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

Physiological cell culture media alter nucleotide metabolism in T lymphocytes and increase their sensitivity to methotrexate | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Physiological cell culture media alter nucleotide metabolism in T lymphocytes and increase their sensitivity to methotrexate Claudia Cano-Estrada, Neus Ontiveros, Paula Escudero-Ferruz, Veronika Baresova, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8681623/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. T-lymphocytes methotrexate physiological media nucleotide metabolism cancer folic acid Full Text Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8681623","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584610527,"identity":"ff91bd38-cb50-4b18-9c2d-b96287268bc2","order_by":0,"name":"Claudia Cano-Estrada","email":"","orcid":"","institution":"Universitat Autònoma de Barcelona: Universitat Autonoma de Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Cano-Estrada","suffix":""},{"id":584610528,"identity":"33d16e36-a3b3-4c4b-bd70-b56d026eb469","order_by":1,"name":"Neus Ontiveros","email":"","orcid":"","institution":"Universitat Autònoma de Barcelona: Universitat Autonoma de Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Neus","middleName":"","lastName":"Ontiveros","suffix":""},{"id":584610529,"identity":"af40333e-1460-4799-b93d-fef4855a2430","order_by":2,"name":"Paula Escudero-Ferruz","email":"","orcid":"","institution":"IMO: Instituto de Microcirugia Ocular","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"","lastName":"Escudero-Ferruz","suffix":""},{"id":584610530,"identity":"061f987c-6078-4012-85c8-42cff07a36c2","order_by":3,"name":"Veronika Baresova","email":"","orcid":"","institution":"Charles University: Univerzita Karlova","correspondingAuthor":false,"prefix":"","firstName":"Veronika","middleName":"","lastName":"Baresova","suffix":""},{"id":584610531,"identity":"2dcacd01-a14d-4242-935d-372550cb9680","order_by":4,"name":"Marie Zikanova","email":"","orcid":"","institution":"Charles University: Univerzita Karlova","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"","lastName":"Zikanova","suffix":""},{"id":584610532,"identity":"2953c0d2-ac1b-4aa6-8075-9208eff5883e","order_by":5,"name":"Daniel Iglesias-Serret","email":"","orcid":"","institution":"Universitat de Barcelona","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Iglesias-Serret","suffix":""},{"id":584610533,"identity":"4ae8b87d-12fa-4970-ab63-e89badf29fa5","order_by":6,"name":"José M. 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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. 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