Multifactorial resistance to 5,10-dideazatetrahydrofolic acid in cell lines derived from human lymphoblastic leukemia CCRF-CEM.
5,10-dideaza-5,6,7,8-terrahydrofolic acid (DDATHF) is a potent antiproliferative agent in cell culture systems and in vivo in a number of murine and human xenograft tumors. In contrast to classical antifolates, which are dihydrofolate reductase inhibitors, DDATHF primarily inhibits GAR transformylase, the first folate-dependent enzyme along the pathway of de novo purine biosynthesis. The (6R) diastereomer of DDATHF (Lometrexol), currently undergoing clinical investigation, was used to develop CCRF-CEM human leukemia sublines resistant to increasing concentrations of the drug. Three cell lines were selected for ability to grow in medium containing 0.1 microM, 1.0 microM, and 10 microM of (6R)DDATHF, respectively. Impaired polyglutamylation was identified as a common mechanism of resistance in all three cell lines. A progressive decrease in the level of polyglutamylation was associated with diminished folylpolyglutamate synthetase activity and paralleled increasing levels of resistance to the drug. However, the expression of folylpolyglutamate synthetase RNA was not altered in the resistant cell lines compared to the parent cells. The most resistant cell subline also displayed an increased activity of gamma-glutamyl hydrolase. The sublines were scrutinized for other possible mechanisms of resistance. No alterations in drug transport or in purine economy were found. Modest increases were found in the activity of methylene tetrahydrofolate dehydrogenase but no alterations of other folate-dependent enzymes were observed. Increases in accumulation and conversion of folic acid to reduced forms, particularly 10-formyltetrahydrofolate, was also seen. The resistant cell lines were sensitive to dihydrofolate reductase inhibitors, methotrexate and trimetrexate, for a 72-h exposure period but showed cross-resistance to methotrexate for 4 and 24 h exposures. Cross-resistance was also shown toward other deazafolate analogues for both short- and long-term exposures.
Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510.
G
G
Pizzorno
B A
BA
Moroson
A R
AR
Cashmore
O
O
Russello
J R
JR
Mayer
J
J
Galivan
M A
MA
Bunni
D G
DG
Priest
G P
GP
Beardsley
D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002999:Clone Cells; D004351:Drug Resistance; D005493:Folic Acid Antagonists / Q000494:pharmacology; D005574:Formate-Tetrahydrofolate Ligase / Q000378:metabolism; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D007945:Leukemia, Lymphoid; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000378:metabolism; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D010088:Oxidoreductases / Q000378:metabolism; D010453:Peptide Synthases / Q000378:metabolism; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D051026:Phosphoribosylglycinamide Formyltransferase; D013237:Stereoisomerism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013763:Tetrahydrofolates / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000378:metabolism; D014407:Tumor Cells, Cultured; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism
D000970:Antineoplastic Agents; D005493:Folic Acid Antagonists; D013763:Tetrahydrofolates; C045894:lometrexol; D010088:Oxidoreductases; D045103:5,10-Methylenetetrahydrofolate Reductase (FADH2); D042965:Methylenetetrahydrofolate Reductase (NADPH2); D013762:Tetrahydrofolate Dehydrogenase; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D013940:Thymidylate Synthase; D019877:Hydroxymethyl and Formyl Transferases; D012696:Glycine Hydroxymethyltransferase; D051026:Phosphoribosylglycinamide Formyltransferase; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D000217:Acyltransferases; D011623:gamma-Glutamyl Hydrolase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D005574:Formate-Tetrahydrofolate Ligase