Resistance mechanisms for nucleoside analogues : With focus on metabolism and apoptosis

The aim of the thesis was to elucidate the mechanisms underlying resistance to nucleoside analogues used in the treatment of leukemias, with focus on cellular metabolism and induction of apoptosis. Cladribine (CdA), Clofarabine (CAFdA), Fludarabine (Fara-A) and Nelarabine (Ara-G) are nucleoside analogues with activity against various types of leukemias. CAFdA is a relatively new nucleoside analogue and we showed that CAFdA nucleotides were accumulated to a higher extent than CdA nucleotides in samples from patients with chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). CAFdA is more efficiently phosphorylated by deoxycytidine kinase (dCK) and CAFdA nucleotides are more slowly eliminated than CdA nucleotides. As earlier indicated in patients, there is an absence of cross-resistance between CdA and Fara-A. In an acute myeloid leukemia cell line the mechanism of resistance to CdA was a deficiency in dCK. Fara-A resistant cells had another contributing factor to resistance, the deoxynucleoside triphosphate pools being altered, indicating a mutation or altered regulation of ribonucleotide reductase (RR). Further studies in a lymphoid leukemia cell line supported these findings, and we also demonstrated that Fara-A resistant cells had increased RR activity and protein levels of the R2 subunit of RR. A real time quantitative PCR (RQ-PCR) method was established to measure mRNA levels of enzymes important in the metabolism of dCK, deoxyguanosine kinase (dGK) and high Km 5'-nucleotidase (5'-NT). The RQPCR method was compared to semi-quantitative PCR and enzyme activity measurements and tested with samples from pediatric patients with acute lymphocytic or myeloid leukemias, and was shown to be a convenient and reliable tool in the measurement of these enzymes. The major cause of resistance to CdA at the apoptotic level was a disturbed sensitivity to increased Ca 2+ levels in the cytosol. Increased Ca 2+ levels may induce changes in mitochondrial membrane potential (alpha psi mito). Accordingly, the increased Ca 2+ levels and the following drop in alpha psi mito are important events for CdA-induced apoptosis. In another study we demonstrated that Ara-G-resistance was associated with perturbations in apoptotic events. Resistance to Ara-G correlated with upregulation of the anti-apoptotic protein BcI-xL and downregulation of the Fas receptor. Thus, our data demonstrate that CAFdA is more effectively accumulated in samples from CILL and AML patients. Important for CdA- and CAFdA- resistance is dCK and important for Fara-A-resistance in addition to dCK is RR. Apparent is also that abberations in apoptosis induced by nucleoside analogues may contribute to resistance