The cell biology of multiple drug resistance.

Multidrug resistance (MDR) is a significant problem in the treatment of cancer. Chemotherapeutic drugs distribute through the cyto- and nucleoplasm of drug-sensitive cells but are excluded from the nucleus in drug-resistant cells, concentrating in cytoplasmic organelles. Weak base chemotherapeutic drugs (e.g., anthracyclines and vinca alkaloids) should concentrate in acidic organelles. This report presents a quantification of the pH for identified compartments of the MCF-7 human breast tumor cell line and demonstrates that (a) the chemotherapeutic Adriamycin concentrates in acidified organelles of drug-resistant but not drug-sensitive cells; (b) the lysosomes and recycling endosomes are not acidified in drug-sensitive cells; (c) the cytosol of drug-sensitive cells is 0.4 pH units more acidic than the cytosol of resistant cells; and (d) disrupting the acidification of the organelles of resistant cells with monensin, bafilomycin A1, or concanamycin A is sufficient to change the Adriamycin distribution to that found in drug-sensitive cells, rendering the cell vulnerable once again to chemotherapy. These results suggest that acidification of organelles is causally related to drug resistance and is consistent with the hypothesis that sequestration of drugs in acidic organelles and subsequent extrusion from the cell through the secretory pathways contribute to chemotherapeutic resistance.

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Defective acidification in human breast tumor cells and implications for chemotherapy.

The ability of neoplastic cells to survive exposure to various chemotherapeutic drugs represents the main obstacle to successful cancer chemotherapy. These essays summarize the results of molecular biological, pharmacological, biochemical, cytogenetic, immunological, and pathological studies on mult

Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells

Alkalinization of normally acidic intracellular compartments or acidification of a mildly alkaline cytoplasm by biochemical or genetic manipulation has been demonstrated to inhibit both endocytosis and secretion (Tartakoff, 1983a; Cosson et al., 1989; Mellman et al., 1986; Davoust et al., 1987; Cosson et al., 1989; van Deurs et al., 1989; Maxfield & Yamashiro, 1991; Hansen et al., 1993). These results provide the basis for the conclusion that the maintenance of pH gradients between acidic vesicular compartments and a mildly alkaline cytoplasm is an essential biochemical requirement for the correct functioning of the endocytotic and secretory machinery. Tumor cells have been shown to have an abnormally acidic cytoplasmic pH (Warburg, 1956; Simon & Schindler, 1994). Here we report that the intracellular vesicular compartments in tumor cells (MCF-7) derived from a human breast cancer fail to acidify. This failure results in a significant decrease in the pH gradient (0.9 pH unit) between the vesicular luminal compartments and the cytoplasm. These defects are correlated with a disruption in the organization and function of the trans-Golgi network (TGN) and the pericentriolar recycling compartment (PRC). In marked distinction, drug-resistant tumor cells (MCF-7adr) derived from the MCF-7 line that are resistant to the most widely employed chemotherapeutic drug, adriamycin, appear normal in both acidification and organization of the PRC and TGN. Treatment of drug-resistant MCF-7adr cells with nigericin and monensin, ionophores demonstrated to disrupt vesicular acidification (Tartakoff, 1983b), leads to a resensitization of these cells to adriamycin. Drug sensitivity is proposed to result from an acidification defect within vesicles of the recycling and secretory pathways. A functional consequence of this defect is the diminished capacity of cells to remove cytotoxic drugs from the cytoplasm by sequestration of protonated drugs within the vesicles, followed by drug secretion through the activity of the secretory and recycling pathways.

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Defective pH Regulation of Acidic Compartments in Human Breast Cancer Cells (MCF-7) Is Normalized in Adriamycin-Resistant Cells (MCF-7adr)†

Daunorubicin transport was compared in P388 murine leukemia and in P388/Adramycin (ADR), an anthracycline-resistant subline. We can demonstrate an energy-dependent outward transport system in P388/ADR which limits drug accumulation. Although no calcium requirement for the outward transport process could be shown, several calcium antagonists inhibited outward transport of daunorubicin in P388/ADR and modified the drug resistance pattern. But these agents failed to alter calcium fluxes in either cell line, suggesting that their mode of action in these studies, was not related to interactions with calcium-dependent processes. Accumulation differences could not account for the level of daunorubicin resistance observed in the P388/ADR cell line, nor could resistance be wholly circumvented by calcium antagonists.

Anthracycline Resistance in P388 Murine Leukemia and Its Circumvention by Calcium Antagonists

The doxorubicin (Adriamycin) transport was investigated in murine Ehrlich ascites tumour cells by measuring the initial rate of cellular net uptake in vitro at 37°C (pH 7.3). Transport characteristics were compared with previously published data on doxorubicin transport in human red blood cells. The apparent permeability coefficient in ascites cells (2.4 × 10−5 cm sec−1) and in red cells was of the same order of magnitude when calculated from the initial influx into cells suspended in a salt solution (37°C, pH 7.3). Doxorubicin was strongly adsorbed to the cell surface of ascites cells in contrast to the doxorubicin adsorption to red cells when the cells were suspended in a salt solution. The adsorbed doxorubicin could be removed by washing the ascites cells either with DNA or with human albumin salt solutions indicating that the adsorption to cell surface components was reversible. Cell membrane modifiers, 1-alcohols, local anaesthetics, phloretin, HgCl2, para-chloromercuribenzoate, affected doxorubicin ...

Else K. Hoffmann

Papers

Doxorubicin (Adriamycin) transport in Ehrlich ascites tumour cells: comparison with transport in human red blood cells.