Pharmacogenetics studies the genetic basis of inter-individual differences in drug response. The goal of pharmacogenetic testing is to individualize drug treatment by identifying relevant factors which influence drug efficacy and lower the therapy-related toxicity in individual patients. Thiopurines are prodrugs used in the treatment of haematological malignancies and autoimmune diseases. Due to their narrow therapeutic index, they frequently exhibit life-threatening side-effects. They require metabolic activation, in order to achieve their cytotoxic effect. The amount of cytotoxic and inactive metabolites largely depends on the activity of thiopurine S-methyltransferase (TPMT), a highly polymorphic drug metabolizing enzyme. TPMT genotyping is the basis for dosage adjustment protocols to prevent thiopurine-related side-effects and improve treatment outcome. In presented dissertation, the correlation of TPMT genetic polymorphisms and enzyme activity in the Slovenian population was evaluated. The genotypic frequencies of the most common polymorphisms TPMT*3A (4.1 %), TPMT*3C (0.5 %) and TPMT*3B (0.3 %) were in accord with the values observed in other Caucasian populations. Heterozygous individuals with a single mutant allele exhibited lower enzyme activity than wild-type individuals. Statistic analysis was employed to determine the cut-off value (9.82 pmol/107 RBC h-1) between intermediate and high TPMT activity groups. The correlation between TPMT genotype and enzyme activity was 91.6 %. With the aid of relevant analytical methods, the pharmacogenetic basis for the identification of patients with altered response to thiopurines requiring dosage adjustment protocols was established. Due to the incomplete TPMT genotype-to-phenotype correlation observed in several studies, we examined the potential factors involved in additional regulation of TPMT activity and unfavourable thiopurine toxicity. Using a model cell line for acute lymphoblastic leukemia (ALL), we delineated the molecular mechanism of the effect of methyl donor S-adenosylmethionine (SAM) on TPMT activity. We found that the addition of exogenous SAM prevents 6-MP induced cytotoxicity, delays the onset of 6-MP triggered apoptosis and lowers the concentration of cytotoxic metabolites. The potential effect of SAM on thiopurine toxicity was confirmed in the retrospective study, where we established the correlation between hematotoxic events in ALL patients and the presence of TPMT genetic polymorphisms, as well as low-activity mutations in the MTHFR gene, coding for an important enzyme involved in the biosynthesis of SAM. By identifying SAM as an important modulator of TPMT activity and 6-MP cytotoxicity, novel rationalization of therapy may apply. Further, to delineate the endogenous function of TPMT, we generated human HEK293, HepG2 and Jurkat cell lines exhibiting stable TPMT overexpression. The gene delivery system termed artificial viral particles (AVP) was used to transfect the cells with a plasmid containing TPMT cDNA insert, enabling stable overexpression of the recombinant fusion EGFP-TPMT protein and elevated TPMT activity. In addition, we found that TPMT overexpression in HEK293 and HepG2 cells results in faster intracellular SAM turnover, while the concentration of intracellular S-adenosylhomocysteine (SAH) is decreased, maintaining the cellular methylation potential. The effect of TPMT overexpression on global DNA methylation was not observed in the absence of DNA methyltransferase inhibition, while significantly lower amounts of reduced glutathione were found in HepG2 cells with stable TPMT overexpression. These results show that TPMT might be involved in endogenous processes affecting the cellular SAM turnover, transmethylation reactions and oxidative status. TPMT activity is one of the major factors influencing thiopurine drug metabolism, drug efficacy and unwanted toxicity. In addition to TPMT genetic polymorphisms, other factors involved in the regulation of endogeous methyl donor SAM may influence TPMT activity, and consequently, thiopurine toxicity and drug response. Further studies will clarify the endogenous role of TPMT in intracellular metabolism, which is crucial for the identification of different phenotypes resulting from significant inter-individual differences in TPMT activity.
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