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Evidence for Cytoplasmic P-Glycoprotein Location Associated with Increased Multidrug Resistance and Resistance to Chemosensitizers¹

Bone Marrow Transplant Program [M. R. A., B. W. F., W. S. D.], Arizona Cancer Center [M. R. A., A. E. C., B. W. F., W. S. D.], Departments of Pharmacology/Toxicology [M. R. A., B. W. F., W. S. D.], Radiation Oncology [A. E. C.], and Pathology [W. T. B.], The University of Arizona, Tucson, Arizona 85724

A new human myeloma cell line, 8226/MDR₁₀V, was selected from a P-glycoprotein-positive cell line, 8226/Dox₄₀, in the continuous presence of doxorubicin and verapamil. MDR₁₀V 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 MDR₁₀V compared to the Dox₄₀ cells. Despite higher resistance to cytotoxic agents, MDR₁₀V expresses 40% less P-glycoprotein in the plasma membrane compared to Dox₄₀; 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 MDR₁₀V cells as compared to Dox₄₀ cells. The cytoplasmic location of P-glycoprotein in the MDR₁₀V cells is associated with a redistribution of doxorubicin. In Dox₄₀ cells, doxorubicin is concentrated in the nucleus, whereas in MDR₁₀V 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 MDR₁₀V cell line compared to the Dox₄₀ cell line. This finding is in agreement with the intracellular doxorubicin fluorescence studies showing less doxorubicin in the nuclei of MDR₁₀V cells compared to Dox₄₀ cells. Verapamil is less effective in increasing doxorubicin accumulation in the nuclei of MDR₁₀V cells compared to Dox₄₀ 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 MDR₁₀V cells compared to Dox₄₀ 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.

¹ Supported in part by National Cancer Institute Grants CA43043 and CA17094 (to W. S. D.).

² Present address: Diagnostic Development Branch, National Center for Human Genome Research, NIH, Bethesda, MD 20892.

³ To whom requests for reprints should be addressed, at Arizona Cancer Center, P.O. Box 245024, Tucson, AZ 85724-5024. Phone: (520) 626-4196; Fax: (520) 626-2415; E-mail: dalton@azcc.arizona.edu.

Received 8/ 1/96. Accepted 10/ 3/96.

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