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Summary Doxorubicin accumulation defects in multidrug reistant tumour cells are generally small in comparison to the resistance factors. Therefore additional mechanisms must be operative. In this paper we show by a quantitative approach that doxorubicin resistance in several P-glycoprotein-positive non-small cell lung cancer and breast cancer multidrug resistant cell lines can be explained by a summation of accumulation defect and alterations in the efficacy of the drug once present in the cell. This alteration of efficacy was partly due to changes in intracellular drug localisation, characterised by decreased nuclear/cytoplasmic doxorubicin fluorescence ratios (N/C-ratios). N/C-ratios were 2.8-3.6 in sensitive cells, 0.1-0.4 in cells with high (>70-fold) levels of doxorubicin resistance and 1.2 and 1.9 in cells with low or intermediate (7.5 and 24-fold, respectively) levels of doxorubicin resistance. The change of drug efficacy was reflected by an increase in the total amount of doxorubicin present in the cell at equitoxic (IC50) concentrations. N/C ratios in highly resistant P-glycoprotein-containing cells could be increased with the resistance modifier verapamil to values of 1.3-2.7, a process that was paralleled by a decrease of the cellular doxorubicin amounts present at IC50. At the low to moderate residual levels of resistance, obtained with different concentrations of verapamil, a linear relationship between IC50 and cellular doxorubicin amounts determined at IC50 was found. This shows that at this stage of residual resistance, extra reversal by verapamil should be explained by further increase of drug efficacy rather than by increase of cellular drug accumulation. A similar relationship was found for Pglycoprotein-negative MDR cells with low levels of resistance. Since in these cells N/C ratios could not be altered, verapamil-induced decrease of IC50 must be due to increased drug efficacy by action on as yet unidentified targets. Although the IC50 of sensitive human cells cannot be reached with resistance modifiers, when using these relationships it can be shown by extrapolation that cellular and nuclear doxorubicin amounts at IC50 at complete reversal of resistance were the same as in sensitive cells. It is concluded that doxorubicin resistance factors for multidrug resistant cells can for a large part, and in the case of P-glycoprotein-containing cells probably fully, be accounted for by decreased amounts of drug at nuclear targets, which in turn is characterised by two processes only: decreased cellular accumulation and a shift in the ratio nuclear drug/cytoplasmic drug.
There is a large discrepancy between the changes in drug accumulation and the changes in drug cytotoxicity that accompany development of anthracycline resistance in multidrug-resistant cells. In our study, a quantitative relationship has been established between reversal of multidrug resistance by resistance modifiers and a concomitant decrease in intracellular levels of doxorubicin measured at equitoxic concentrations (IC50) in CHRC5 and 2780AD multidrug-resistant cells. (IC50 = concentration required for 50% growth inhibition.) We have demonstrated that resistance modifiers like verapamil and Ro 11-2933/001 act by increasing the effectiveness of intracellular doxorubicin, apparently by inducing redistribution of the drug from the cytoplasm to the nucleus of a multidrug-resistant cell, as shown by quantitative fluorescence microscopy. At complete reversal of resistance, as measured directly or inferred by extrapolation, the amount of intracellular doxorubicin at the IC50 as well as the ratio of nuclear doxorubicin to cytoplasmic doxorubicin were the same as those in sensitive cells. These results offer an explanation for the frequently observed discrepancies between drug accumulation and cytotoxicity and also show quantitatively that a decrease in drug accumulation and a change in intracellular drug distribution together are the only determinants of doxorubicin resistance in the multidrug-resistant cells studied.
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