Impaired drug uptake in methotrexate resistant Crithidia fasciculata without changes in dihydrofolate reductase activity or gene amplification.

Crithidia fasciculata cells grown in defined medium are sensitive to methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR). When cells are challenged with 2-5 microM MTX, cell division ceases after 3-4 divisions and the cells become rounded and immotile for approximately 60 h, with a 40% decrease in cell viability occurring during this period. The cells then recover normal morphology and cell division resumes. Cells which undergo this treatment can be transferred directly into high levels of the drug (1-2 mM). The resistance phenotype is stable in the absence of the drug. Resistance correlates with impaired uptake of [3H]MTX, which in wild-type cells is taken up by a carrier-mediated process. There is no indication of gene amplification at the DNA level or at the level of DHFR activity, as occurs in the case of MTX-resistant Leishmania major. Several lines of MTX-resistant L. major which show gene amplification also exhibit impaired uptake of [3H]MTX.