This research deals with production, and subsequent destruction of PDGF- inducible mRNAs. We have two broad objectives. Our first objective is to identify nuclear and cytoplasmic components of a PDGF signal transduction pathway which does not involve protein kinase C or the cis acting """"""""Serum- Response Element"""""""". Towards this end, we will carry on with structure/function analysis of the promoter for """"""""JE""""""""- a gene which is induced by PDGF through activation of the kinase C-independent pathway. Transient transfections have shed little insight into functional elements of the JE promoter. Accordingly, we will develop new tactics. We will ligate the JE promoter to a reporter gene which can be detected by fluorescence activated cell sorting (FACS). Through FACS, we hope to conduct structure/function analysis of the JE promoter in stable transfectants without resorting to dominant selectable marker genes. At the cytoplasmic level, we will evaluate the role of c-raf protein as an intermediate in JE induction. These experiments will involve cell microinjection and in situ hybridization. The second broad objective is to define cis and trans acting elements which mediate selective degradation of PDGF-inducible mRNAs. Here we will also develop new technologies. Artificial mRNAs will be microinjected into the cytoplasm of Balb/c-3T3 cells to define cis acting elements which regulate mRNA stability. We have adapted """"""""RNA gel-band shifts"""""""" to the problem or trans acting factors involved in message stability. Using RNA band shift, we are isolating and characterizing a protein which binds specifically to 3' A/U rich motifs within PDGF-inducible mRNAs. We will explore the potential of yeast genetics as an alternative vehicle to the isolation of trans acting factors.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA022042-17S1
Application #
2086996
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1977-08-01
Project End
1996-01-31
Budget Start
1994-02-01
Budget End
1996-01-31
Support Year
17
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
149617367
City
Boston
State
MA
Country
United States
Zip Code
02215
Freter, R R; Alberta, J A; Hwang, G Y et al. (1996) Platelet-derived growth factor induction of the immediate-early gene MCP-1 is mediated by NF-kappaB and a 90-kDa phosphoprotein coactivator. J Biol Chem 271:17417-24
Guha, A; Dashner, K; Black, P M et al. (1995) Expression of PDGF and PDGF receptors in human astrocytoma operation specimens supports the existence of an autocrine loop. Int J Cancer 60:168-73
Freter, R R; Alberta, J A; Lam, K K et al. (1995) A new platelet-derived growth factor-regulated genomic element which binds a serine/threonine phosphoprotein mediates induction of the slow immediate-early gene MCP-1. Mol Cell Biol 15:315-25
Wang, C; Stiles, C D (1994) Platelet-derived growth factor alpha receptor gene expression: isolation and characterization of the promoter and upstream regulatory elements. Proc Natl Acad Sci U S A 91:7061-5
Alberta, J A; Rundell, K; Stiles, C D (1994) Identification of an activity that interacts with the 3'-untranslated region of c-myc mRNA and the role of its target sequence in mediating rapid mRNA degradation. J Biol Chem 269:4532-8
Freter, R R; Irminger, J C; Porter, J A et al. (1992) A novel 7-nucleotide motif located in 3' untranslated sequences of the immediate-early gene set mediates platelet-derived growth factor induction of the JE gene. Mol Cell Biol 12:5288-300
Hall, D J (1990) Regulation of c-myc transcription in vitro: dependence on the guanine-rich promoter element ME1a1. Oncogene 5:47-54
Oquendo, P; Alberta, J; Wen, D Z et al. (1989) The platelet-derived growth factor-inducible KC gene encodes a secretory protein related to platelet alpha-granule proteins. J Biol Chem 264:4133-7
Hall, D J; Stiles, C D (1987) Platelet-derived growth factor-inducible genes respond differentially to at least two distinct intracellular second messengers. J Biol Chem 262:15302-8
Zumstein, P; Stiles, C D (1987) Molecular cloning of gene sequences that are regulated by insulin-like growth factor I. J Biol Chem 262:11252-60

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