Changes in subcellular doxorubicin distribution and cellular accumulation alone can largely account for doxorubicin resistance in SW-1573 lung cancer and MCF-7 breast cancer multidrug resistant tumour cells.

Doxorubicin accumulation defects in multidrug resistant 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 P-glycoprotein-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.

Quantification by laser scan microscopy of intracellular doxorubicin distribution.

Changes in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560-590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW-1573 and 1.8 and 0.4 in its MDR sublines SW-1573/2R120 and SW-1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively. The doxorubicin distribution was independent of the doxorubicin concentration within a range from 1-32 microM. Furthermore, the progressive mean of the nuclear/cytoplasmic doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8).

Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P-glycoprotein.

Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/- cells with increasing selection pressure to 1.2-2.6 for cells with a resistance factor of 7-17. N/C ratios could be restored partly with verapamil only in Pgp/+ cells. N/C ratio measurements may define a general Pgp-independent type of defense of mammalian cells against certain anticancer agents which may precede Pgp expression in early doxorubicin resistance.

Quantitative determination of factors contributing to doxorubicin resistance in multidrug-resistant cells.

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.

Quantification of epithelial area by image processing applied to endometrial carcinomas: a comparison with ovarian tumors.

In endometrial carcinomas, the epithelial area measured by interactive morphometry is an important feature in the classification of tumors of varying histologic grades. This report describes an image analysis technique for the fully automated estimation of the area percentages of epithelium and stroma in tissue sections of the endometrium obtained from hysterectomy specimens. The method is evaluated using endometrial carcinomas with varying degrees of malignancy. From standard paraffin sections stained with pararosanilin Feulgen and naphthol yellow, a blue-yellow image pair was recorded. The blue image was used to determine the total tissue area and the yellow image was used to determine the epithelial area. Image processing of the blue image was comprised of correction for shading, segmentation of the tissue area, and restoration of the segmented image by removing small artefacts and closing small tears in the tissue. Image processing of the yellow image was based on the fact that epithelial nuclei are generally more tightly packed than stromal nuclei and consists of the following steps: correction for shading, gaussian blurring, segmentation of nuclei, and editing the segmented image by removing small objects and closing small spaces between the epithelial nuclei. These image processing steps are compared with those used for quantification of the epithelial area in ovarian tumors. The performance of the method was evaluated using 120 image pairs from 30 endometrial carcinomas of varying histologic grades. The epithelial area percentages, as assessed by digital image processing, strongly correlate to control percentages that were established by interactive morphometry (r = .987).