and point-mutated mouse c-Ha-ras gene under the control of heterologous promoters, and the cells transplanted into SENCAR mice to test for development of papillomas. Another aim of this project is to investigate factors involved in post-transcriptional regulation of the mouse Ha-ras proto-oncogene. We have proposed experiments to compare the stability of Ha-ras transcripts in normal epidermis and in tumors, and to identify structural features of Ha-ras message which may determine its susceptibility to decay in the cytoplasm.
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