Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation [Immunology]

August 4th, 2015 by Rushworth, D., Mathews, A., Alpert, A., Cooper, L. J. N.

Methotrexate (MTX) is an antifolate which 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 a MTX−resistant DHFR − DHFR L22F, F31S (DHFR FS) to rescue healthy cells. 5−fluorouracil resistant TYMS T51S, G52S (TYMSSS) is resistant to MTX and improves MTX resistance of DHFRFS in primary T cells. Here we find that a known mechanism of MTX−induced increase in DHFR expression persists with DHFRFS and cis-expressed transgenes. We also find that TYMSSS expression of cis−expressed transgenes is similarly decreased in an MTX−inducible manner. MTX−inducible changes in DHFRFS and TYMSSS expression changes are lost when both genes are expressed together. In fact, expression of the DHFRFS and TYMSS 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 interleukin − 12 (IL−12).
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation [Immunology]