Species-specific differences in translational regulation of dihydrofolate reductase.

We have observed that rodent cell lines (mouse, hamster) contain approximately 10 times the levels of dihydrofolate reductase as human cell lines, yet the sensitivity to methotrexate (ED(50)), the folate antagonist that targets this enzyme, is similar. Our previous studies showed that dihydrofolate reductase protein levels increased after methotrexate exposure, and we proposed that this increase was due to the relief of feedback inhibition of translation as a consequence of methotrexate binding to dihydrofolate reductase. In the current report, we show that unlike what was observed in human cells, dihydrofolate reductase (DHFR) levels do not increase in hamster cells after methotrexate exposure. We provide evidence to show that although there are differences in the putative mRNA structure between hamster and human mRNA in the dihydrofolate reductase binding region previously identified, "hamsterization" of this region in human dihydrofolate reductase mRNA did not change the level of the enzyme or its induction by methotrexate. Further experiments showed that human dihydrofolate reductase is a promiscuous enzyme and that it is the difference between the hamster and human dihydrofolate reductase protein, rather than the DHFR mRNA, that determines the response to methotrexate exposure. We also present evidence to suggest that the translational up-regulation of dihydrofolate reductase by methotrexate in tumor cells is an adaptive mechanism that decreases sensitivity to this drug.

Regulation of human dihydrofolate reductase activity and expression.

Dihydrofolate reductase (DHFR) enzyme catalyzes tetrahydrofolate regeneration by reduction of dihydrofolate using NADPH as a cofactor. Tetrahydrofolate and its one carbon adducts are required for de novo synthesis of purines and thymidylate, as well as glycine, methionine and serine. DHFR inhibition causes disruption of purine and thymidylate biosynthesis and DNA replication, leading to cell death. Therefore, DHFR has been an attractive target for chemotherapy of many diseases including cancer. Over the following years, in order to develop better antifolates, a detailed understanding of DHFR at every level has been undertaken such as structure-functional analysis, mechanisms of action, transcriptional and translation regulation of DHFR using a wide range of technologies. Because of this wealth of information created, DHFR has been used extensively as a model system for enzyme catalysis, investigating the relations between structure in-silico structure-based drug design, transcription from TATA-less promoters, regulation of transcription through the cell cycle, and translational autoregulation. In this review, the current understanding of human DHFR in terms of structure, function and regulation is summarized.