5,10-Dideazatetrahydrofolic acid (DDATHF) transport in CCRF-CEM and MA104 cell lines.
5,10-Dideazatetrahydrolic acid (DDATHF) is representative of a new class of antifolates acting through inhibition of de novo purine synthesis. We report here the transport characteristics of the diastereomers of DDATHF, which differ in configuration at C6, and comparison studies with other folate and antifolate analogs. (6R)-DDATHF showed high affinity for the influx system of CCRF-CEM cells with a Km of 1.07 microM and an influx Vmax of 4.04 pmol/min/10(7) cells. Comparative studies with methotrexate yielded an influx Km of 4.98 microM and a Vmax of 6.64 pmol/min/10(7) cells, and with 5-formyltetrahydrofolate an influx Km of 2.18 microM and a Vmax of 6.84 pmol/min/10(7) cells. Uptake of (6R)-DDATHF was competitively inhibited by (6S)-DDATHF, methotrexate (MTX), and 5-formyltetrahydrofolate, all with Ki values similar to their influx Km. The (6S)-DDATHF diastereomer had an influx Km of 1.04 microM, similar to that of (6R)-DDATHF; however, the Vmax of 1.72 pmol/min/10(7) cells was 2.3-fold lower than for (6R)-DDATHF. The transport properties of DDATHF were also studied in a mutant cell line (CEM/MTX), resistant to MTX based on impaired drug transport. In this system (6R)-DDATHF showed an influx Km of 1.49 microM and a decreased influx Vmax of 0.60 pmol/min/10(7) cells. A similar effect was shown for MTX (Km of 7.48 microM, Vmax of 1.02 pmol/min/10(7) cells). The number of binding sites in CCRF-CEM cells was similar for (6R)-DDATHF, (6S)-DDATHF, and MTX, 0.74, 0.71, and 0.76 pmol/10(7) cells, respectively. These values were slightly higher in the CEM/MTX cell line (1.07 and 1.09 pmol/10(7) cells for (6R)-DDATHF and MTX, respectively). Treatment of CCRF-CEM cells with either the N-hydroxysuccinimide ester of MTX or the corresponding N-hydroxysuccinimide ester of (6R)-DDATHF caused substantial inhibition (> 90%) of the influx of (6R)-[3H]DDATHF and [3H]MTX, respectively. These results suggest strongly that DDATHF and MTX share a common influx mechanism through the reduced folate transport system. The internalization of DDATHF by monkey kidney epithelial MA104 cells, which express a high affinity folate receptor, was also studied. Competitive binding studies using purified folate receptor and radiolabeled 5-methyltetrahydrofolate showed that (6S)- and (6R)-DDATHF both had I50 values lower than 5-methyltetrahydrofolate (12 nM). Further studies indicate that both DDATHF isomers are actively intracellularly concentrated through this route and are also rapidly converted to high chain length polyglutamates. Transport via this system was inhibited in folate-depleted cells by 10 nM folic acid.(ABSTRACT TRUNCATED AT 400 WORDS)
Pizzorno
G
G
Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510.
Cashmore
A R
AR
Moroson
B A
BA
Cross
A D
AD
Smith
A K
AK
Marling-Cason
M
M
Kamen
B A
BA
Beardsley
G P
GP
D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D004351:Drug Resistance; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007945:Leukemia, Lymphoid; D008727:Methotrexate / Q000378:metabolism; D011956:Receptors, Cell Surface; D013237:Stereoisomerism; D013379:Substrate Specificity; D013763:Tetrahydrofolates / Q000378:metabolism*; D014316:Tritium; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists; D011956:Receptors, Cell Surface; D013763:Tetrahydrofolates; D014316:Tritium; C045894:lometrexol; D008727:Methotrexate