| Classical antifolates, like methotrexate (MTX), are used to inhibit dihydrofolate reductase (DHFR) resulting in the death of rapidly dividing cells. As a result, MTX is currently the treatment of choice for a wide variety of cancers, auto-immune diseases, and ectopic pregnancy. However, problems with MTX therapy can include acquired resistance, toxicity, and teratogenesis. Therefore, there is a need to understand the mechanism of action of MTX, but unfortunately, many of these studies involve mammalian sacrifice. Cell lines are often a good alternative, however, these are not always the best predictor of treatment outcomes in a whole organism. Thus, this thesis examined the possibility of using an alternative invertebrate model, Drosophila melanogaster, to investigate drug resistance, toxicity and drug interactions.;Point mutations in Drosophila DHFR, L30Q and L22R, were able to confer MTX resistance when transferred to CHO cells, 20-fold and ∼200-fold resistance, respectively. When the drug was used to treat flies, there was a reduction in female fecundity as well as the appearance of developmental abnormalities in the progeny. When ovaries from control and MTX-treated Drosophila females were analyzed, transcript levels were perturbed in sequences associated with cell cycle, transport, defense response, or various aspects of metabolism. L30Q DHFR conferred protection against reduced female fecundity and teratogenesis in transgenic Drosophila, and transcript analysis of ovaries revealed that L30Q DHFR expression also eliminated much of the altered transcript abundance profile caused by MTX.;Thus, this thesis has convincingly established a robust non-mammalian animal model in which the mechanism of methotrexate action can be examined. As well, it has developed methods so that the possibility of "rescue" from the toxic and teratogenic effects of antifolate treatment in a non-mamalian system can be further investigated.;The preferred mechanism of MTX resistance in Drosophila appears to be DHFR mutations. However, when the Drosophila enzyme was expressed in Chinese hamster ovary cell lines and selected for high MTX resistant levels, there was evidence of amplification of the transgenes. This suggests that the difference in resistance mechanisms in the two species may be due to differences in genome organization, rather than in the DHFR gene itself. |
