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 anti cancer drugs out of leukemic blast cells. Efflux assays are used to get an impression of functional resistance in those cells. Dyes like rhodamine 123 or 3'3'-diethyloxocarbocyanine iodide are commonly used for this purpose. A major known disadvantage is that dyes do not behave like cytotoxic drugs in efflux experiments. Assays using the self fluorescence of drugs like anthracyclines can not reveal a real impression of intracellular or effluxed drug due to quenching of the drug fluorescence in the nuclei of the cells. We have developed a reproducible and sensitive assay for direct and quantitative determination of drug efflux out of blast cells. This was done by a novel double radioactive labelling using a 3H-labelled drug and 14C-labelled sucrose as extracellular marker. So this assay can be applied to every drug of interest. Quenching of fluorescence is also by-passed with this technique as well as protracting washing or silicon oil procedures. As a model system we used the T-lymphoblastoid cell line CCRF CEM and its resistant sublines vincristine 100 and adriamycin 5000. The results were also transferable to clinical specimens of leukemic patients. In conclusion our assay may be used for precise and direct efflux measurement of a broad range of anti-cancer drugs in clinical MDR evaluation.