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 (MTXPGs) is recognized 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 versus B-lineage lymphoblasts in children with ALL, which is consistent with the worse prognosis of T-lineage ALL when treated with conventional 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 (Emax) model in both groups. The human leukemia cell lines NALM6 (B-lineage) and CCRF/CEM (T-lineage) were used to assess potential mechanisms for these lineage differences in MTX accumulation, revealing i) greater total and long-chain MTXPG accumulation in NALM6 over a wide range of methotrexate concentrations (0.2-100 microM), ii) saturation of MTXPG accumulation in both cell lines, with a higher maximum (Emax 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 NALM6 cells, and v) 4-6 fold lower extracellular MTX concentration and 2-fold lower intracellular MTXPG concentration to produce equivalent cytotoxicity (LC50) in NALM6 versus CEM. There was a significant relationship between FPGS mRNA and enzyme activity in lymphoblasts from children with newly diagnosed ALL, and blast FPGS mRNA and activity increased after methotrexate treatment. These data indicate higher FPGS and lower DHFR levels as potential mechanisms contributing to greater MTXPG accumulation and cytotoxicity in B-lineage lymphoblasts.
Galpin
A J
AJ
Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, College of Pharmacy, University of Tennessee, Memphis 38101, USA.
Schuetz
J D
JD
Masson
E
E
Yanishevski
Y
Y
Synold
T W
TW
Barredo
J C
JC
Pui
C H
CH
Relling
M V
MV
Evans
W E
WE
D000964:Antimetabolites, Antineoplastic / Q000378:metabolism*; D001483:Base Sequence; D002051:Burkitt Lymphoma / Q000188:drug therapy / Q000201:enzymology*; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000188:drug therapy / Q000201:enzymology*; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D012333:RNA, Messenger; D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D013762:Tetrahydrofolate Dehydrogenase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D008727:Methotrexate