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
- Amy J. Galpin1,
- John D. Schuetz1,3,
- Eric Masson1,
- Yuri Yanishevski1,
- Timothy W. Synold1,
- Julio C. Barredo4,
- Ching-Hon Pui2,3,
- Mary V. Relling1,3 and
- William E. Evans1,3
- Departments of 1Pharmaceutical Sciences (A.J.G., J.D.S., E.M., Y.Y., T.W.S., M.V.R., W.E.E.) and 2Hematology-Oncology (C.-H.P.), St. Jude Children’s Research Hospital, and the 3Center for Pediatric Pharmacokinetics and Therapeutics (M.V.R., W.E.E., C.-H.P., J.D.S), Colleges of Pharmacy and Medicine, University of Tennessee, Memphis, Tennessee 38101, and 4Division of Pediatric Hematology-Oncology, Medical University of South Carolina, Charleston, South Carolina (J.C.B.)
Abstract
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 (E max) 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 μm), ii) saturation of MTXPG accumulation 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 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.
Footnotes
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Send reprint requests to: Dr. William E. Evans, Pharmaceutical Sciences, St. Jude Children’s Research Hospital, 332 N. Lauderdale Street, Memphis, TN 38101. E-mail: william.evans{at}stjude.org
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This study was supported in part by National Institutes of Health/National Cancer Institute Grant R37-CA36401, Leukemia Program Project Grant CA20180, and Cancer Center CORE Grant CA21765; a State of Tennessee Center of Excellence grant; and the American Lebanese Syrian Associated Charities.
- Abbreviations:
- MTX
- methotrexate
- ALL
- acute lymphoblastic leukemia
- CEM
- human T-lineage leukemia cell line (CEM/CCRF)
- DHFR
- dihydrofolate reductase
- FBS
- fetal bovine serum
- FPGS
- folylpolyglutamate synthetase
- GAT
- glycine, adenosine, thymidine
- HDMTX
- high-dose methotrexate
- LC50
- concentration producing 50% cell kill
- LC90
- concentration producing 90% cell kill
- LDMTX
- low-dose methotrexate
- MTXPG
- methotrexate polyglutamate
- MTXPGx
- methotrexate, wherex is number of glutamyl residues
- NALM6
- human B-lineage leukemia cell line
- PCR
- polymerase chain reaction
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- Received February 14, 1997.
- Accepted April 10, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
