A NOVEL METHOD FOR DIRECT AND FLUORESCENCE INDEPENDENT DETERMINATION OF DRUG EFFLUX OUT OF LEUKEMIC BLAST CELLS

Multi drug resistance (MDR) is often due to an increased efflux of ant i cancer drugs out of leukemic blast cells, Efflux assays are used to get an impression of functional resistance in those cells, Dyes like r hodamine 123 or 3'3'-diethyloxocarbocyanine iodide are commonly used f or this purpose, A major known disadvantage is that dyes do not behave like cytotoxic drugs in efflux experiments, Assays using the self flu orescence of drugs like anthracyclines can not reveal a real impressio n of intracellular or effluxed drug due to quenching of the drug fluor escence in the nuclei of the cells, We have developed a reproducible a nd sensitive assay for direct and quantitative determination of drug e fflux out of blast cells, This was done by a novel double radioactive labelling using a H-3-labelled drug and C-14-labelled sucrose as extra cellular marker. So this assay can be applied to even/drug of interest . Quenching of fluorescence is also by-passed with this technique as w ell as protracting washing or silicon oil procedures, As a model syste m we used the T-lymphoblastoid cell line CCRF CEM and its resistant su blines vincristine 100 and adriamycin 5000. The results were also tran sferable to clinical specimens of leukemic patients, In conclusion our assay may be used for precise and direct efflux measurement of a broa d range of anti-cancer drugs in clinical MDR evaluation, (C) 1997 Else vier Science Ltd.