Screening of colon tumor cells and tissues for folylpolyglutamate synthetase activity

Polyglutamylation of (anti)folates catalyzed by folylpolyglutamate synthetase (FPGS) determines the retention of these compounds in the cell. This feature is essential for the activity of folates (e.g., folinic acid) and antifolates (e.g., methotrexate) in the treatment of cancer. A FPGS assay was developed using murine liver and was based on published methods, but had a novel analytic procedure. Tritiated glutamate and aminopterin served as substrates for FPGS, and after the reaction the mixture of substrates and products was separated by thin-layer chromatography. Results were verified by standard anion-exchange high performance liquid chromatography for folates. The assay was applied to measure the activity of FPGS in several cancer cell lines and human and murine (tumor) tissues. Cancer cell lines had a much higher activity (varying from 82 to 656 pmol diglutamate formed per hour per 10(6) cells) than murine bone marrow cells (35 pmol/h/10(6) cells). Murine gut mucosa had a very low FPGS activity compared to murine liver (7 vs. 24 pmol diglutamate/h/g wet weight), but the activity in murine colon tumors was comparable to or higher than that in liver (28-52 pmol diglutamate/h/g wet weight). A screening of 11 human colon tumors or metastases demonstrated that there was a large variation in FPGS activity in this tumor type, but overall the activity was higher in tumor tissue than in normal colon mucosa. The latter feature may increase the selectivity of antifolate-based chemotherapy of colon tumors. The FPGS assay described in this paper allows large-scale screening of cell lines and tissues, because of its rapid separation procedure by thin-layer chromatography.