Evidence for cytoplasmic P-glycoprotein location associated with increased multidrug resistance and resistance to chemosensitizers
Abbaszadegan, M.R.; Cress, A.E.; Futscher, B.W.; Bellamy, W.T.; Dalton, W.S.
Cancer Research 56(23): 5435-5442
1996
ISSN/ISBN: 0008-5472 PMID: 8968098 Document Number: 467498
A new human myeloma cell line, 8226/MDR-10V, was selected from a P-glycoprotein-positive cell line, 8226/Dox-40, in the continuous presence of doxorubicin and verapamil. MDR-10V cells are 13-fold more resistant to doxorubicin and 4-fold more resistant to vincristine than the parent cell line, Dox-40. Chemosensitizers are also less effective in reversing resistance in the MDR-10V compared to the Dox-40 cells. Despite higher resistance to cytotoxic agents, MDR-10V expresses 40% less P-glycoprotein in the plasma membrane compared to Dox-40; however, total cellular P-glycoprotein is the same in both cell lines. Confocal immunofluorescence microscopy shows 2.5-fold more P-glycoprotein in the cytoplasm of MDR-10V cells as compared to Dox-40 cells. The cytoplasmic location of P-glycoprotein in the MDR-10V cells is associated with a redistribution of doxorubicin. In Dox-40 cells, doxorubicin is concentrated in the nucleus, whereas in MDR-10V cells, 90% of doxorubicin is found in the cytoplasm. In the presence of equivalent intracellular doxorubicin, there was a decrease in DNA-protein crosslinks in the MDR-10V cell line compared to the Dox-40 cell line. This finding is in agreement with the intracellular doxorubicin fluorescence studies showing less doxorubicin in the nuclei or MDR-10V cells compared to Dox-40 cells. Verapamil is less effective in increasing doxorubicin accumulation in the nuclei of MDR-10V cells compared to Dox-40 cells. Processing of P-glycoprotein from the endoplasmic reticulum to the medial Golgi was identical between the two cell lines as determined by endoglycosidase H sensitivity of newly sensitized P-glycoprotein. No mutations were found in MDR1 cDNA from MDR-10V cells compared to Dox-40 cells. These results suggest that resistance to chemosensitizing agents plus cytotoxic drugs is associated with a redistribution of P-glycoprotein from the plasma membrane to the cytoplasm, which in turn reduces the amount of cytotoxic drug reaching the nucleus.