Cytotoxicity of
trimetrexate (TMQ), a lipophilic
dihydrofolate reductase inhibitor, was examined in
antifolate-resistant human
T-cell leukemia cell lines developed in oxidized or reduced
folate. An approximately 60-fold
methotrexate (MTX)-resistant subline was developed in oxidized
folate (
pteroylglutamic acid:
PGA) (CCRF-CEM/MTX60-
PGA) from human
T-cell leukemia cell line CCRF-CEM; this line exhibited impaired membrane transport of the
drug. Further enhancement of MTX resistance resulted in selection of an approximately 5000-fold MTX-resistant subline (CCRF-CEM/ MTX5000-
PGA), which showed increased
dihydrofolate reductase activity due to gene amplification in addition to further impairment of MTX transport. An approximately 140-fold MTX-resistant subline, and then a 1500-fold MTX-resistant subline were developed in reduced
folate (10 nM
leucovorin) (CCRF-CEM/MTX140-LV and CCRF-CEM/MTX1500-LV); they exhibited increased
dihydrofolate reductase due to gene amplification accompanied by increased intracellular
drug accumulation of MTX. While CCRF-CEM/MTX140-LV and CCRF-CEM/MTX1500-LV cells showed cross-resistance to TMQ, CCRF-CEM/MTX60-
PGA and CCRF-CEM/MTX5000-
PGA cells were at least as sensitive to TMQ as the parent cells. TMQ was more potent against approximately 200-fold N10-propargyl-5,8-dideazafolic-acid (CB3717)-resistant human
T-cell leukemia MOLT-3 sublines developed in
PGA (MOLT-3/
CB3717(200)-
PGA) or
leucovorin (MOLT-3/
CB3717(200)-LV), as compared to the parent cells; MOLT-3/
CB3717(200)-
PGA and MOLT-3/
CB3717(200)-LV cells were resistant to
CB3717 by virtue of impaired transport, only the former possessing gene amplification of
thymidylate synthase. The cytotoxicity of TMQ in both MOLT-3/
CB3717(200)-
PGA and MOLT-3/
CB3717(200)-LV cells was reduced by addition of
leucovorin in a dose-dependent manner, suggesting intracellular
folate deficiency as a cause of TMQ sensitivity. These results demonstrate that TMQ overcomes transport-impaired
antifolate resistance, irrespective of gene amplification of
dihydrofolate reductase or
thymidylate synthase. Types of
folate used during the development of
antifolate resistance seem to be important in relation to the mechanism of TMQ responsiveness as well as that of
antifolate resistance.