The role of 5'-nucleotidases and Deoxynucleoside Kinases in Responses to Nucleoside Analogues

The efficacy of nucleoside analogues (NAs) in treating several hematological malignancies, solid tumors and viral infections is limited primarily by side-effects and the development of drug resistance. The aims of the present thesis were to elucidate mechanism(s) involved in tissue-specific toxicity associated with NA therapy, as well as the mechanisms underlying resistance to these drugs. The mRNA levels and activities of different cytosolic and mitochondrial deoxynucleoside kinases (dNKs) and 5'-nucleotidases (5'NTs) exhibit a distinct pattern for each of a variety of mouse tissues. Heart and skeletal muscle, as well as adipose tissue demonstrate low levels of both the anabolic and catabolic enzymes, which may explain at least some of the adverse side-effects of NA treatment. A novel approach based on high-performance liquid chromatography (HPLC) revealed that each of 14 different mouse and rat tissues exhibits a unique profile of dNK and 5'NT activities, with 2-3-fold species differences for certain of these tissues. These observations have important implications for the choice of an animal model for characterization of NA toxicity. The cytotoxicity of gemcitabine (a cytidine-containing NA employed clinically to treat patients with cancer) towards human leukemia and melanoma cell lines rendered deficient in either deoxycytidine kinase (dCK) or deoxyguanosine kinase (dGK) with siRNA was compared to the corresponding toxicity towards cells that express these enzymes. Both types of deficient cells were more sensitive to gemcitabine, suggesting that, at least in this system, the toxicity of this drug is not correlated to the levels of activating enzymes. The activites of various 5'-NTs in peripheral blood cells isolated from CLL patients show considreble inter-individual variation. Degradation of the phosphorylated forms of cladribine and fludarabine, two important anti-leukemic NAs, was characterized in these cells. Significant correlations between the rate of cladribine monophosphate degradation and the activity of cytosolic 5'-nucleotidase 1 (CN1), as well as between the rate of fludarabine monophosphate degradation and the activity of cytosolic 5'nucleotidase 2 (CN2) were observed. This investigation provides new insights regarding the patterns of expression of anabolic and catabolic enzymes that hopefully can be used to improve NA therapy in the future. LIST OF PUBLICATIONS This thesis is based on the following papers: I. Svetlena N. Rylova, Saeedeh Mirzaee, Freidoun Albertioni, Staffan Eriksson. Expression of deoxynucleoside kinases and 5'-nucleotidases in mouse tissues: Implications for mitochondrial toxicity. Biochemical Pharmacology, 2007, 74, 169-75. II. Saeedeh Mirzaee, Staffan Eriksson, Freidoun Albertioni. Differences in cytosolic and mitochondrial 5’-nucleotidase and deoxynucleoside kinase activities in SpragueDawley rat and CD-1 mouse tissues: Implication for the toxicity of nucleoside analogs in animal models. Toxicology, 2010, 267, 15964. III. Anna Fyrberg, Saeedeh Mirzaee, Freidoun Albertioni, Kourosh Lotfi. RNA interference targeting nucleoside analog activating enzymes in leukemic and solid malignancies enhances the effect of gemcitabine (Submitted). IV. Saeedeh Mirzaee, Xin Wang, Narges Bayat, Anna Fyrberg, Kourosh Lotfi, Karin Karlsson, Gunnar Juliusson, Staffan Eriksson, Freidoun Albertioni. Activity profiles of 5’-nucleotidases in blood cells from untreated patients with B-cell chronic lymphocytic leukemia and in phytohemag-glutinin stimulated cells from healthy subjects: correlation to nucleoside analogues therapy (Manuscript).