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National Yang-Ming University
Thesis Advisors: Emine E. Abali, PhD and Joseph R. Bertino, MD
Program: Cellular & Molecular Pharmacology
RWJMS Research Tower
5th floor Conference Room
Friday, April 30, 2010
Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate into tetrahydrofolate using NADPH as the cofactor, plays an essential role in the de novo synthesis of purines, pyrimidines and certain amino acids. Thus, DHFR has been a critical target for chemotherapy. Methotrexate (MTX), an antifolate that targets DHFR, has been used for decades in the clinic. 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 translational repression caused by DHFR protein-mRNA interaction as a consequence of methotrexate binding to DHFR. In this study, we further investigated the mechanism of MTX-induced upregulation of DHFR and strategies to regulate DHFR levels in order to overcome the inherent resistance related to this adaptive mechanism of cancer cells to MTX.
Here, we demonstrated that divergent translational regulation of human and hamster DHFR is due to the differences in their protein properties rather than differences in their mRNAs. We also presented 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.
Compounds such as methotrexate that target the folate binding site of dihydrofolate reductase are in clinical use to treat a wide variety of human diseases including cancers, autoimmune diseases and infectious diseases. Limitations to their use in cancer include natural resistance and acquired resistance. Here, we report that concomitant use of inhibitors targeting the NADPH binding site of DHFR may overcome some of the limitations of antifolate cancer treatment, and describe a novel mechanism of action of the NADPH analog, NADP-S, to lower cellular DHFR levels and inhibit cancer cell growth, alone or in combination with MTX.
Finally, we demonstrated that DHFR mRNA is associated with polyribosomes in the presence and absence of MTX and that there is no mobilization of DHFR mRNA in polyribosome fractions indicating that MTX-mediated DHFR upregulation is not regulated at the translational initiation stage.