Abstract

The ultimate goal of this research project is to determine the genetic and molecular basis of signal transduction mechanisms of the tumor promoting phorbol ester TPA in cultured mouse cells. We are approaching the proposed goals through the use of somatic cell genetics in combination with molecular and biochemical techniques.
Specific aims of our investigation are the following: 1. Continued characterization of the TPA-nonresponsive 3T3-L1 variants in terms of (a) A new role of collagen (TIE-5) gene expression in mitogenic response, (b) Expression of the known proto-oncogenes and growth-related genes in TPA-nonresponsive VT-1 cells upon TPA-treatment, (c) Identification of new protein components whose phosphorylation is enhanced upon cell's mitogenic response and cDNA cloning, and (d) Involvement of cytoskeletal components in the translocation of protein kinase C and 80K protein upon TPA stimulation. 2. Comprehensive analysis of the as yet unidentified TIE (TPA Inducible Early) genes in terms of (a) Sequence analysis of the three TIE gene (TIE-4, -10B and -44), (b) Expression patterns of the three TIE genes in 3T3-L1 cells upon stimulation by TPA and growth factors and in various mouse tissues, (c) Functional analysis of the three TIE genes using antisense expression vector, (d) Biochemical characterization and subcellular localization of the three TIE gene products, and (e) Genomic cloning and regulation of TIE gene expression. 3. Alternate approach for isolation of the genes for signal transduction using gene transfer technique for variant cells. 4. Activation of DNA topoisomerase I (Topo I) by protein kinase C-mediated phosphorylation as a significant nuclear event of signal transduction. 5. Chromosomal mapping of growth-related early genes using cell hybrid DNA panel and flow-sorted human chromosomes. 6. Future attempts to isolate more variants affected in mitogenic response. The cell and molecular genetic approaches to the dissection of transmembrane signalling mechanisms are unique, and the information and materials to be generated should prove useful for our further understanding the regulation of cell growth and differentiation and the malignant cell transformation during tumor development.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024375-15
Application #
3272263
Study Section
Mammalian Genetics Study Section (MGN)
Project Start
1977-07-01
Project End
1995-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
15
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
University of Arizona
Department
Type
Schools of Arts and Sciences
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721

  Publications

Stuiver, I; Hendrix, M J; Shimizu, Y et al. (1996) The phorbol ester TPA regulates collagen gene expression at the transcriptional level. Cell Struct Funct 21:259-69
Samuels, D S; Shimizu, N (1994) The predominant form of mammalian DNA topoisomerase I in vivo has a molecular mass of 100 kDa. Mol Biol Rep 19:99-103
Samuels, D S; Shimizu, Y; Nakabayashi, T et al. (1994) Phosphorylation of DNA topoisomerase I is increased during the response of mammalian cells to mitogenic stimuli. Biochim Biophys Acta 1223:77-83
Gardner, D P; Shimizu, N (1994) Loss of cytotoxic effect of epidermal growth factor (EGF) on EGF receptor overexpressing cells is associated with attenuation of EGF receptor tyrosine kinase activity. J Cell Physiol 158:245-55
Hsieh, J C; Jurutka, P W; Nakajima, S et al. (1993) Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical and functional evaluation of the serine 51 recognition site. J Biol Chem 268:15118-26
Pavlath, G K; Shimizu, Y; Shimizu, N (1993) Cytoskeletal active drugs modulate signal transduction in the protein kinase C pathway. Cell Struct Funct 18:151-60
Samuels, D S; Shimizu, N (1992) DNA topoisomerase I phosphorylation in murine fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate and in vitro by protein kinase. J Biol Chem 267:11156-62
Hsieh, J C; Jurutka, P W; Galligan, M A et al. (1991) Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 51, a residue crucial to its trans-activation function. Proc Natl Acad Sci U S A 88:9315-9
Stuiver, I; Shimizu, Y; Shimizu, N (1991) Phorbol-ester-mediated expression of the collagen type I pro-alpha 2 gene in mouse 3T3-L1 cells and its absence in a phorbol 12-myristate 13-acetate-non-responsive variant. Biochem J 278 ( Pt 2):369-73
Samuels, D S; Shimizu, Y; Shimizu, N (1989) Protein kinase C phosphorylates DNA topoisomerase I. FEBS Lett 259:57-60

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