Fujimoto et al. BMC Cancer 2010, 10:443 http://www.biomedcentral.com/1471-2407/10/443 to be about 90% less than the a-thrombin level of the initial cell cultures from which the concentrates were derived. Subsequent separate cultures of MKN45/PAR1 cells underwent a 6 hr exposure to one of either of the high molecular weight concentrates. The cultures exposed to the concentrates derived from initial cultures exposed to 15 nM a-thrombin for 12 hr were incubated for 6 hr either in the presence or absence of TN-C-neu- tralizing antibody (Clone BC24, 25 μg/ml). After incuba- tion each culture exposed to concentrates was lysed, and the lysate subjected to SDS-PAGE, Western blotting and probing to quantify levels of phosphorylated EGFR. Results PAR1 mRNA is expressed in Gastric Cancer Cell Lines The expression of PAR1 mRNA in seven gastric cancer cell lines was evaluated by RT-PCR and was found to be present in MKN28, MKN74, and NUGC3 cell lines (Fig- ure 1A). Although, although a faint band was detected for KATOIII cells, no RT-PCR signal for PAR1 was detected for the NUGC2, MKN45, and TMK-1 cell lines (Figure 1A). Expression of PAR1 in MKN45 cells Since MKN45 cells did not express PAR1, we selected this gastric carcinoma-derived cell line as a ‘host’ cell Page 4 of 14 for PAR1 expression, in order to evaluate the functional properties of PAR1 in a gastric carcinoma cell back- ground. As shown in Figure 1B, transfection of MKN45 cells with pcDNA3.1-PAR1 bearing the human full PAR1 coding sequence and a neomycin-resistance gene yielded a receptor-expressing MKN45 cell line, selected in the presence of G418. PAR1-expressing MKN45 cells proliferate in response to a PAR1-activating peptide and a-thrombin a-thrombin clearly stimulated replication of MKN45/ PAR1 over a 4 day time frame, with a greater than 2- fold increase in cell number relative to non-thrombin- treated MKN45/PAR1 at 96 hr (Figure 3). The prolifera- tion of MKN45/mock was not stimulated either by a- thrombin or TFLLR-NH2 (Figure 2). Even in the absence of a-thrombin, MKN45/PAR1 outgrew the MKN45/mock at 96 hr, suggesting the presence of receptor-activating proteinases in the growth medium. Similarly, TFLLR-NH2 triggered an approximately 3-fold increase in cell number, in treated compared with untreated MKN45/PAR1 (Figure 4). Of particular signif- icance, the PAR1-selective antagonist, SCH79797, in a concentration-dependent manner, was able to block the proliferative actions of both a-thrombin and TFLLR- NH2 (Figures 3 and 4). The data thus indicated a PAR1- Figure 1 The expression of PAR1 mRNA and protein in gastric cancer cell lines. (A) The expression of PAR1 mRNA in 7 gastric cancer cell lines by RT-PCR. PAR1 mRNA was confirmed in MKN28, MKN74 and NUGC3. (B) Parent MKN45 cells and MKN45 cells transfected with pcDNA3.1- empty-vector alone (designated as MKN45/mock) have no detectable expression of PAR1 protein, and MKN28 and MKN45 cells transfected with PAR1 cDNA bearing pCDNA3.1 vector, which was treated 750 μg/ml G418 to select permanent PAR1 expressing clone, (designated as MKN45/ PAR1) was confirmed to continuously express high level PAR1 protein by immunoblotting (upper panel). The bottom panel showed a protein band of a house keeping gene, GAPDH as a control. (The final dilution of PAR1 antibody was 1:500, GAPDH was 1:2000.)