Establishment of 5-FU-tolerant gastric cancer cell lines

The human gastric cancer cell lines MKN45 and MKN74 were obtained from the RIKEN Cell Bank. Cells were cultured at 37 °C in RPMI 1640 medium supplemented with 10% fetal bovine serum and 5% CO₂, 80% humidity. Cells were subsequently cultured in the presence of continuous (3 to 5 day) 5-FU exposure followed by increasing concentrations of 5-FU for approximately one year. The timing of the passages and drug concentrations were adjusted based on the growth rate. Here, MKN45 and MKN74 cells were established as the 5-FU-tolerant lines MKN45/5FU and MKN74/5FU, respectively^{11 (link)}. Except for the colony profiling and western blot described below, we used MKN45/5FU cells for all subsequent studies. Growth suppression and colony formation assays were carried out to confirm the cell line characteristics as previously described^{8 (link)}. In addition to 5-FU, CIS, DTX, sorafenib, and gefitinib were used in the assays to eliminate cross-resistance.