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[CANCER RESEARCH 52. 3157-3163, June 1. 1992| Drug Transport Mechanisms in HL60 Cells Isolated for Resistance to Adriamycin: Evidence for Nuclear Drug Accumulation and Redistribution in Resistant Cells1 David Marquardt and Melvin S. Center2 Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901 ABSTRACT HL60 cells isolated for resistance to Adriamycin are multidrug resist ant and defective in the cellular accumulation of drug. These cells do not contain detectable levels of P-glycoprotein. At the present time the mechanism by which HL60/Adr cells reduce drug levels is not known. To gain insight into the molecular basis of this system we have analyzed transport pathways and the distribution of daunomycin in drug-resistant HI/60 cells. Using a cell fractionation technique we find that the major portion of daunomycin accumulates in the nucleus of both sensitive and resistant cells. Further studies reveal, however, that under efflux condi tions drug is retained in the nuclei of sensitive cells but rapidly removed from the nuclei of the resistant isolate. Essentially identical results are obtained when daunomycin distribution and transport are analyzed by fluorescence microscopy. A number of agents which alter transport processes have been tested for their effect on drug accumulation in resistant cells. Thus we find that brefeldin A, which disassembles Golgi, and various lysosomotropic agents such as chloroquine and methylamine do not affect drug levels. In contrast the protonophores nigericin and monensin induce an increase in drug accumulation and inhibit efflux. The results of this study thus suggest that resistance in IIL60/Adr cells is related to a mechanism whereby drug is transported to the nucleus and thereafter rapidly redistributed to the extracellular space. The mo lecular basis of this transport pathway is not known. INTRODUCTION Multidrug resistance in many experimental isolates is related to the presence of a surface membrane P-glycoprotein (1-4). Cell lines containing this protein are defective in the cellular accumulation of drug, and this appears to be related to the presence of enhanced levels of an energy-dependent drug efflux system (5, 6). Current evidence indicates that P-glycoprotein functions in this efflux pathway as a transporter which binds drug (7, 8) and exports this material to the exterior of the cell. Hydrolysis of ATP by the P-glycoprotein ATPase activity (9) may provide the energy which drives this reaction. Recently HL60 cells selected for anthracycline resistance have been isolated and characterized (10, 11). These cells ex hibit a multidrug-resistant phenotype, are defective in the cell ular accumulation of drug, and contain enhanced levels of a drug efflux system (10-12). Despite these properties isolates of these cells do not overexpress mdrl (13, 14) and do not contain detectable levels of P-glycoprotein (12, 15). Proteins contained in cell membranes of these isolates which may contribute to drug resistance have been described, however. Thus it has been observed that with cell surface labeling techniques a M, 150,000-160,000 protein (PI50) can be detected in resistant but not sensitive cells (10, 11). Other studies show that devel opment of resistance is accompanied by a major increase in the Received 9/9/91; accepted 3/25/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by Research Grant CA37585 from the National Cancer Institute. Department of Health and Human Services, and by a grant from Bristol-Myers Squibb. 2 To whom requests for reprints should be addressed, at Division of Biology, Ackert Hall. Kansas State University. Manhattan, KS 66506-4901. phosphorylation levels of P150 and that this protein may ac tually represent a hyperphosphorylated form of a protein con tained in sensitive cells ( 12). It has also been found that resistant isolates contain increased levels of a M, 190,000 membrane protein (PI90) which is capable of binding ATP (13). Recent studies have shown that antiserum against a synthetic peptide which corresponds to a specific P-glycoprotein sequence reacts with both P-glycoprotein and P190 (16). This peptide contains sequences which may be part of a nucleotide binding site in Pglycoprotein and P190 (16). The mechanism by which HL60/Adr cells reduce cellular drug accumulation is not known. Studies carried out thus far have not identified a drug-binding protein which may act as a drug transporter in the efflux pathway (13). It thus seems to be indicated that HL60/Adr cells reduce drug levels by a mecha nism distinct from that of isolates containing P-glycoprotein. In the present study we have examined drug transport pathways in HL60 cells isolated for resistance to Adriamycin. MATERIALS AND METHODS Materials. [3H]Daunomycin (1.6 Ci/mmol) was purchased from New England Nuclear. Brefeldin A was from Epicentre Technologies (Mad ison, WI). All other agents were from Sigma. Isolation of Resistant Cells. HL60 cells isolated for resistance to Adriamycin (HL60/Adr) were prepared as described previously (11). The isolate used in these studies is multidrug resistant and exhibits an 80-fold increase in resistance to the selecting agent (12). Fractionation of Cells Which Have Accumulated pHjDaunomycin. Cells growing in RPMI-10% fetal bovine serum were centrifuged and thereafter suspended in fresh media at a concentration of 1 x 106/ml. The cells were incubated with [3H]daunomycin (600 cpm/ng) for var ious time periods at 37°Cin a COi incubator and thereafter centrifuged and suspended in cold 0.01 M Tris-HCI (pH 7.6)-1.0 mivi MgCl2. The cells were homogenized with 15 strokes of a glass homogenizer and centrifuged at 500 x g for 5 min. The pellet containing the nuclei was collected, and the supernatant was centrifuged at 10,420 x g for 45 min to pellet membranes. Total radioactivity associated with nuclei, cytoplasm, and membranes was thereafter determined. Fluorescence Microscopy. Sensitive and resistant cells were centri fuged and suspended in fresh RPMI-10% fetal bovine serum at a concentration of 1 x 106/ml. The cells were incubated with daunomycin (0.5 /jg/ml) for 40 min at 37°Cand thereafter centrifuged and placed in fresh drug-free media. After incubation at 37°Cfor various time periods aliquots were taken and cytospun onto a glass slide. The cells were examined by either phase-contrast or fluorescence microscopy using a Nikon UFX-IIA microscope. For certain experiments live cells in drug-free media were placed directly on a microscope slide and examined by phase contrast or fluorescence microscopy. Analysis of Drug Efflux in Cells Loaded with Drug in the Presence of Azide. Cells growing in RPMI-10% fetal bovine serum were centrifuged and suspended in efflux media containing 50 mM Tris-HCI (pH 7.6), 0.14 M NaCl, 5.0 mM KCI, 1.0 mM CaCl2, 0.5 mM MgCl2, 2 mM glutamine, 1 x minimum essential medium amino acids and vitamins, and 2% fetal bovine serum. Sodium azide was added to 10 mM, and the cells were incubated with daunomycin (1 Mg/ml) for 30 min at 37°C. At the end of this time period the cells were centrifuged and suspended in EF media containing 15 HIM glucose. Aliquots were taken and examined microscopically as described above. 3157 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1992 American Association for Cancer Research. DRUG RESISTANCE IN HL60 CELLS Effect of Various Agents on Drug Efflux. Cells were loaded with [3H] daunomycin in EF media in the absence of glucose and presence of azide as described above. At the end of this time period the cells were centrifuged and suspended in EF media containing 15 HIMglucose. A single agent was added, and after incubation for various time periods the cells were centrifuged and radioactivity contained in the pellet was determined. Analysis of |3H]Daunomycin Metabolism. Cells in complete RPMI were incubated with [3H]daunomycin at 37°Cfor periods of up to 2 h, and the cell pellet was thereafter extracted with chloroform:methanol (2:1) for 90 min on ice. Under these conditions [3H]daunomycin is completely recovered from the cell. The chloroform:methanol extract was evaporated to dryness, and the dried material was dissolved in 0.01 M Tris-HCl (pH 7.6). This sample was thereafter chromatographed on silica gel thin-layer sheets in a buffer containing 65% chloroform-30% methanol-5% water. After drying the sheet was cut into 0.5-cm strips, and the radioactivity in each was determined and compared with a [3H] daunomycin marker. RESULTS Accumulation and Distribution of |'H|Daunomycin in Sensitive and Resistant Cells. Sensitive and HL60/Adr cells in complete RRMI were incubated with [3H]daunomycin, and fractions containing nuclei, cytoplasm, and membranes were prepared as described in "Materials and Methods." Analysis of distribution of [3H]daunomycin demonstrates that about 80% of the drug is contained in the nuclei of both sensitive and resistant cells (Fig. 1). The two cell types also contain similar levels of drug in the cytoplasmic and membrane fractions (Fig. 1). Analysis of drug accumulation under identical conditions demonstrates that resistant cells accumulate about one-half the amount of [3H]daunomycin as sensitive cells (not shown). In view of these findings studies were carried out to examine rates of [3H] daunomycin efflux from the nuclear, membrane, and cyto plasmic fractions. In these experiments cells were loaded with [3H]daunomycin in complete RPMI and thereafter suspended in drug-free media. At various time periods the amount of drug associated with various cell fractions was determined (Fig. 2). * 100 _ 3 O O o 80 60 Û co 40 20 O o Nuclei Cytosol Membrane Fig. 1. Intracellular distribution of l'I I|ilaiinonni-in. Sensitive and resistant HL60 cells growing in RPMI-10% fetal bovine serum were centrifuged and suspended in fresh media. The cells (5 x 10s ml) were thereafter incubated for 45 min at 37'C with [3H]daunomycin (600 cpm/ng; final concentration, 0.4 jig/ml). At the end of the incubation period cell fractionation was carried out, and the distribution of radioactively labeled drug was determined as described in "Mate rials and Methods." The amount of drug contained in nuclei, cytoplasm, and membranes is presented as the percentage of total (3H]daunomycin accumulated. In three identical experiments the difference in drug distribution has varied by no more than 10%. •¿, sensitive cells; D, resistant cells. 0 JO «0 Time (min) Fig. 2. Drug efflux from cell fractions. Sensitive (A) and resistant (B) cells were centrifuged, suspended in fresh RPMI-10% fetal bovine serum, and there after incubated with [3H]daunomycin for 40 min. At the end of this incubation period the cells were centrifuged and suspended in complete RPMI, and an aliquot of cells was fractionated into nuclei, membranes, and cytoplasm as described in "Materials and Methods." The remaining cells were allowed to continue incubation, and after 10, 30, and 60 min cell fractionation was carried out. The amount of [3H]daunomycin contained in the various cell fractions during the efflux is compared to that contained in the same fractions prepared prior to the efflux period. O, nuclei; •¿ cytoplasm; D, membranes. The results demonstrate a rapid and major loss of drug from all fractions of the resistant cell (Fig. IB). Cytoplasmic levels of drug remain relatively constant during a 30- and 60-min efflux period. In resistant cells this may represent drug which accumulates as it passes from the nucleus to the cytoplasmic space. Of particular interest is the finding that drug which is located in the nucleus of resistant cells (Fig. 2B) is rapidly removed, whereas in sensitive cells there is essentially a com plete nuclear retention of drug (Fig. 2A). Loss of drug does occur, however, from the cytoplasmic and membrane fractions of sensitive cells, but this is less than that occurring in these same fractions from resistant cells (Fig. 2). Analysis of Drug Distribution and Efflux Using Fluorescence Microscopy. To gain additional evidence for a nuclear drug accumulation in the HL60/Adr isolate, detailed studies were carried out to analyze by fluorescence microscopy daunomycin distribution in sensitive and resistant cells. In these experiments cells were incubated with daunomycin, centrifuged, and there after suspended in drug-free media. After incubation for various time periods the cells were examined microscopically. The results demonstrate that after a 40-min incubation period with daunomycin major levels of drug are contained in the nuclei of both sensitive (Fig. 3/1) and resistant cells (Fig. 3B). Incubation of resistant cells in drug-free media for 40 min results in a loss of daunomycin from the nuclear compartment (Fig. 3D), whereas under these same conditions drug is retained in the nuclei of sensitive cells (Fig. 3C). Phase-contrast microscopic examination of cells during these experiments does not indicate any detectable differences in sensitive and resistant cells, nor is there any preferential loss of resistant cells during the efflux period (Fig. 3, E and F). Additional experiments have been carried out in which resistant cells were loaded with daunomy cin in glucose-free media containing sodium azide. The cells were centrifuged suspended in media containing glucose and after incubation for various time periods aliquots were exam ined by fluorescence microscopy (Fig. 4). Control cells exam ined immediately after being placed in glucose media contain a high nuclear localization of drug (Fig. 4A). Continued incuba tion of cells for time periods of 20, 40, and 60 min results in a progressive loss of daunomycin from the nuclei of resistant cells 3158 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1992 American Association for Cancer Research. DRUG RESISTANCE IN HL60 CELLS ..... ... rx * •¿ * '"'.i ^ •¿ •¿ . •¿' . V- v *•! "I *•*•' S - f^ « .• y f - «R Fig. 3. Analysis of drug transport using fluorescence microscopy. Sensitive (A, C, and E) and resistant (A, O, and F) cells growing in RPMI-10% fetal bovine serum were incubated with daunomycin (0.5 jig/ml) for 40 min at 37°C.The cells were centrifuged and resuspended in complete RPMI, and a sample was cytospun onto a glass slide and taken for microscopy (A and B) as described in "Materials and Methods." The remaining cells were incubated for 40 min (C and D), after which the cells were examined by fluorescence microscopy. All samples were examined by phase contrast, and the results of the 40-min time point for sensitive and resistant cells are given in fand F, respectively. In separate experiments Hoechst staining of cytospun cells was used to verify that fluorescent structures identified in these experiments actually represent intact nuclei. 3159 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1992 American Association for Cancer Research. DRUG RESISTANCE IN HL60 CELLS (Fig. 4, B-D). In parallel experiments with sensitive cells there was essentially complete retention of drug in the nucleus during the 60-min efflux period (not shown). Studies have also been carried out in which sensitive and resistant cells were loaded with drug in complete RPMI and thereafter incubated under efflux conditions. Cells from the flask were placed directly on a microscope slide and examined by fluorescence microscopy (Fig. 5). Prior to incubation under efflux conditions fluores cence is distributed throughout the entire cell (Fig. 5, A and B). Based on the cell fractionation studies (Fig. 1) it would be predicted, however, that most of the drug is located in the nuclei of sensitive and resistant cells. Analysis of the live cells under efflux conditions demonstrates a progressive loss of drug from the resistant isolate (Fig. 5, B, D, and F). In contrast only low levels of drug are removed from sensitive cells (Fig. 5, A, C, and E). These studies also demonstrate that as drug is removed from HL60/Adr cells there is a localization and accumulation of daunomycin at a perinuclear site (Fig. 5F). At the present time the identity of the structure which contains daunomycin is not known. Extensive studies have been carried out to analyze the metab olism of intracellular daunomycin. Using the thin-layer Chro matographie technique described in "Materials and Methods" we have not detected any change in the drug after incubation with sensitive or resistant cells. These results strongly suggest that the loss of daunomycin fluorescence from the nuclei of resistant cells is not due to any major structural change in the drug. Effect of Various Agents on Drug Accumulation in Resistant Cells. The results described above suggest that drug which reaches the nucleus of resistant cells is rapidly removed and redistributed to the extracellular space. To examine the possible mechanisms involved in this process we have analyzed the effect of a variety of agents on drug accumulation in resistant cells. Thus we have found that the incubation of resistant cells in the presence of brefeldin A (18 ^M) for periods of up to 2 h has no effect on cellular drug levels (not shown). This agent has pre viously been shown to be highly active in blocking protein transport by a mechanism which involves the disassembly of the Golgi apparatus (17, 18). Similarly, drug levels are not altered by the incubation of resistant cells for periods of up to 2 h with chloroquine (90 ^M), methylamine (10 mivi), or am monium chloride (20 HIM) (not shown). These agents are ca pable of alkalinizing and disrupting the function of a variety of intracellular membrane vesicles (19). In contrast to these agents the protonophores nigericin and monensin (19, 20) induce an increase in drug accumulation (Fig. 6A) and inhibit efflux (Fig. 6B). Nigericin at a final concentration of 6 pg/m\ induces a complete block of [3H]daunomycin efflux from HL60/Adr cells (Fig. 6B). We have consistently found that nigericin is consid erably more active than monensin in inducing drug accumula tion and inhibiting efflux. At the concentration used neither nigericin or monensin affects drug accumulation in sensitive cells (not shown). Studies have also been carried out to examine the effect of nigericin on drug efflux from the nucleus. Using fluorescent microscopic techniques we find that in the presence Fig. 4. Analysis of drug efflux using fluorescence microscopy. Resistant cells were loaded with daunomycin (I ng/ml) in the presence of azide as described in "Materials and Methods." The cells were centrifuged and suspended in EF media containing 15 HIMglucose, and a sample was taken for microscopy I (). Additional aliquots were taken for microscopy after incubation of the cells at 37'C for 20 (B), 40 (C). and 60 (D) min. 3160 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1992 American Association for Cancer Research. DRUG RESISTANCE IN HL60 CELLS Fig. 5. Direct analysis of drug transport using fluorescence microscopy. Sensitive (A, C, and E) and resistant (fi, D, and f) cells growing in RPMl-10% fetal bovine serum were incubated with daunomycin (0.5 ng/ml) for 40 min. The cells were centrifuged and resuspended in complete RPMI, and a sample was placed directly on a microscope slide and examined by fluorescence microscopy (A and fi). The remaining cells were incubated for 40 (C and D) and 60 (£and F) min. and samples were examined directly by fluorescence microscopy. Although fluorescence is distributed throughout sensitive and resistant cells during the initial 40-min incubation with daunomycin (A and fi), fractionation studies (Fig. 1) indicate that drug is located primarily in the nucleus. 3161 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1992 American Association for Cancer Research. DRUG RESISTANCE IN HL60 CELLS •¿a S 'a 30 60 »0 120 Time (min) 150 20 4o so ao Time (min) Fig. 6. Effect of nigericin and monensin on drug accumulation and efflux. Resistant cells (5 X I0'/ml) in complete RPMI were incubated for 5 min in the absence or presence of either nigericin (6 /Jg/ml) or monensin (6 ng/ml), after which |'H|daunomycin was added to the cells. Incubations were continued at 37°C,and at various time periods aliquots were taken and, after centifugation, radioactivity contained in the cell pellet (A) was determined. A separate experi ment was carried out to examine the effect of nigericin and monensin on drug efflux (B). In this study resistant cells were loaded with [3H|daunomycin in the presence of azide as described in "Materials and Methods." The cells were ccntrifuged, suspended in EF media containing IS HIMglucose, and thereafter incubated in the absence or presence of either nigericin or monensin at a final concentration of 6 >ig/ml. At the various times indicated the radioactivity asso ciated with the cell pellet was determined. D, control cells incubated in the absence of agent: O, cells incubated with nigericin: •¿ cells incubated with monensin. of nigericin there is essentially a complete retention of drug in the nuclei of resistant cells (not shown). DISCUSSION Previous studies have shown that HL60 cells isolated for resistance to anthracyclines are defective in the cellular accu mulation of drug (10, 11). Results obtained thus far suggest that reduced drug levels are related to an enhanced efflux system (10, 11). In the present study we have extended our previous findings and have analyzed intracellular pathways for drug transport in the resistant cell. Using both cell fractionation and fluorescence microscopy we have shown that incubation with daunomycin results in a major accumulation of drug in the nuclei of both sensitive and resistant cells. The nuclei of sensi tive cells retain the drug, where it would be expected to exert a cytotoxic effect. In contrast resistant cells contain a mechanism whereby drug which reaches the nucleus is redistributed to the extracellular space. Extensive studies suggest that this mecha nism does not involve any structural change in the drug. At the present time the exact pathway taken by the drug as it moves from the nucleus to the exterior of the cell is not known. Direct examination of live resistant cells by fluorescence microscopy indicates that under efflux conditions drug localizes at a site which appears to be in close proximity to the nucleus. The nature of this site and its involvement in efflux is not known at the present time, however. Hindenburg et al. (21) have previ ously analyzed daunomycin distribution in an independent iso late of HL60/AR cells. Examination of these cells using digi tized video fluorescence microscopy and confocal microscopy indicates that daunomycin localizes at a perinuclear site which possibly represents a prelysosomal sorting compartment (21, 22). Similarly, Willingham et al. (23) have utilized fluorescence microscopy to examine daunomycin distribution in drug-resist ant KB cells. An examination of both fixed and live cells indicated no nuclear drug accumulation but instead a major localization of daunomycin in Golgi and lysosomes (23). The results described in the present study therefore suggest that HL60/Adr cells contain a drug transport pathway which is distinct from previously analyzed multidrug-resistant human cells. This difference as determined by microscopic studies is futher supported by the finding that neither brefeldin A (17, 18) or lysosomotropic agents (19) alter drug accumulation, thus suggesting that neither the Golgi apparatus or acidic vesicles such as lysosomes are involved in drug transport. In contrast to these agents nigericin and to a lesser extent monensin have been found to enhance drug accumulation and inhibit efflux. The mechanism by which these agents inhibit drug efflux is not known. The ability of these agents to alkalinize intracellular vesicles (19) would not seem to account for their effect on drug accumulation. These agents are capable, however, of altering cytosolic pH (20, 24) and may function in this way to block the efflux pathway. Nigericin and monensin have been found to enhance drug accumulation and inhibit efflux in other drugresistant isolates (25, 26). Previously it has been shown that verapamil induces an increase in drug levels in anthracyclineresistant HL60 cells (10, 13). This activity does not appear to be related to the ability of verapamil to compete for a drugbinding site on a transporter in HL60/Adr cells (13). Attempts to modulate Ca2+ levels with specific ionophores or chelators have not altered drug levels. It seems to effect of verapamil on drug accumulation Ca2+ channel blocking activity. Recently bafilomycin AI, an agent which selectively be indicated that the is not related to its we have found that inhibits vacuolar H+- ATPase activity at concentrations less than 10 fiM (27), en hances drug levels and blocks efflux in both HL60/Adr and in HL60 cells isolated for resistance to vincristine (28). These latter cells overexpress mdrl and contain high levels of Pglycoprotein (13, 16). Vacuolar H+-ATPases are capable of regulating the internal pH of various membrane vesicles and organdÃ-es (29). In view of the results obtained with various lysosomotropic agents it would not seem that vacuolar H+ATPases function in resistance by maintaining an acidic envi ronment in certain intracellular vesicles. Possibly the involve ment of the enzyme in regulating the pH of other organdÃ-es such as endoplasmic reticulum is important in drug transport. The results obtained in the present study and those described previously (13) suggest that movement of drug from HL60/Adr resistant cells does not involve the participation of acidic vesi cles or the presence of a protein which can physically bind and transport drug. The mechanism for drug transport thus appears to be distinct from cells containing P-glycoprotein, since drug binding by this protein seems to be an intermediate in the efflux pathway (7, 8). Proteins which may contribute to drug resistance in HL60/ Adr cells have been previously described. These cells have been found to contain a M, 150,000 plasma membrane protein (PI 50) which represents a modified (hyperphosphorylated) form of a protein contained in drug-sensitive cells ( 12). Recently a second protein (M, 190,000) has been detected which is overexpressed in HL60/Adr cells and is located primarily in the endoplasmic reticulum (13, 16). P 190, which can bind ATP (13), is reactive with an antibody prepared against a synthetic peptide which corresponds to the deduced sequence of P-gly coprotein (16). The sequence of this peptide is also contained in HAM-2 (histocompatibility antigen modifier), a transporter protein involved in antigen presentation (30). It is thus indicated that P 190, P-glycoprotein, and HAM-2 share a minor sequence homology. P 190 may thus be a member of a family of proteins which are involved in a variety of transport processes. It would 3162 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1992 American Association for Cancer Research. DRUG RESISTANCE IN HL60 CELLS thus be expected that this protein is involved in the events leading to the redistribution of drug from nuclear material. REFERENCES 1. Juliano, R. L., and Ling, V. A surface glycoprotein modulating drug perme ability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta, 455: 152-162, 1976. 2. Peterson, R. H. 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J., Cho, S., and Attaya, M. Transport protein genes in the murine MHC: possible implications for antigen processing. Science (Washington DC), 250: 1723-1726, 1990. 3163 Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1992 American Association for Cancer Research. Drug Transport Mechanisms in HL60 Cells Isolated for Resistance to Adriamycin: Evidence for Nuclear Drug Accumulation and Redistribution in Resistant Cells David Marquardt and Melvin S. Center Cancer Res 1992;52:3157-3163. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/52/11/3157 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. 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