title: Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation.
abstract: Methotrexate (MTX) is an anti-folate that inhibits de novo purine and thymidine nucleotide synthesis. MTX induces death in rapidly replicating cells and is used in the treatment of multiple cancers. MTX inhibits thymidine synthesis by targeting dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS). The use of MTX to treat cancer also causes bone marrow suppression and inhibits the immune system. This has led to the development of an MTX-resistant DHFR, DHFR L22F, F31S (DHFR(FS)), to rescue healthy cells. 5-Fluorouracil-resistant TYMS T51S, G52S (TYMS(SS)) is resistant to MTX and improves MTX resistance of DHFR(FS) in primary T cells. Here we find that a known mechanism of MTX-induced increase in DHFR expression persists with DHFR(FS) and cis-expressed transgenes. We also find that TYMS(SS) expression of cis-expressed transgenes is similarly decreased in an MTX-inducible manner. MTX-inducible changes in DHFR(FS) and TYMS(SS) expression changes are lost when both genes are expressed together. In fact, expression of the DHFR(FS) and TYMS(SS) cis-expressed transgenes becomes correlated. These findings provide the basis for an unrecognized post-transcriptional mechanism that functionally links expression of DHFR and TYMS. These findings were made in genetically modified primary human T cells and have a clear potential for use in clinical applications where gene expression needs to be regulated by drug or maintained at a specific expression level. We demonstrate a potential application of this system in the controlled expression of systemically toxic cytokine IL-12.
authors.affiliation: From the Division of Pediatrics, Children's Cancer Hospital, The University of Texas MD Anderson Cancer Center (MDACC), Houston, Texas 77030 and the The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030.
authors.forename: David
authors.initials: D
authors.lastname: Rushworth
authors.affiliation: the The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030.
authors.forename: Amber
authors.initials: A
authors.lastname: Mathews
authors.affiliation: the The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030.
authors.forename: Amir
authors.initials: A
authors.lastname: Alpert
authors.affiliation: From the Division of Pediatrics, Children's Cancer Hospital, The University of Texas MD Anderson Cancer Center (MDACC), Houston, Texas 77030 and the The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas 77030 ljncooper@mdanderson.org.
authors.forename: Laurence J N
authors.initials: LJ
authors.lastname: Cooper
mesh_terms: D019943:Amino Acid Substitution; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D006801:Humans; D019169:Jurkat Cells; D008727:Methotrexate / Q000494:pharmacology*; D020125:Mutation, Missense; D013601:T-Lymphocytes / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
chemical_list: D000964:Antimetabolites, Antineoplastic; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D008727:Methotrexate
keywords: chemoresistance; co-expression; dihydrofolate reductase; drug-inducible; gene therapy; immunology; immunotherapy; methotrexate; post-transcriptional regulation; thymidylate synthase