The plasma membrane ATPase P-glycoprotein (P-gp) is believed to confer multidrug resistance (MDR) on cells by the active transport of chemotherapeutic drugs out of the cell. Also, it is present in normal tissues and may play a physiologic role in the protection of cells from dietary xenobiotics. Using adriamycin-resistant cells of the breast cancer cell line MCF-7 previously developed in this laboratory, we have investigated the regulation of P-gp by examining the accumulation and efflux of adriamycin and vinblastine. We have found that the activity of P-gp is affected by the level of intracellular iron. When the cells are loaded with iron using the lipophilic metal chelator 8-hydroxyquinoline or by culturing the cells in the presence of hemin, drug accumulation in the resistant cell lines increases to levels similar to that seen in the wild type cell. Drug efflux, normally quite rapid in the resistant cells, is abolished. Conversely, using metal chelators to lower the level of intracellular iron increases P-gp function. The regulation of P-gp by iron is a novel observation whose molecular mechanism is unknown. Since iron is involved in metal-catalyzed oxidation of macromolecules, including proteins, the possibility exists that the iron state of the cells affects P-gp through a redox-dependent mechanism. Oxidative modification of proteins causes several alterations in the amino acid side chains of susceptible residues, including the introduction of carbonyl groups. Oxidative inactivation of several enzymes has been demonstrated. The extent of oxidative modification in a protein or cellular protein extract may be assayed with the carbonyl reagent 2,4-dinitrophenylhydrazine. We propose to examine P-gp for oxidative modification using this assay. In order to discover whether MDR may be associated with oxidation, we further propose to investigate the relative susceptibility of the wild type and resistant MCF-7 cells to oxidative damage caused by iron, using the carbonyl assay and by examining the oxidative modification of DNA bases. A thorough understanding of the relationship between the redox state of the cell and the regulation of P-gp is important in determining the role of P-gp in the prevention of cancer.
