Fluorescence-based assessment of LRP activity: a comparative study.

ABSTRACT

BACKGROUND: Broad resistance to anticancer drugs is a major cause of failure in cancer treatment. The Lung Resistance-related Protein (LRP) is a protein associated with drug resistance, which is involved in nucleo-cytoplasmic transport and is known to predict a poor response to chemotherapy in acute myeloid leukaemia. The only method allowing the detection of LRP activity is based on radio-labelled daunorubicin incorporation. Our goal was to develop a fluorescence-based assay to analyse LRP function. MATERIALS AND METHODS: We used human colon carcinoma cell lines treated with sodium butyrate (NaB) in order to induce LRP expression. Daunorubicin efflux in isolated nuclei was measured by flow cytometry, the localization and quantification of Daunorubicin analysed by confocal laser scanning microscopy (CLSM) and the diffusion coefficient of this drug estimated by Fluorescence Correlation Spectrometry (FCS). RESULTS: According to the method using [14C] Doxorubicin cells incubated with NaB displayed an efflux of Daunorubicin out of isolated nuclei demonstrated by flow cytometry or CLSM. The FCS method was able to evaluate kinetics of Daunorubicin molecules in nucleus and cytoplasm and showed a higher dispersion of Daunorubicin kinetics with cells previously NaB-treated. This argument is in favour of an increase of nucleo-cytoplasmic exchange. CONCLUSION: Using CLSM we showed that LRP was able to modify anticancer drug repartition in the cells. LRP activity assessment needs either isolated nuclei if flow cytometry is employed, or FCS, and only a few cells may be analysed.