AN ASSAY FOR THE DETERMINATION OF REDUCED METHOTREXATE ACCUMULATION IN CELLS DISPLAYING LIMITED VIABILITY IN-VITRO

Amongst the mechanisms known to mediate resistance to methotrexate (MT X), a major component in the treatment of childhood leukemia, reduced drug accumulation appears to have direct clinical relevance. However, due to the poor viability of patient-derived acute lymphoblastic leuke mia cells in vitro, determination of this parameter in clinical sample s is associated with a number of difficulties. We have therefore devel oped an assay for reduced MTX accumulation, which controls for the met abolic state of the cell population under study by utilizing accumulat ion of the nucleoside thymidine as an independent indicator of this pa rameter. To establish this assay, we have utilized pediatric leukemic cell populations maintained as xenografts in nude mice, which, despite displaying sensitivity to MTX, demonstrated reduced accumulation of M TX when assayed using standard methodology, When accumulation of MTX b y such cell populations was expressed, however, relative to their accu mulation of thymidine, MTX accumulation was shown to be equal to that of drug-sensitive CCRF-CEM cells maintained in long-term culture. In c ontrast, significantly less MTX was accumulated, in this assay, by xen ografted cell populations with demonstrated resistance to MTX. Identic al results were obtained using either fresh or cryopreserved cells. Th e data thus indicate that by controlling for variable metabolic status of leukemic cells, it is possible to accurately assess MTX accumulati on in leukemic samples displaying limited viability in culture.