DIFFERENCES IN FOLYLPOLYGLUTAMATE SYNTHETASE AND DIHYDROFOLATE-REDUCTASE EXPRESSION IN HUMAN B-LINEAGE VERSUS T-LINEAGE LEUKEMIC LYMPHOBLASTS - MECHANISMS FOR LINEAGE DIFFERENCES IN METHOTREXATE POLYGLUTAMYLATION AND CYTOTOXICITY

Cellular accumulation of methotrexate polyglutamates (MTX-PGs) is reco gnized as an important determinant of the cytotoxicity and selectivity of methotrexate in acute lymphoblastic leukemia (ALL). We identified a significantly lower cellular accumulation of MTXPGs in T-lineage ver sus B-lineage lymphoblasts in children with ALL, which is consistent w ith the worse prognosis of T-lineage ALL when treated with conventiona l antimetabolite-based therapy. Maximum MTXPG accumulation in leukemic blasts in vivo was 3-fold greater in lymphoblasts of children with B- lineage ALL (129 children) compared with those with T-lineage ALL (20 children) (p < 0.01) and was characterized by a saturable (E-max) mode l in both groups. The human leukemia cell lines NALM6 (B-lineage) and CCRF/CEM (T-lineage) were used to assess potential mechanisms for thes e lineage differences in MTX accumulation, revealing i) greater total and long-chain MTXPG accumulation in NALM6 over a wide range of methot rexate concentrations (0.2-100 mu M), ii) saturation of MTXPG accumula tion in both cell lines, with a higher maximum (E-max) in NALM6, iii) 3-fold higher constitutive FPGS mRNA expression and enzyme activity in NALM6 cells, iv) 2-fold lower levels of DHFR mRNA and protein in NALM 6 cells, and v) 4-6 fold lower extracellular MTX concentration and 2-f old lower intracellular MTXPG concentration to produce equivalent cyto toxicity (LC50) in NALM6 versus CEM. There was a significant relations hip between FPGS mRNA and enzyme activity in lymphoblasts from childre n with newly diagnosed ALL, and blast FPGS mRNA and activity increased after methotrexate treatment. These data indicate higher FPGS and low er DHFR levels as potential mechanisms contributing to greater MTXPG a ccumulation and cytotoxicity in B-lineage lymphoblasts.