Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells.
A 170,000-Da glycoprotein (P170 multidrug transporter) becomes specifically labeled in multidrug-resistant human KB carcinoma cells by the photolabile lipophilic membrane probe 5-[125I]iodonaphthalene-1-azide ([125I]INA) when photoactivation of the probe is triggered by energy transfer from intracellular doxorubicin or rhodamine 123. In contrast, in drug-sensitive cells, drug-induced specific labeling of membrane proteins with [125I]INA was not observed. Instead, multiple membrane proteins became labeled in a nonspecific manner. This phenomenon of drug-induced specific labeling of P170 by [125I]INA is observed only in living cells, but not in purified membrane vesicles or lysed cells. It is generated by doxorubicin and rhodamine 123, drugs that are chromophores and to which the cells exhibit resistance; but it is not observed with other drugs or dyes. Verapamil, a calcium channel blocker which reverses resistance to doxorubicin, also abolishes doxorubicin-induced specific [125I]INA labeling of P170. These results reveal that a specific interaction between P170 and doxorubicin takes place in living cells and demonstrate that P170 is directly involved in the mechanism of drug resistance in vivo. They also provide a possible means to label functional domains in the multidrug transporter. The results demonstrate that photosensitized [125I]INA labeling is a technique which provides sufficient spatial and time resolution to detect specific intracellular interactions between chromophores and proteins in vivo.
Raviv
Y
Y
Laboratory of Cell Biology and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Pollard
H B
HB
Bruggemann
E P
EP
Pastan
I
I
Gottesman
M M
MM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance; D006801:Humans; D007457:Iodine Radioisotopes; D007624:KB Cells; D008027:Light; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*; D014466:Ultraviolet Rays
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C015298:iodonaphthylazide; D004317:Doxorubicin