A substantial body of correlative data has indicated that the serine protease, plasminiogen activator (PA) is highly-regulated in normal cells, expressed at elevated levels in malignant cells and catalytically involved in specific cell behavior patterns. In order to understand the cellular and genetic mechanisms involved in PA regulation and provide direct experimental evidence for its catalytic role in normal and malignant cell behavior, cell function will be linked directly to structural aspects of PA and PA- associated proteins and to PA gene expression. Using the well-defined primary culture system of normal chick embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV), we propose to examine the cellular regulation of PA in terms of its catalytic activity, its structure, its genomic regulation and the involvement of PA-related protein. The procurement of molecular, immunological and nucleotide probes will form the initial experimental approaches. A newly-acquired monoclonal antibody, specific for avian PA, will be used to purify microgram to milligram quantities of PA. In addition to allowing for PA structural studies, the purified PA also will be employed to produce a monospecific polyclonal antibody to PA. The latter probe will be used to directly monitor PA production (PA zymogen and active PA) in normal and transformed cells and PA expression in vectors carrying RSVCEF cDNA for the eventual isolation of a PA cDNA. Once acquired these three probes (PA, anti-PA IgG and PA cDNA) will be used to examine the cellular regulation and catalytic manifestation of PA. A coordinate search for a specific PA receptor on the surface of normal and transformed cells also will be carried out as this molecule could have a profound influence on PA expression at close cell-cell and cell-matrix contacts. The cellular balance of zymogen PA, active PA, and PA receptors will be quantitated and related directly to transformed cell behavior through the use of specific antibodies, the binding of purified PA to PA-deficient cells, and the selective employment of the conditional, temperature-sensitive mutant of RSV. The genomic expression of PA will be examined under a variety of culture conditions using the cDNA probe and finally PA genomic DNA will be isolated and its structure related to any cell physiological events and factors that affect PA expression.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA016740-13
Application #
3164493
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1978-08-01
Project End
1992-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
13
Fiscal Year
1988
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Ramos-DeSimone, N; Moll, U M; Quigley, J P et al. (1993) Inhibition of matrix metalloproteinase 9 activation by a specific monoclonal antibody. Hybridoma 12:349-63
Brooks, P C; Lin, J M; French, D L et al. (1993) Subtractive immunization yields monoclonal antibodies that specifically inhibit metastasis. J Cell Biol 122:1351-9
Chen, J M; Aimes, R T; Ward, G R et al. (1991) Isolation and characterization of a 70-kDa metalloprotease (gelatinase) that is elevated in Rous sarcoma virus-transformed chicken embryo fibroblasts. J Biol Chem 266:5113-21
Berkenpas, M B; Quigley, J P (1991) Transformation-dependent activation of urokinase-type plasminogen activator by a plasmin-independent mechanism: involvement of cell surface membranes. Proc Natl Acad Sci U S A 88:7768-72
Moll, U M; Youngleib, G L; Rosinski, K B et al. (1990) Tumor promoter-stimulated Mr 92,000 gelatinase secreted by normal and malignant human cells: isolation and characterization of the enzyme from HT1080 tumor cells. Cancer Res 50:6162-70
Quigley, J P; Sullivan, L M; DeMarinis, C M et al. (1988) Functional role of specific secreted and cell surface molecules in tumour cell invasion and metastasis. Ciba Found Symp 141:22-47
Quigley, J P; Gold, L I; Schwimmer, R et al. (1987) Limited cleavage of cellular fibronectin by plasminogen activator purified from transformed cells. Proc Natl Acad Sci U S A 84:2776-80
Sullivan, L M; Quigley, J P (1986) An anticatalytic monoclonal antibody to avian plasminogen activator: its effect on behavior of RSV-transformed chick fibroblasts. Cell 45:905-15
Gordon, J R; Quigley, J P (1986) Early spontaneous metastasis in the human epidermoid carcinoma HEp3/chick embryo model: contribution of incidental colonization. Int J Cancer 38:437-44
Fairbairn, S; Gilbert, R; Ojakian, G et al. (1985) The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants. J Cell Biol 101:1790-8