The extracellular matrix (ECM) has such a significant influence on normal growth and differentiation that is likely to play a role in diseases involving abnormal growth and development, such as cancer. The long-term objective of this research is to define alterations in cell- extracellular matrix interactions which play a role in the development of this disease. The ECM of cultured chicken embryo fibroblasts, infected with temperature-sensitive (ts) mutants of Rous Sarcoma virus, is modified during the early stages of transformation by the synthesis and deposition of a small protein (Mr about 21,000). Sufficient amounts of the 21K protein have been purified to determine its function in the matrix and potential role in transformation. The protein promotes the detachment of transforming cells from the ECM and stimulates proliferation of quiescent cells. Amino acid sequence data and other properties indicate that the protein is related, but not identical, to tissue inhibitor of metalloproteinases (TIMP/erythroid potentiating activity (EPA). We have detected five metalloproteinase inhibitor activities (Mr about 20,000-26,000) in cultured chicken embryo fibroblasts; these are designated ChIMPs (Chicken Inhibitor of MetalloProteinases). ChIMP-3 is the 21K protein. This proposal is designed to analyze the structure of ChIMP-3 using biochemical, molecular and physical techniques and to test the hypothesis that the protein promotes cell detachment from the ECM during oncogenic transformation. Electrophoresis, chromatography and mass spectrometry will be used to define post-translational modifications. Polyclonal antibodies to ChIMP-3 will be used to aid in its characterization and to describe its distribution in the matrix by immunofluorescence microscopy. The potential interaction of ChIMP-3 with other matrix components will be established by electrophoresis, autoradiography and binding studies. The role of ChIMP-3 in expression of the transformed phenotype will be investigated by inhibition studies with antisense oligodeoxynucleotides. Site-directed mutagenesis of ChIMP-3 will be used to determine if the biological properties of this protein are mediated through its metalloproteinase inhibitory activity.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA039919-07A2
Application #
3179269
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1984-09-30
Project End
1996-07-31
Budget Start
1992-09-30
Budget End
1993-07-31
Support Year
7
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Type
Schools of Pharmacy
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Hawkes, Susan P; Li, Hongxia; Taniguchi, Gary T (2010) Zymography and reverse zymography for detecting MMPs and TIMPs. Methods Mol Biol 622:257-69
Hawkes, S P; Li, H; Taniguchi, G T (2001) Zymography and reverse zymography for detecting MMPs, and TIMPs. Methods Mol Biol 151:399-410
Bass, K E; Li, H; Hawkes, S P et al. (1997) Tissue inhibitor of metalloproteinase-3 expression is upregulated during human cytotrophoblast invasion in vitro. Dev Genet 21:61-7
Leco, K J; Apte, S S; Taniguchi, G T et al. (1997) Murine tissue inhibitor of metalloproteinases-4 (Timp-4): cDNA isolation and expression in adult mouse tissues. FEBS Lett 401:213-7
Kishnani, N S; Staskus, P W; Yang, T T et al. (1995) Identification and characterization of human tissue inhibitor of metalloproteinase-3 and detection of three additional metalloproteinase inhibitor activities in extracellular matrix. Matrix Biol 14:479-88
Leco, K J; Khokha, R; Pavloff, N et al. (1994) Tissue inhibitor of metalloproteinases-3 (TIMP-3) is an extracellular matrix-associated protein with a distinctive pattern of expression in mouse cells and tissues. J Biol Chem 269:9352-60
Pavloff, N; Staskus, P W; Kishnani, N S et al. (1992) A new inhibitor of metalloproteinases from chicken: ChIMP-3. A third member of the TIMP family. J Biol Chem 267:17321-6
Yang, T T; Hawkes, S P (1992) Role of the 21-kDa protein TIMP-3 in oncogenic transformation of cultured chicken embryo fibroblasts. Proc Natl Acad Sci U S A 89:10676-80
Staskus, P W; Masiarz, F R; Pallanck, L J et al. (1991) The 21-kDa protein is a transformation-sensitive metalloproteinase inhibitor of chicken fibroblasts. J Biol Chem 266:449-54
Henrich, C J; Hawkes, S P (1989) Molecular weight dependence of hyaluronic acid produced during oncogenic transformation. Cancer Biochem Biophys 10:257-67