The long term objective of the work is to understand at the molecular level how tumor promoters in particular the phorbol ester 12otetradecanoylphorbol13acetate (TPA), interact with cell and virus genes to produce a transformed phenotype in cells exposed to an initiating carcinogenic stimulus. This proposal continues to test the general hypothesis that TPA stimulates the transcription of specific genes by altering the activity and/or specificity of RNA polymerase II, and to examine the mechanism of this effect. The proposed experiments will attempt to identify components of the regulatory pathway as well as targets associated directly with the process of transcription. Successful conclusion of the experiments will provide an in vitro test for the regulation of cloned cell sequences responsive to TPA in vivo, and will lay the groundwork for complete in vitro reconstruction of a biologically important transmembrane signaling pathway. The proposed experiments will characterize the requirements for stimulation of in vitro transcription of the type 5 human adenovirus (Ad5) immediate early gene (E1A) by activated endogenous protein kinase C in whole cell extracts and by purified Ckinase in reconstructed reactions using purified RNA polymerase II plus cell fractions containing partially purified transcription factors. Experiments will attempt to identify a phosphoprotein target for Ckinase in in crude extracts; in purified fractions that stimulate transcription activity; on isolate transcription complexes from Ad5infected cells and from in vitro transcription reactions; and from the chromain of TPAtreated cells. Phosphoproteins from labeled transcription complexes will be characterized by two dimensional polyacrylamide gel electrophoresis and autoradiography. The effect of Ckinase on in vitro transcription of other TPAinducible genes will be tested.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA037761-06
Application #
3175561
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1984-07-01
Project End
1990-12-31
Budget Start
1989-07-01
Budget End
1990-12-31
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
St. John's University
Department
Type
Schools of Arts and Sciences
DUNS #
City
Queens
State
NY
Country
United States
Zip Code
11439
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James, C B; Carter, T H (1992) Activation of protein kinase C inhibits adenovirus VA gene transcription in vitro. J Gen Virol 73 ( Pt 12):3133-9
Weigel, N L; Carter, T H; Schrader, W T et al. (1992) Chicken progesterone receptor is phosphorylated by a DNA-dependent protein kinase during in vitro transcription assays. Mol Endocrinol 6:8-14
Carter, T; Vancurova, I; Sun, I et al. (1990) A DNA-activated protein kinase from HeLa cell nuclei. Mol Cell Biol 10:6460-71
Carter, T H; Kopman, C R; James, C B (1988) DNA-stimulated protein phosphorylation in HeLa whole cell and nuclear extracts. Biochem Biophys Res Commun 157:535-40
Carter, T; James, C; Chan, E et al. (1987) Induction of integrated adenovirus E1A and E1B genes in transformed human cells by phorbol ester tumor promoters. Cancer Res 47:803-8