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.