My major research efforts are aimed at characterizing the cis- and trans-regulatory elements controlling transcription of the Ha- ras message during two-stage skin carcinogenesis, and the factors which effect the stability of that message. We propose to investigate regulation of Ha-ras gene expression at both the transcriptional and post-transcriptional level. We will 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. Results of these studies will tell us whether the 5' upstream flanking sequence of the Ha- ras gene has undergone a sequence change or organizational change. We will investigate transcriptional regulation in cis of the mouse c-Ha-ras gene in vitro, by identifying and characterizing cis-transcriptional 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 propose to investigate elements which may regulate expression of the Ha-ras gene in trans: 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. We will also examine Ha-ras regulation in vivo, by transfecting mouses kin cells with the normal and point-mutated mouse c-Ha-ras gene under the control of heterologous promoters, and transplanting the cells into SENCAR mice to test for development of papillomas. The last aim of this proposal will be 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.
