Pharmacological and molecular investigations on the mechanisms underlying resistance of human leukaemia cells to the antimetabolites methotrexate, 6-mercaptopurine and 6-thioguanine

Sammanfattning: The goal of this thesis has been to elucidate the mechanisms underlying resistance to methotrexate (MTX), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), the antimetabolites widely used in treatment of childhood leukemia, as well as to determine the influence of 7-hydroxymethotrexate (7-OHMTX), the major metabolite of MTX, on the therapeutic action of MTX. Resistant sublines of leukemic cell lines were developed by long-term exposure to stepwise increasing concentrations of these different agents. The mechanism underlying resistance to MTX in cells exposed to this drug was a pronounced reduction (> 10-fold) in reduced folate carrier (RFC) mediated uptake of MTX. In CCRF-CEM cells, this reduction was associated with transcriptional silencing of the RFC gene, due to attenuated or even abolished binding of various transcription factors to their cis-acting elements, including the CRE, E-box, AP1, Mzf-1 and GC-box. In contrast, resistance to 7-OHMTX was due solely to a dramatic decrement (> 95%) in folylpolyglutamate synthetase activity, which also conferred a greater than 100-fold increase in resistance to short-term exposure to MTX. The levels of mRNA species originating from approximately 17000 genes present in MTX- and 7-OHMTX-resistant MOLT4 cells were compared. In the MTX-resistant subline, the levels of mRNA encoding proteins involved in DNA and RNA metabolism and in transport were altered most; whereas in the 7-OHMTX-resistant cells, mRNA species associated with metabolism and cell proliferation were affected more profoundly. In these 7-OHMTX-resistant cells, the 10-fold reduction in the level of the mRNA for adenosine deaminase (the major enzyme of purine catabolism), complete absence of mRNA for cystathionine