- MiffyLv 71 decade agoFavorite Answer
Human dihydrofolate reductase (hDHFR) is a ubiquitous cytosolic enzyme that catalyzes the reduction of 5,6-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF).
THF is an essential co-factor in several metabolic pathways, including purine andthymidylate biosynthesis. As a result of its importance in cellular proliferation, hDHFR has longbeen a key pharmacological target for the treatment of various types of cancer.
Methotrexate (MTX) is a folate analog that acts as a slow, tight binding competitive inhibitor of hDHFR, thereby inhibiting cellular THF synthesis and cellular proliferation.
MTX is widely used for the treatment of cancer.
Since MTX is toxic to healthy haematopoietic stemcells during chemotherapy, MTX-resistant hDHFRshave the potential to protect patients from immuno-suppression.
Gene therapy strategies have previouslybeen used to transfer moderately MTX-resistant hDHFRs into mouse and human bone marrowprogenitor cells, ensuring stem cell survival upon exposure to MTX.
The wealth of information abouthuman treatment with MTX indicates the recognized potential of the MTX/hDHFR system for developinghuman cell selection markers.
These novel MTX-resistant hDHFR mutants, including a variety of combinatorial mutants thatdisplay an important decrease in MTX binding, arecomparable to the weakest-binding hDHFR mutantreported to date, while offering greater residualspecific activity.
As a result, very efficient protection of a relevant mammalian cell model, (DHFR-deficient CHO DUKX B11 cells) was obtained. Importantly, the same mutants provided protectionfrom high levels of MTX in isolated murinehematopoietic stem cells.
Selection was efficient in as little as 4 days in liquid medium.Source(s): Methotrexate-resistant combinatorial mutants of human dihydrofolate reductase (hDHFR)