Moleculaire veranderingen in het gereduceerde folaat carrier-gen, door multi-farmacon resistentie-eiwit gemedieerde uitstroom en folaat-compartimentatie: invloed op de folaat-homeostase van de cel en de aktiviteit van antifolaat op leukemie bij kinderen
2000 - 12 / 2004
Background': There is accumulating evidence that the cancer patient's cellular folate status is the most important parameter determining toxicity and anti-tumor activity of folate-based chemotherapeutic drugs. Cellular folate homeostasis is considered to be regulated at the level of membrane transport and polyglutamylation of reduced folate co-factors. The net uptake of (anti)folates depends on (1) (anti)folate influx, mediated by the reduced folate carrier (RFC), and (2) (anti)folate efflux, recently identified to be mediated by members of the multidrug resistance protein (MRP) family, in particular MRP1 and MRP2. Intracellular accumulation and retention of (anti)folates is established by polyglutamylation, mediated by folylpolyglutamate synthetase (FPGS), an enzyme compartmentalized in the cytosol and in mitochondria. Recent studies by our laboratories demonstrated that changes in folate homeostasis could be provoked by mutations in the RFC gene (Jansen et al, J. Biol. Chem. 1998), overexpression of MRP1 and MRP2 (Jansen, Proc. Am. Assoc. Cancer Res. 1999), defective folate efflux (Jansen et al, Mol. Pharmacol. 1999, in press), and by differential FPGS activities (Rots et al, Blood 1999a/b). Expanded intracellular folate pools conferred resistance to antifolates, including methotrexate (MTX) and other polyglutamatable antifolates, by competition for polyglutamylation and via the bypassing of target enzyme inhibition. In contrast, low intracellular folate levels coincided with enhanced antifolate drug activity. Aim of the study: The aim of this study is to identify the clinical relevance of parameters controlling folate homeostasis, i.e. molecular alterations in the RFC gene, MRP-mediated efflux and folate compartmentation. This can be used to improve dosing of antifolates in childhood leukemia and thus potentially improve treatment results. Plan of investigation: For mechanistic studies we will use in vitro model systems displaying either (1) a constitutive expanded intracellular folate pool due to overexpression of a structurally altered RFC, and (2) over/under-expression of MRP. Mechanistic studies will include: (a) the use of a genomic PCR-SSCP assay (Single Strand Conformational Polymorphism) to (a1) identify mutation(s) in the RFC gene, giving rise to structurally altered RFC proteins which retain transport of natural folates but not of antifolate drugs, and (a2) to address the importance of polymorphic variations in the RFC gene in relation to (reduced) folate/MTX transport; (b) characterization of MRP-mediated efflux of (anti)folates along with analyzing the effect of MRP inhibitors, which will include the use of inside-out vesicles. (c) subcellular fractionations to isolate intact mitochondria for the determination of: (c1) the kinetics of mitochondrial transport and polyglutamylation of natural folates and a selected group of antifolate compounds, and (c2) the changes in cytosolic and mitochondrial folate pools after cellular exposure to antifolates. In order to explore the hypothesis defined in these model systems, these studies will be extended, wherever possible, with clinical specimens including childhood leukemia cells (B-lineage ALL, relapsed B-lineage ALL cells and T-lineage ALL) displaying different (clinical) sensitivities to MTX. We will use an in situ thymidylate synthase inhibition assay to determine the sensitivity of these cells for MTX as well as for a selected group of second generation antifolates that are (non)-dependent on polyglutamylation. Drug sensitivity in these cells will also be determined as a function of exogenous and intracellular folate concentrations. These data will be correlated with levels of RFC and various MRPs, and folate compartmentation in the cytosol and mitochondria. Collectively, the results of these studies will be evaluated in a multivariate analysis for a predictive assessment of antifolate drug sensitivity in relation to cell biological (DNA-ploidy, chromosomal aberrations and immunophenotype) and clinical features (white blood cell count, age and sex) known to have prognostic value in ALL. Expected results and clinical relevance for cancer research: A multiparameter study of factors governing folate homeostasis can be exploited for a rationalized design of antifolate-containing chemotherapy of different subtypes of childhood leukemia and management of drug toxicity.