3E). When an expression level of 1.00, which corresponds to a value 5-fold of the mean expression level in non-cancerous gastric foveolar epithelium, was used as a cutoff value for the expression status in gastric cancer (i.e., low expression group, 0�C0.99; high expression group, 1.00�C3.00), 88 www.selleckchem.com/products/DAPT-GSI-IX.html (24.4%) of the 360 primary gastric cancers were included in the high expression group (Figure (Figure3F).3F). To examine whether CRKL overexpression is associated with CRKL amplification in gastric cancer, we performed a FISH analysis for the CRKL gene in the 360 primary gastric cancers and compared the prevalence of CRKL amplification between the low expression group and the high expression group. As expected, the percentage of gastric cancer cells with CRKL amplification was significantly higher in the high expression group (9.

1%; 8/88 cases) than in the low expression group (2.2%; 6/272 cases) (P=0.028, chi-square test). This result suggests that CRKL amplification contributes to CRKL overexpression in primary gastric cancer. We further investigated whether the levels of CRKL expression is associated with clinicopathological features in primary gastric cancer patients, the high CRKL expression was observed significantly more often in male and differentiated-type gastric cancer (Table (Table2).2). These results suggested that CRKL protein is overexpressed partly due to CRKL amplification in a subset of primary gastric cancers and is associated with the gender and histopathology.

Table 2 Association between CRKL expression and clinicopathological factors in 360 patients with primary gastric cancer Decrease in the viability of CRKL-expressing MKN74 cells treated with BMS354825 Finally, we tested the possibility of using CRKL as a therapeutic target in MKN74 cells with CRKL amplification. Since Philadelphia chromosome-positive leukemia expressing BCR-ABL is responsive to BMS354825 (a dual Src/BCR-ABL kinase inhibitor) and AMN107 (a highly selective BCR-ABL kinase inhibitor) [22,24], we checked the response of MKN74 cells to both inhibitors. Cell viability was significantly decreased in BMS354825-treated (0.01�C1.0��M) MKN74 cells, compared with cells treated with the solvent only, while it was not significantly decreased in AMN107-treated cells (Figure (Figure4A).4A).

When the status of CRKL phosphorylation was examined in the MKN74 cells using western blot analysis with an anti-phospho CRKL antibody, CRKL phosphorylation was found to be inhibited more effectively by BMS354825 than by AMN107 (Figure (Figure4B).4B). These results suggested that BMS354825 has the potential to suppress the viability of MKN74 GSK-3 cells expressing CRKL, likely via the inhibition of CRKL phosphorylation. To further characterize the role of CRKL in the BMS354825-induced suppression of MKN74 cell viability, we examined the effect of BMS354825 on gastric cancer cells without CRKL amplification.