Effect of fibronectin amount and conformation on the strength of endothelial cell adhesion to HEMA/EMA copolymers

P-Glycoprotein (P-gp) and multidrug resistance protein (MRP) are plasma membrane associated proteins which can confer multidrug resistance (MDR) to cancer cells by lowering the intracellular amount of drug. Although clinical trials with MDR-reverting agents have been initiated, not much attention has been paid to blood components which may modulate the activity of P-gp or MRP. The present investigation was performed to identify and characterize blood components which may influence the drug content and the drug cytotoxicity of MDR cells. Human plasma, from healthy volunteers, was tested for its effects on the daunorubicin (DNR) accumulation and cytotoxicity in the MDR cell lines SW-1573/2R160 (2R160) and GLC4/ADR containing P-gp and MRP, respectively. The data were compared to the effects observed in wild-type cells. MDR-modifying plasma components were isolated by extraction procedures and characterized using ultrafiltration, high-performance liquid chromatography (HPLC) and mass spectrometry. An increase in the proportion of plasma in the culture medium led to a reduction of the ratio between the DNR content of wild-type and corresponding MDR cells. At 100% plasma we observed an increase in the cellular DNR content of 2R160 cells, which was 10-30% (median 18%) of the maximum possible increase induced by well-known MDR-reverting agents, such as verapamil (for GLC4/ADR cells: 10-20%, median 15%). The DNR cytotoxicity in MDR cells also increased with an increasing amount of plasma included in the culture media. There was neither an increase in the cellular DNR content nor an effect on the DNR cytotoxicity in wild-type cells. Plasma extract analysis by HPLC showed a major peak which increased the DNR content of MDR cells. The HPLC column retention time of this fraction was identical to that of a standard of cortisol and it was further confirmed to be cortisol using mass spectrometry. Moreover, inclusion of a standard of cortisol in culture media induced a similar effect. We analyzed the data for one of the plasma pools and found that blood cortisol was responsible for the MDR-modulating effect only for 35% of the effect of 100% plasma. Other plasma components were responsible for the remaining modulation effect on MDR cells. In conclusion, the DNR pumping activity of P-gp and MRP is inhibited by human plasma, resulting in 10-30% of the maximum possible increase in cellular drug content. Based on cellular pharmacokinetic calculations this percentage will most likely increase at clinical levels of drug resistance (reaching 40-50%). In one sample blood cortisol accounted for 35% of the effect of plasma on the DNR content in MDR 2R160 cells. These data show the need for additional studies to test plasma samples for their MDR modulating effects before the administration of MDR-reverting agents in chemotherapy. The data suggest that the effectiveness of chemotherapeutic drugs may be enhanced when administered in accordance with the circadian peak of endogenous corticoids.     

Mechanisms of multidrug resistance in tumor cells and analytical methods for its detection

We reviewed mechanisms of multidrug resistance (MDR) phenotype in tumor cells and evaluated analytical methods for detection of clinical MDR. A well-recognized mechanism of MDR phenotype is the induction and increased expression of P-glycoprotein (P-gp) which is a 170 kDa cellular transmembrane protein encoded by a multidrug-resistance 1 gene (MDR1) and works as a drug efflux pump. Cellular MDR phenotype through P-gp/MDR1 can be detectable at protein level by: (1) using immunohistochemical method, flow cytometric assay and Western blot analysis with monoclonal antibodies against human P-gp, and (2) measuring Rhodamine 123 dye-efflux as a functional assay of P-gp. Molecular knowledge and recent technical progress enable to determine MDR1 gene expression by RT-PCR-based analytical methods as well as conventional quantification methods of gene expression such as Northern blot analysis. In the evaluation of P-gp/MDR1 expression in clinical samples, in which amount of materials was limited, utilization of simple and sensitive methods like competitive RT-PCR assay might be efficacious for its quantitative detection in clinical laboratories. Evidences which showed the positive correlation between the expression of P-gp/MDR1 and clinical resistance or refractoriness of tumor cells to anticancer drugs involved in MDR have been accumulated and support the clinical importance of its detection to circumvent resistance with alternate use of non-MDR drugs.     

A new anthracycline with potent anti-leukemic activity overcomes P-glycoprotein multidrug resistance

In this study, we assessed the ability of a new anthracycline, moflomycin, to circumvent multidrug resistance. Moflomycin showed superior anti-proliferative activity compared to daunorubicin and doxorubicin on two resistant cell lines: leukemic HL-60 cell line resistant to daunorubicin (HL-60/DR) and breast cancerous cell line resistant to doxorubicin (MCF-7/AR). The effect of moflomycin on cell proliferation was correlated with an increased uptake and a decreased cellular efflux. The data obtained in the presence of the P-gp inhibitor, verapamil, confirmed the absence of interaction between P-gp and moflomycin. Our results indicate that moflomycin exhibits an important reduction in cross-resistance with daunorubicin and doxorubicin resulting from its ability to circumvent P-gp.     

Antibody affinity maturation using bacterial surface display

A drug-resistant cell line (EAC/Dox) was developed by repeated exposure of Ehrlich ascites carcinoma cells to Doxorubicin (Dox) in vivo in male albino Swiss mice (6-8 weeks old). The weekly i.p. injections of Dox to mice (2 or 4 mg/kg/week for 4 months) gave rise to Dox-resistant cell line EAC/Dox, which displayed typical multidrug resistant (MDR) features of cross-resistance to a number of structurally and functionally unrelated drugs like doxorubicin, vinblastine and cisplatin. Moreover, the EAC/Dox cell line had lower drug accumulation than drug-sensitive (EAC/S) cells. Study of Western blots and immunofluorescence revealed that P-glycoprotein 170 kDa (P-gp) was absent in EAC/Dox cells. The drug resistance appeared to be due to the presence of a higher level of reduced glutathione (GSH) and glutathione S-transferase (GST) in EAC/Dox cells than in drug-sensitive (EAC/S) cells. The two structurally similar hydroxamic acid derivatives, i.e. oxalyl bis(N-phenyl)hydroxamic acid (X1) and succinyl bis(N-phenyl)hydroxamic acid (X2), having very low in vitro toxicity (IC50 value 250 microg/ ml), were investigated for their efficacy to reverse MDR. The compound X1 was able to reverse the effect of MDR and reduce GST in EAC/Dox cells. The compound X2 had no ability to reverse the effect of MDR. Further study on the mechanism of glutathione depletion and the resistance modifying property of X1 on other cell lines is warranted.     

The multidrug resistance in human leukemias. Minireview

Leukemia/lymphoma cells, clinically refractory to therapy are often associated with expression of P-glycoprotein (P-gp), which is encoded by the multidrug resistance (MDR) gene, mdr1. Cell lines expressing mdr1 exhibit resistance to several structurally unrelated lipophilic drugs, such as anthracyclines, vinca alkaloids, and epopodophyllotoxins. This MDR can be conferred to drug-sensitive cells mdr1 cDNA transfer. In resistant cells, MDR is characterized by overexpression of P-gp and by the enhanced efflux, and P-gp fluorescence probe, rhodamine 123 (Rh 123). This can be circumvented by addition of certain non-cytotoxic drugs, such as verapamil and cyclosporin A.     

Overexpression of ecto-5'-nucleotidase promotes P-glycoprotein expression in renal epithelial cells

P-glycoprotein (P-gp), responsible for multidrug resistance (MDR) of tumoral cells, is also expressed in apical membranes of normal epithelial cells, among which are proximal tubular cells. Ecto-5'-nucleotidase (5'Nu), co-located with P-gp in renal brush border membranes, could be instrumental in the expression of MDR phenotype. P-gp activity [assessed by rhodamine 123 (R123) and [3H]vinblastine (3H-VBL) accumulation] was evaluated in MDCK cell lines in which human 5'Nu was expressed at different levels after retroviral infection: MDCK-5'NU/- cells with a low 5'Nu activity (Vmax < 2 pmol/mg protein/min) and MDCK-5'NU/+ cells, which expressed a high level of 5'Nu (Vmax 150 +/- 18.5 pmol/mg protein/min). MDCK-5'NU/- cells did not display functional expression of MDR. In MDCK-5'NU/+ cells, R123 and 3H-VBL accumulation was significantly lower than in MDCK-5'NU/- cells and was dramatically enhanced by P-gp inhibitors. This high P-gp activity in MDCK-5'NU/+ cells was confirmed by their resistance to colchicine (measured by LDH release and MTT assay) as compared to MDCK-5'NU/- and was accounted for by increased membrane expression of P-gp assessed by Western blot. Neither AMP nor adenosine, the substrate and the product of 5'Nu, respectively, affected P-gp activity. Inhibition of 5'Nu with alpha beta-methylene-adenosine-diphosphate (alpha beta MADP) or with a blocking anti-5'Nu antibody (1E9) did not blunt MDR expression in MDCK-5'NU/+ cells. Conversely, the anti-5'Nu antibody 5F/F9, which did not block the enzymatic site, induced a decrease of P-gp activity. Further, incubation of MDCK-5'NU/- cells with conditioned medium from MDCK-5'NU/+ cells, which contained significant amounts of released 5'Nu, induced MDR phenotype. In conclusion: (i) expression of ecto-5'Nu promotes multidrug resistance (MDR) activity in renal epithelial cells by enhancement of P-gp expression; (ii) this effect does not involve enzymatic activity of 5'Nu; (iii) supernatants of cells that express 5'Nu conferred P-gp activity to 5'Nu negative cells.     

Co-ordinate loss of protein kinase C and multidrug resistance gene expression in revertant MCF-7/Adr breast carcinoma cells

The aim of this study was to investigate the link between protein kinase C (PKC) and multidrug resistance (mdr) phenotype. The expression of both was studied in doxorubicin-resistant MCF-7/Adr cells as they reverted to the wild-type phenotype when cultured in the absence of drug. The following parameters were measured in cells 4, 10, 15, 20 and 24 weeks after removal of doxorubicin; (1) sensitivity of the cells towards doxorubicin; (2) levels of P-glycoprotein (P-gp) and MDR1 mRNA; (3) levels and cellular localization of PKC isoenzyme proteins alpha, theta and epsilon; and (4) gene copy number of PKC-alpha and MDR1 genes. Cells lost their resistance gradually with time, so that by week 24 they had almost completely regained the drug sensitivity seen in wild-type MCF-7 cells. P-gp levels measured by Western blot mirrored the change in doxorubicin sensitivity. By week 20, P-gp had decreased to 18% of P-gp protein levels at the outset, and P-gp was not detectable at week 24. Similarly, MDR1 mRNA levels had disappeared by week 24. MCF-7/Adr cells expressed more PKCs-alpha and -theta than wild-type cells and possessed a different cellular localization of PKC-epsilon. The expression and distribution pattern of these PKCs did not change for up to 20 weeks, but reverted back to that seen in wild-type cells by week 24. MDR1 gene amplification remained unchanged until week 20, but then was lost precipitously between weeks 20 and 24. The PKC-alpha gene was not amplified in MCF-7/Adr cells. The results suggest that MCF-7/Adr cells lose MDR1 gene expression and PKC activity in a co-ordinate fashion, consistent with the existence of a mechanistic link between MDR1 and certain PKC isoenzymes.     

The chemosensitizing potential of GF120918 is independent of the magnitude of P-glycoprotein-mediated resistance to conventional chemotherapeutic agents in a small cell lung cancer line

GF120918, at 250 ng/ml, increased the sensitivity of a P-glycoprotein (P-gp)-mediated multidrug resistant (MDR) small cell lung cancer cell line (H69/LX4) to the P-gp substrates, paclitaxel, taxotere, vinblastine, vinorelbine, daunorubicin and etoposide to levels which were either greater (in the case of etoposide) or close to that of the parent cell line (H69/P). This was achieved in spite of the great variation in the levels of resistance of the MDR cell line for the various anti-cancer drugs tested. These data suggest that GF120918 is a potent antagonist of P-gp mediated multidrug resistance, even in the case of high levels of resistance, as was the case with paclitaxel and taxotere (2560 and 2215 fold more than the sensitive parent cell line respectively).     

In vitro effect of GF120918, a novel reversal agent of multidrug resistance, on acute leukemia and multiple myeloma cells

Resistance to chemotherapy in multiple myeloma (MM) and acute myeloid leukemia (AML) is frequently caused by multiple drug resistance (MDR), characterized by a decreased intracellular drug accumulation. MDR is associated with expression of P-glycoprotein (P-gp). GF120918, an acridine derivative, enhances doxorubicin cell kill in resistant cell lines. In this study, the effect of GF120918 on MDR cell lines and fresh human leukemia and myeloma cells was investigated. The reduced net intracellular rhodamine-123 (Rh-123) accumulation in the MDR cell lines RPMI 8226/Dox1, /Dox4, /Dox6 and /Dox40 as compared with wild-type 8226/S was reversed by GF120918 (0.5-1.0 microM), and complete inhibition of rhodamine efflux was achieved at 1-2 microM. This effect could be maintained in drug-free medium for at least 5 h. GF120918 reversal activity was significantly reduced with a maximum of 70% in cells incubated with up to 100% serum. GF120918 significantly augmented Rh-123 accumulation in vitro in CD34-positive acute leukemia (AML) blasts and CD38-positive myeloma (MM) plasma cells obtained from 11/27 de novo AML and 2/12 refractory MM patients. A significant correlation was observed between a high P-gp expression and GF120918 induced Rh-123 reversal (P=0.0001). Using a MRK16/IgG2a ratio > or = 1.1, samples could be identified with a high probability of GF120918 reversal of Rh-123 accumulation. In conclusion, GF120918 is a promising MDR reversal agent which is active at clinically achievable serum concentrations.     

Reversal of a novel multidrug resistance mechanism in human colon carcinoma cells by fumitremorgin C

We selected a human colon carcinoma cell line in increasing concentrations of mitoxantrone to obtain a resistant subline, S1-M1-3.2, with the following characteristics: profound resistance to mitoxantrone; significant cross-resistance to doxorubicin, bisantrene, and topotecan; and very low levels of resistance to Taxol, vinblastine, colchicine, and camptothecin. This multidrug resistance (MDR) phenotype, which was not reversed by verapamil or another potent P-glycoprotein (Pgp) inhibitor, CL 329,753, was dependent, in part, upon an energy-dependent drug efflux mechanism. Pgp and the multidrug resistance protein (MRP) were not elevated in the resistant cells relative to the drug-sensitive parent, suggesting that resistance was mediated by a novel pathway of drug transport. A cell-based screen with S1-M1-3.2 cells was used to identify agents capable of circumventing this non-Pgp, non-MRP MDR. One of the active agents identified was a mycotoxin, fumitremorgin C. This molecule was extremely effective in reversing resistance to mitoxantrone, doxorubicin, and topotecan in multidrug-selected cell lines showing this novel phenotype. Reversal of resistance was associated with an increase in drug accumulation. The compound did not reverse drug resistance in cells with elevated expression of Pgp or MRP. We suggest that fumitremorgin C is a highly selective chemosensitizing agent for the resistance pathway we have identified and can be used as a specific pharmacological probe to distinguish between the diverse resistance mechanisms that occur in the MDR cell.     

Cyclosporin A and PSC 833 prevent up-regulation of MDR1 expression by anthracyclines in a human multidrug-resistant cell line

We have previously demonstrated that within 24 h of exposure of the CEM/A7R cell line to epirubicin (EPI), MDR1 gene expression is induced. The aim of the current study was to investigate the role of cyclosporin A (CyA) and PSC 833, two biochemical modulators of the classical multidrug-resistant phenotype, in this model. CEM/A7R cells were exposed to EPI in the presence or absence of various concentrations of CyA or PSC 833. MDR1 expression was assessed using Northern blot analysis and quantitated using a phosphorimager. P-glycoprotein (P-gp) expression was analyzed by the determination of MRK16 binding using flow cytometry. P-gp function was measured in an assay of [3H]daunomycin accumulation. The coincubation of CyA or PSC 833 with EPI prevented the increase in MDR1 gene expression induced by EPI alone. This effect of the two modulators was dose dependent. Neither modulator alone had any significant effect on the expression of MDR1. In these experiments, changes in MDR1 expression correlated with changes in P-gp levels (based on MRK16 binding) and P-gp function. Thus, both PSC 833 and CyA appear to prevent the induction of MDR1 gene expression caused by the short-term exposure of CEM/A7R cells to EPI.     

Effect of the multidrug inhibitor GG918 on drug sensitivity of human leukemic cells

The drug GG918 has been specifically developed for overcoming MDR phenotype and is now in use in clinical trials. In this study, the effects of GG918 on leukemic cell were investigated using a 3 day MTT assay. Results showed that, in a highly resistant P-gp(+) leukemic cell line, 0.1 microM of GG918 gives rise to a 40-fold sensitization to daunorubicin (DNR) (residual resistance: 2.1), a 57-fold sensitization to mitoxantrone (residual resistance: 1.5), and a 3.3-fold sensitization to idarubicin (residual resistance: 2.9). When human AB serum was added to the incubation medium, 1 microM of GG918 was needed to observe the full P-gp modulation potency described above. The effect of 1 microM of GG918 was tested on 27 samples of poor prognosis acute leukemia (25 AML, two ALL). DNR sensitization (using the MTT assay) and modulation of rhodamine 123 uptake were monitored and used as criteria for comparing the in vitro modulation potency of this new compound to the potency of 10 microM of verapamil, which was used as reference. A good correlation (r = 0.8, P = 0.001) was observed between the results of the two tests. Eleven out of the 26 cases tested were MDR1(+) (42%), and showed a higher IC50 for DNR than the negative cases (861 +/- 1284 nM vs 187 +/- 246 nM, P = 0.05). GG918 was able to modulate the in vitro resistance to DNR in eight cases (seven MDR1(+), no MDR1(-), one non-tested). Verapamil did not increase DNR toxicity in four of these eight cases, but was more efficient in one other MDR1(+) case. In conclusion, the DNR sensitivity of the majority of the fresh AML samples expressing P-gp could be modulated in vitro by 1 microM of GG918.     

Poor expression of MDR1 transgene in HeLa cells by bicistronic Moloney murine leukemia virus-based vector

Cotransfer of a therapeutic gene together with the human MDR1 gene provides an opportunity to increase the number of transduced marrow cells, expressing the therapeutic gene, by in vivo selection for MDR1. We have used an Lg-MDR1-IRES-neo (LgMIN) retroviral vector, containing MDR1 and neo genes, separated by the EMCV IRES. Human HeLa or canine CTAC cells, transduced with GALV env pseudotyped LgMIN at an MOI of less than 0.01 to ensure 1 proviral copy/genome, were selected with either G418 for neo expression or colchicine for MDR1 expression. The titer determined on HeLa cells with G418 selection was eight-fold higher than that with colchicine selection. In contrast, the same viral supernatant exhibited only a 1.4-fold difference between neo- and MDR1-based viral titer values for CTAC cells. The transduced HeLa cells, with one intact proviral copy per genome, exhibited a 55-fold higher resistance to G418 but only a 4-fold higher resistance to colchicine and a 2-fold higher resistance to Taxol compared with nontransduced cells. About 23% of the transduced cell population did not express vector-derived P-glycoprotein (P-gp) as detected by anti-human P-gp MAb MRK-16. This could explain the difference in viral titers obtained on CTAC cells but not that obtained on HeLa cells. The vector-mediated increase in expression of P-gp was about 20-fold higher in CTAC cells as compared with HeLa cells. These results indicated suppression of expression of vector-derived MDR1 in HeLa cells, in contrast with CTAC cells. To investigate further the possible reasons for this difference, genomic DNA was isolated from the G418-resistant individual colonies of infected cells and analyzed by PCR for full-length proviral MDR1. For transduced CTAC and HeLa cells, selected at a G418 concentration of 1 mg/ml, PCR detected aberrant forms of MDR1 in 17 to 25% of colonies tested. The aberrant forms consisted of MDR1 genes with 2- and 0.7-kb deletions. DNA sequencing across the 2-kb and the 0.7-kb deletion junction suggests cryptic splicing in the producer cell line as the origin of these deletions. The 2-kb deletion corresponds to MDR1 mRNA cryptic splicing via donor (codon 113) and acceptor (codon 773). The 0.7-kb deletion corresponds to splicing via the same donor and a different acceptor (codon 344). When transduced HeLa cells were selected at a higher concentration of G418 (3 mg/ml), the aberrant forms were detected at an increased frequency of about 50% of colonies tested. These results indicate that vector-derived MDR1 is a poor selective marker in HeLa cells but not in CTAC cells and that deletions, which inactivated the MDR1 gene in a bicistronic Mo-MuLV vector, may provide an advantage for expression of the second transgene in HeLa cells.     

Regulation of the function of P-glycoprotein by epidermal growth factor through phospholipase C

Many multidrug-resistant (MDR) cell lines overexpress the epidermal growth factor receptor (EGFR) as well as P-glycoprotein (P-gp). However, the role of the increased EGFR in P-gp-mediated drug resistance remains unclear. Since recent studies suggest that activation of phospholipase C (PLC) could increase the phosphorylation of P-gp, and activation of the EGFR would also activate PLC, we investigated whether the effect of epidermal growth factor (EGF) on the phosphorylation of P-gp was mediated through PLC. Treatment of the human MDR breast cancer cell line, MCF-7/AdrR, with EGF increased the phosphorylation of P-gp by 20-50%. The increased phosphorylation of P-gp was accompanied by stimulation of PLC activity, as measured by the production of inositol, 1,4,5-trisphosphate and diacylglycerol, products of phosphatidylinositol-4,5-bisphosphate hydrolysis. Treatment of MDR cells with EGF also had detectable effects on P-gp function. For example, following incubation of MCF-7/AdrR cells with ECF, we observed a consistent decrease in total vinblastine (VBL) accumulation. Kinetic analysis revealed this change to be due to an increase in membrane efflux. The latter was measured by the initial uptake velocity, which was inhibited by EGF. VBL uptake measured at 0-320 sec was inhibited by 20-40%, which was associated with a similar increase in VBL efflux. EGF had no effect on drug accumulation, uptake, or efflux in sensitive MCF-7 cells. These data indicate that EGF can modulate the phosphorylation and function of P-gp, and suggest that this effect may be initiated by the activation of PLC.     

Altered methylation of the human MDR1 promoter is associated with acquired multidrug resistance

One of the most important forms of drug resistance in acute myeloid leukemia is the multidrug resistance (MDR) phenotype, which is characterized by the expression of the MDR1 gene product, P-glycoprotein. Although a number of factors affect MDR1 gene expression, the genetic events that "switch on" the human MDR1 gene in tumor cells that were previously P-glycoprotein negative have remained elusive. Here, we report evidence that the methylation status of the human MDR1 promoter may serve as a basis for this "switch." Based on Southern analysis using methylation-sensitive and methylation-insensitive restriction enzymes, a tight correlation was found between MDR phenotype and demethylation of the 5' region of the MDR1 gene in a human T cell leukemia cell line. Similar results were obtained from the analysis of P-glycoprotein-positive and P-glycoprotein-negative samples of chronic lymphocytic leukemia. Treatment of the cell lines with the demethylating agent 5'-azadeoxycytidine altered the methylation pattern of the MDR1 promoter in P-glycoprotein-negative cells to resemble that of P-glycoprotein-positive cells and activated the promoter such that MDR1 mRNA was now detectable. Treatment also resulted in an increased resistance to epirubicin and decreased daunomycin accumulation, both of which were reversible by verapamil, a characteristic of the classical MDR phenotype in cells expressing P-glycoprotein. These results suggest that the MDR phenotype may be acquired as a result of changes in methylation of the MDR1 promoter.     

Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines

This study highlights the usefulness of laser scanning confocal microscopy in the examination of subcellular disposition of anthracyclines in tumour cell lines. The distribution of anthracycline compounds has been studied in two pairs of parental and multidrug resistant (MDR) cell lines. For the parental EMT6 mouse mammary tumour cell line EMT6/P treated with doxorubicin (DOX) the anthracycline fluorescence was shown to be predominantly nuclear but with some particulate cytoplasmic fluorescence and very low levels of plasma membrane staining. In the same experiments much fainter fluorescence was seen for the EMT6/AR1.0 MDR subline which hyperexpresses P-glycoprotein. The loss of nuclear fluorescence was comparatively greater than loss of cytoplasmic fluorescence. For the human large cell lung cancer line COR-L23/P cellular DOX disposition was markedly nuclear with nuclear membrane staining and diffuse cytoplasmic fluorescence. For the MDR line COR-L23/R, which lacks P-glycoprotein expression, DOX fluorescence was reduced in the nucleus compared with the parental line, but an intense area of perinuclear staining was seen consistent with localisation to the Golgi apparatus. The morpholinyl-substituted analogue MR-DOX achieved very similar subcellular distribution in both parental and MDR lines, consistent with its retention of activity in the latter. The presence of verapamil during anthracycline exposure increased the intensity of fluorescence in the MDR lines, particularly in the nucleus. Relatively little effect was seen in the parental lines. Confocal microscopy provides high resolution images of the subcellular distribution of anthracyclines in parent and MDR cell lines. Differences in drug disposition in various cell lines may provide insights into the mechanism of multidrug resistance and suggest strategies for its therapeutic circumvention.