![]() Using an in vitro translation assay, our laboratory ( Ercikan et al., 1993) and Chu et al. Based on studies demonstrating an interaction between DHFR protein and its cognate mRNA, a model was proposed to account for increases in DHFR protein levels in response to MTX exposure, namely that the binding of human DHFR protein to its cognate mRNA results in decreased translation and that the addition of MTX disrupts this autoregulation ( Chu et al., 1993 Ercikan et al., 1993). The first suggestion that translational regulation may play a role in DHFR induction came from studies showing that DHFR mRNA levels remain the same in the presence or absence of MTX ( Bastow et al., 1984 Cowan et al., 1986). Later studies showed that the half-life of DHFR protein was unaltered in the presence of MTX ( Domin et al., 1982 Cowan et al., 1986), indicating that protection from degradation was not the explanation for the increase. Because the increase in DHFR is associated with MTX bound to the protein, it was suggested that MTX protected DHFR from degradation ( Bertino et al., 1970). The rapid increase in DHFR protein levels seen upon exposure to MTX was unaffected by the transcriptional inhibitor actinomycin D but was blocked by the translational inhibitor cycloheximide ( Hillcoat et al., 1967a, b) ruling out an increase in transcription as the mechanism. This rapid adaptive response differs from the increase in DHFR because of gene amplification in cells after long-term MTX selection ( Alt et al., 1978). After this initial report, several studies of human cells in vivo and in vitro showed that DHFR protein levels rapidly increase, or are “induced,” in response to MTX treatment ( Bertino et al., 1962, 1963 Hillcoat et al., 1967a Hillcoat and Bertino, 1969). Within days after exposure to MTX, DHFR activity increases approximately 6-fold in blast cells of leukemic patients ( Bertino et al., 1962). MTX, a tight binding inhibitor of DHFR, is used for the treatment of acute lymphocytic leukemia, non-Hodgkin's lymphoma, osteosarcoma, choriocarcinoma, breast, and head and neck cancer, as well as a variety of other nonmalignant diseases ( Chu and Allegra, 1996 Bertino et al., 1997). Inhibition of DHFR results in a depletion of the reduced folate pools, inhibition of DNA synthesis, and cell death. Tetrahydrofolate and its one-carbon adducts are essential cofactors in the synthesis of thymidylate, purines, and some amino acids ( Blakley, 1984). 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.ĭihydrofolate reductase (DHFR, 5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.51.3) catalyzes the reduction of dihydrofolate to tetrahydrofolate using NADPH as a cofactor. 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 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. 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. 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. 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.
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