REGULATION OF TRANSCRIPTION BY A TUMOR PROMOTER

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The long-term objective of the proposed study is to understand at the molecular level how tumor promoters, and in particular the phorbol ester tumor promoter 12-o-tetradecanoyl-phorbol-13-acetate (TPA), interact with cellular and viral genes to produce a transformed phenotype in cells exposed to an initiating carcinogenic stimulus. This proposal will test the hypothesis that TPA stimulates the transcription of specific genes by altering the activity or specificity of RNA polymerase II, and will examine the mechanism of this effect. Successful conclusion of the proposed experiments will provide the rationale for attempting to identify and clone regions of the mammalian genome that contain genes that are responsive to TPA in vivo, and will also provide an in vitro test for the cloned regulatory sequences. Cloned viral and cellular genes will be transcribed in vitro by soluble whole cell extracts and partially-purified RNA polymerase II to yield "runoff" products characteristic of initiation at specific promoter sequences. Extracts from TPA-treated and control HeLa cells will be compared for transcriptional activity using human adenovirus type 5 and SV40 early and late genes, the thymidine kinase gene of herpes simplex, avian and mouse mammary tumor virus long terminal repeat (LTR) sequences, and human interferon and beta-globin genes. Kinetic analysis of transcription will include the effect of total- and substrate-DNA concentration and of varying protein concentration. Mixing experiments will assess the presence of soluble regulatory factors in the whole cell extracts, and subfractionation of the crude extracts will be attempted to isolate such factors. The relationship of TPA action to that of an early regulatory product encoded by the adenovirus Ela region will be studied by assessing the ability of TPA to suppress various phenotypes of deletion mutants, and their revertants, in the Ela region. These phenotypes include stimulation of early viral transcription in Hela cells, and transformation of rat embryo cells (CREF) in culture.