Physiological cell culture media alter nucleotide metabolism in T lymphocytes and increase their sensitivity to methotrexate

Most cancer cells have an increased synthesis of purine nucleotides to fulfil their enhanced division rate. The de novo purine synthesis requires folic acid in the form of N10 formyltetrahydrofolate (10-formyl-THF). However, regular cell culture media contain very high non-physiological concentrations of folic acid, which have an impact on cell metabolism. In a previous study, several human cell lines maintained in RPMI containing physiological levels of folic acid (FA) (25 nM) accumulated 5-aminoimidazole-4-carboxamide riboside 5’-monophosphate (ZMP), an intermediary of the de novo purine biosynthetic pathway, but not with the artificially high levels (2200 nM) present in regular media [1]. Here, we investigate how culturing human cell lines in a new medium formulated to mimic physiological levels of all nutrients including vitamins (Plasmax-PV) [2] affects nucleotide content and cell death induced by the antifolate drug methotrexate (MTX). In Plasmax-PV, most of the cell lines do not show any nucleotide alteration, but Jurkat cells show accumulation of ZMP and dUMP, suggesting a disruption of both purine and pyrimidine biosynthetic pathways. Notably, a low dose of MTX induces apoptosis of Jurkat cells cultured in Plasmax-PV more effectively than in standard RPMI medium. Thymidine supplementation of Plasmax-PV completely blocked MTX-induced apoptosis, confirming an impairment of pyrimidine biosynthesis in human lymphocytes. In summary, our findings highlight the critical impact of the culture medium composition on T-cell sensitivity to MTX. These results suggest that physiological culture media can be useful to optimize MTX doses for improving personalized therapy.