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DEVELOPMENT OF AN IN VITRO MODEL FOR THE SCREENING OF BIOLOGICALLY ACTIVE KERATINOCYTE GROWTH FACTOR (KGF/FGF-7) RECEPTOR ANTAGONISTS.
Chang, Hsiang-Lin1, Sugimoto, Yasuro 1,2, Liu, Suling1, Jiang, Jiahua1, Kulp, Samuel1, Brueggemeier, Robert2,3, Lin, Young1,2, 1 2 3
ABSTRACT- KGF is considered to be a stromal-to-epithelial paracrine growth regulator. In the prostate, KGF can mediate the effects of androgens and plays a role in regulating epithelial cell (EC) proliferation. KGF may have similar importance in human breast and breast cancer cells since KGF stimulates aromatase mRNA expression and EC growth, and KGF mRNA expression is elevated by E2. These findings suggest the therapeutic use of a KGF receptor antagonist (KRA) in both prostate and breast cancers. We have synthesized 11 candidate peptide KRAs for which amino acid sequences span the entire KGF molecule. Described here is ongoing work to develop sensitive in vitro assays to screen candidate KRAs for the ability to block the binding of KGF to a type 2 FGF receptor (KGFR) and to inhibit KGF-induced effects on EC function. RT-PCR was used to generate cDNA for the KGFR extracellular domain (ECD) from total RNA isolated from MCF-10A human breast epithelial cell line. The KGFR ECD was cloned into the plasmid vector pEF6/V5-His TOPO®. Plasmid DNA was isolated and sequencing confirmed the fidelity of the KGFR ECD portion of the plasmid. MCF-10A cells and the T47D human breast cancer cell line were stably transfected with the plasmid. Each transfected cell line was seeded into six culture dishes and then selected for the blasticidine resistance (10 
g/ml; 10 days) after cells reached 25% confluence. Four clones from each plate were chosen and propagated in culture. Presence of the KGFR ECD in the transfected cell lines was confirmed by Western immunoblotting of cell lysates using antibody to the V5 epitope which revealed a band of the expected size for the KGFR ECD (~30 kD). Isolation and detection of the KGFR ECD in the conditioned medium of transfected cells is ongoing. An in vitro binding assay will be developed using the KGFR ECD. Binding of 125I-KGF in the presence of candidate KRAs will be evaluated. KRAs that effectively inhibit KGF binding will then be used in whole cell assays to determine functional inhibition of KGF actions. The potent KRA(s) identified by our assays will have potential therapeutic applications for human breast and prostate cancer patients. (Army Breast Cancer Program grant DAMD 17-99-1-9341; Pharmacia & Upjohn-OSU Collaborative Research Grant)
KEY WORDS: KGF, KGF receptor antagonist, stable transfection, cancer epithelial cell