Pharmacology of methotrexate im leukemic blast cells

In childhood acute lymphoblastic leukemia (ALL) cells, methotrexate (MTX) cytotoxicity is directly correlated to the accumulation of intracellular methotrexate polyglutamates (MTXPG). Mechanisms involved in the formation of MTXPGs and their differences between lineage and ploidy ALL subtypes have been well defined previously. Conversely, the intralysosomal degradation of MTXPGs via the enzyme gamma-glutamyl hydrolase (GGH) is poorly characterized. Additionally, it is unknown whether GGH activity changes after MTX treatment. In order to address these questions we analyze GGH enzyme activity in blast cells (3 different cell fractions) obtained at diagnosis and 42 hours after 1 g/m2 MTX treatment. Our preliminary results of GGH enzyme activities in diagnostic blasts from 45 patients contradict reports from the literature but are consistent with our results in ALL cell lines; revealing a significant higher GGH activity in T-lineage cells compared to B-lineage cells (p<0.008). The small number of patients did not allow statistical analyses in regard to ploidy differences. To date, we have analyzed 25 patients` bone marrow samples before and after MTX treatment; and data comparison of paired samples show no difference in median GGH activity (p=0.35). Additionally, we observed a considerable interindividual heterogeneity in GGH activity. Preliminary analyses revealed a lower amount of GGH mRNA in patients with a low GGH activity. In ongoing studies we will further elucidate the role of GGH on MTXPG accumulation in different ALL subtypes, and investigate the cause (e.g. single nucleotide polymorphisms) of the wide range in GGH activity.