Cells were incubated at 37C with 5% CO2 for 48 hours

Cells were incubated at 37C with 5% CO2 for 48 hours. have profound inhibitory effects on development of lung tumors. and Burgess have independently developed single potent neutralizing anti-HGF monoclonal antibodies (mAbs) that can inhibit the various HGF-induced biological activities attributable to both the and subunits (11, 12). These antibodies have been demonstrated to be highly specific to human HGF with no cross-reactivity to mouse HGF. Using human glioblastoma xenograft models, which express both HGF and c-Met in an autocrine manner, both antibodies were able to inhibit tumor growth and regression in nude mice. Additionally, histological analysis Cefonicid sodium revealed that tumors from animals treated with the HGF mAb, L2G7, exhibited decreased cell proliferation and blood vessel area with increased apoptosis (11). HGF/c-Met signaling in the lung is usually primarily through a paracrine mechanism whereby the tumors do not express HGF but rather the surrounding stromal tissue expresses and secretes HGF which then acts on neighboring tumor cells expressing the c-Met receptor (13). This paracrine action of HGF in the lung renders testing these novel HGF mAbs difficult in conventional lung tumor xenografts, since murine HGF produced by the tumor stroma will be unaffected. We recently described a novel transgenic mouse model that overexpresses human HGF in the airways under control of the Clara cell secretory protein promoter and showed that these mice express significantly higher HGF levels in the airway luminal space and have a significantly increased susceptibility to carcinogen-induced lung adenocarcinoma (14). These mice develop lung tumors that mimic aggressive human lung adenocarcinoma with high Cefonicid sodium HGF levels. This model provides a powerful preclinical system to evaluate anti-tumor brokers that target the HGF/c-Met pathway, specifically brokers developed against human HGF. We utilized this animal model to test the therapeutic potential of anti-human HGF antibody, L2G7. The HGF transgenic mouse Cefonicid sodium model is unique for studying effects of an anti-human HGF neutralizing antibody, since the HGF being overexpressed is human, and there is little evidence of murine HGF in the lungs of these animals. We show for the first time that this L2G7 neutralizing human HGF antibody can significantly decrease carcinogen-induced lung carcinogenesis in human HGF transgenic mice and inhibit downstream cancer-related signaling pathways within the tumors. The L2G7 single antibody may have potential as a therapeutic agent in NSCLC. Materials and Methods Reagents L2G7 anti-HGF mAb and 5G8 isotype control were obtained under a Material Transfer Agreement with Galaxy Cefonicid sodium Biotech (Mt. View, CA). Nitrosoamine 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) was from Toronto Research Chemicals (North York, ON, Canada). Recombinant human and mouse HGF was purchased from R&D Systems (Minneapolis, MN). NSCLC cell line, 201T, was established in our laboratory from primary tissue specimen as described previously (15). These cells do not harbor a K-mutation (16). Protein Extraction and Western Analysis Cells were produced to 75% confluency in T75 flasks. Cells were serum-deprived for 48 h followed by addition of recombinant human or mouse HGF (rhHGF or rmHGF) (10 ng/ml), recombinant human EGF (rhEGF) (10 ng/ml), L2G7 (0-300 ng/ml) or 5G8 (0-300 ng/ml) to the cells and protein was harvested at 10 min after HGF or EGF addition to examine phospho-MAPK expression. Cells were washed Cefonicid sodium one time with ice-cold PBS. Protein was extracted by adding 300 l ice-cold RIPA buffer (1X PBS, 1% NP40, 0.5% Rabbit Polyclonal to ARHGEF11 sodium deoxycholate, 0.1% SDS containing 1 protease inhibitor cocktail/10ml buffer (Roche Diagnostics, Indianapolis, IN)). Protein concentration in the supernatant was measured using the BCA-200 Protein Assay Kit (Pierce, Rockford, IL). Equal amounts of.