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


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.


CONCLUSIONS
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.
Durdux
M
M
Laboratoire de Dynamique Cellulaire, EPHE, UMR CNRS 5525, Institut d'Ingéniérie et de l'Information de Santé, IFRT 130 F-38706 La Tronche cedex, France.
Grunwald
D
D
Gautier
T
T
Ronot
X
X
Boutonnat
J
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis; D002087:Butyrates / Q000737:chemistry / Q000494:pharmacology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000737:chemistry / Q000378:metabolism; D003630:Daunorubicin / Q000032:analysis; D005354:Fibrosarcoma / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D020014:K562 Cells; D018613:Microscopy, Confocal; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000032:analysis; D009363:Neoplasm Proteins / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002087:Butyrates; D003630:Daunorubicin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein