Mitochondrial localization and activity of P-glycoprotein in doxorubicin-resistant K562 cells.
It is now well-established that P-glycoprotein 170 (P-gp), an efflux pump involved in multidrug resistance (MDR) is overexpressed at the plasma membrane of doxorubicin-resistant K562 leukemia cells. Nevertheless, several results suggested: (i) that P-gp-mediated drug efflux was not the only mechanism involved in resistance; (ii) that intracellular compartments could accumulate the drug, preventing it from reaching its nuclear targets; (iii) that agents able to reverse multidrug resistance may lead to intracellular drug redistribution. We have studied the localization of P-gp in mitochondria as well as its functional properties in this compartment. Using several monoclonal antibodies (MoAbs) directed against different P-gp epitopes, a protein was detected in the cytoplasm of two doxorubicin-resistant K562 sublines and, by confocal laser scanning microscopy, this protein was shown to co-localize in the Golgi apparatus and in mitochondria, in equivalent proportions. Purified mitochondria were isolated from K562 cell variants; the presence of a protein of about 170 kDa and reacting with several anti-P-gp antibodies was assessed in MDR cells by Western blotting and flow cytometry. Functional assays have shown that mitochondrial P-gp was involved in doxorubicin accumulation inside the organelle but not in its efflux, suggesting an orientation of P-gp in the mitochondrial membrane inverse to that observed in the plasma membrane. A potential role for mitochondrial P-gp in MDR cells would be to protect the nucleus from doxorubicin. This is the first demonstration of the presence and functional activity of P-gp in mitochondria of MDR cells.
Munteanu
Eliza
E
Laboratoire de Physiologie Mitochondriale, EA 3842 Faculté de Médecine, Limoges, France.
Verdier
Mireille
M
Grandjean-Forestier
Fabienne
F
Stenger
Christophe
C
Jayat-Vignoles
Chantal
C
Huet
Sylvie
S
Robert
Jacques
J
Ratinaud
Marie-Hélène
MH
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry; D006801:Humans; D020014:K562 Cells; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D000911:Antibodies, Monoclonal; D004317:Doxorubicin