Cytometry 12:636-644 (1991)

0 1991 Wiley-Liss, Inc.

Flow Cytometric Double Labeling Technique for Screening of Multidrug Resistance1.2 Eric E.O. Gheuens? Dirk R. van Bockstaele, Maarten van der Keur, Hans J. Tanke, Allan T. van Oosterom, and Ernst A. De Bruijn Lab of Cancer Research and Clinical Oncology (E.E.O.G., A.T.v.O., E.A.D.B.), and Laboratory of Experimental Hematology (D.R.v.B.1,Antwerp University, B-2610 Wilrijk, Belgium; Laboratory of Cytochemistry and Cytometry, University of Leyden, Sylvius Laboratories, 2333 AL Leyden, The Netherlands (M.v.d.K., H.J.T.) Received for publication October 31, 1990; accepted April 26, 1991

We investigated the capabilities of flow cytometry in the analysis of a multidrug resistant (MDR) human ovarian cancer cell line 2780ADand its drug sensitive parental A2780. A functional assay using daunorubicin (DNR) as a fluorescent probe was combined with an immunofluorescence assay of P-glycoprotein (P-gp) using the monoclonal antibody MRK-16. Functionally MDR could be demonstrated by the lower DNR-content of MDR cells compared to DNR-content of drug sensitive cells. When incubation was performed with DNR in the presence of verapamil, DNR-content increased in the MDR cells. However the content of the A2780 cells was never attained. Differences in DNR-content were not related

Drug resistance is a major problem in the treatment of cancer. In cancer cell lines resistance to a variety of apparently unrelated cytotoxic agents can be induced in vitro by selection with a single chemotherapeutic drug (multidrug resistance, MDR). The cell lines accumulate the drugs they are resistant to less than their drug sensitive counterparts. This is related to a n increase in the rate of drug efflux. The most consistent finding in multidrug resistant cell lines is the overexpression of a 170 kDa membrane glycoprotein, termed P-glycoprotein (P-gp)(21,23-25). Drugs that are subject to the MDR phenomenon, such as vinca alkaloids, epipodophyllotoxins, anthracyclins, actinomycin D, and colchicin are bound by P-gy, a s are drugs which can reverse the multidrug resistant phenotype, e.g., verapamil, diltiazem, and quinidine (4,5,34,35). The binding is energy dependent: i t requires ATP-hydrolysis. Purification of the protein by affinity chromatography made i t possible to demonstrate the Mg2 -dependent ATPase activity of the molecule, which might be coupled to the active efflux of anticancer drugs (29). +

to differences in DNA-content. In experimental cell lines immunofluorescence data were inversely related with those of DNR-content: MDR cells had high levels of P-gp expression and low levels of DNR-content (and vice versa in drug sensitive cells). Both assays can be easily combined in a multiparametric flow cytometric procedure to evaluate both parameters simultaneously in the same cells. Analysis of clinical samples demonstrates the existence of aberrant subpopulations which would not be detected by using a single parameter assay. Key terms: P-glycoprotein, flow cytometry, monoclonal antibodies, daunorubicin, ovarian cancer

P-glycoprotein is believed to act as a n energy-dependent drug efflux pump, pumping out cytotoxic drugs and hence reducing the intracellular residence time (2,3,13,15,17). In vivo P-gp expression is found intrinsically in many but not all untreated tumors of the adrenal gland, the colon, the kidney, the liver, the pancreas,

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’Presented in part to the Eighty-First Annual Meeting of the American Association for Cancer Research, Washington, D.C., May 1990, and to the International AIO-symposium “Modification of Drug Resistance,” Aachen, June 1990. ’This work was supported in part by grants from the Belgian bank ASLK, the Belgian anticancer society BWK, the “Kom op tegen kanker” (Fight against cancer) campaign under the auspices of the NFWO, and the Saal van Zwanenberg Stichting (NL). Additional financial support was provided by Becton Dickinson ICS Belgium. 3Address reprint requests to Eric E.O. Gheuens, M.D., Lab. of Cancer Res., & Clin. Onc., Antwerp University, Universiteitsplein 1, Building S-4, B-2610 Wilrijk, Belgium.

FLOW CYTOMETRIC DOUBLE LABELING FOR MDR DETECTION

carcinoid tumors, and chronic myelogenous leukemia in blast crisis (intrinsic MDR). In some cases P-gp is expressed in tumors that relapse after chemotherapy (acquired MDR), e.g., in acute lymphocytic leukemia, acute nonlymphocytic leukemia, breast cancer, neuroblastoma, pheochromocytoma, and nodular, poorly differentiated lymphoma (1,9-12,14,18,22,23,28,32,36, 40,41). To establish the clinical importance of MDR in vivo, screening of various malignancies for the presence of P-gp before, during, and after treatment is needed. A suitable detection assay should be sensitive, selective, simple, and fast at lowest cost possible. Because of its single-cell analytical capability, flow cytometry represents a potentially powerful means of detection for the presence of small subpopulations of cells, i.e., detection of an MDR subpopulation in a population of largely sensitive cells. Several flow cytometric techniques for detection of MDR already have been developed either using daunorubicin (DNR) a s a fluorescent marker for intracellular anthracyclin content (18,19,26,33) or using the monoclonal antibodies (MoAbs) C-219 or MRK16 (7,8,16). We combined both methods and used this dual-parameter assay in the determination of MDR expression in a n ovarian cancer cell line.

MATERIALS AND METHODS Cell Culture The multidrug resistant human ovarian carcinoma cell line 2780AD(20,27,38) and its drug sensitive parental line A2780 were kindly provided by R. Ozols through P. Borst at the University of Amsterdam, the Netherlands. From here on these cell lines are referred to as mdr+ and mdr- respectively. Both cell lines grow as monolayers in DMEM substituted with 10% fetal calf serum, aspartic acid (0.1 mM), and glutamic acid (0.3 mM). Cultures were maintained in the absence of antibiotics and checked a t regular intervals for contamination with mycoplasmata using a nucleic acid hybridization assay (Mycoplasma T.C. rapid detection system, GEN-PROBE Inc., San Diego, CAI. The multidrug resistant cell line is continuously exposed to 2 pM doxorubicin (Sigma Chemical Company, St. Louis, MO), which is added fresh to the medium after every subculture.

637

F(ab’)’ fragments) ( 1 5 0 diluted, Dakopatts, Glostrup, Denmark) on ice for 30 min. Controls were stained with 50 pl FITC-RAM-F(ab’I2 fragments in the absence of the primary MoAb. Cells are washed three times in PBS with 1%BSA and finally resuspended to a concentration of lo6 cells/ml. For flow cytometric detection of MDR by measuring the retention of DNR, cell suspensions were incubated with DNR (Sigma Chemical Company, St. Louis, MO) a s described by Krishan and Ganapathi (261, Herweijer et al. (18,191, Nooter et al. (301, and Ross et al. (33). Briefly, incubation was done for 1h with 1 pglml DNR in full growth medium. Cells were then cooled rapidly and kept on ice. Analysis on the flow cytometer was done in the incubation medium. Cells (mdr- and mdr + ) incubated with medium only served a s controls. Verapamil (Knoll, Ludwigshaven, FRG) in a final concentration of 6.6 pg/ml was able to reverse MDR. For the combined assay, cell suspensions were incubated with DNR (1pg/ml) for 1h at 37°C in a waterbath and subsequently incubated with MRK-16 as described before. As controls, cells stained with MRK-16 or DNR only were used together with the appropriate controls for the single labeling techniques. Discrimination of viable cells was performed by using propidium iodide (PI, 10 pM) which was added to the samples at room temperature a few minutes prior to flow cytometric analysis. Since PI fluorescence can be detected through the same 575126 n m bandpass filter on the same photomultiplier tube as DNR this requires no extra equipment for the flow cytometer. Staining for DNA-content was carried out using the detergent-trypsin method described by Vindelev et al. (39). Normal human lymphocytes from a healthy male donor were used a s a diploid marker. These lymphocytes were isolated from a fresh blood sample by density gradient centrifugation (lymphocyte separation medium, Flow Laboratories, Asse-Relegem, Belgium). They were added to cell suspensions from the human ovarian cancer cell lines and all cells were stained with propidium iodide as described by Vindelprv et al.

Flow Cytometry Flow cytometric measurements were performed on a FACStarPLUS(Becton Dickinson, Mountain view, CA) instrument equipped with a 4 Watt argon-ion laser Incubation and Staining Procedures (Spectra Physics model 2025, Mountain View, CAI For flow cytometric analysis, labeling with the tuned to 488 nm with 300 mW power. Green and orMoAb MRK-16 (a kind gift from Dr. Tsuruo) was per- ange fluorescence pulses were collected through 530/30 formed as a modification of the method described by nm and 575/26 nm bandpass filters respectively. PhoEpstein et al. (7,8) and Hamada e t al. (16). Cell sus- tomultiplier pulses were amplified logarithmically. pensions are obtained by a 3-5 min trypsinisation with Overlap between green (FITC) and orange (DNR) flu0.25%trypsin-EDTA (GIBCO Ltd., Paisley, Scotland). orescence was compensated bi-directional (FL1- %FL2: After three washings with PBS, the cells are resus- k 10% and FLB-%FLl: c 30%) using the FACStar pended in PBS with 1% BSA to a concentration of lo6 PLUS Research Software (Becton Dickinson, Mountain cells/ml. Aliquots of 500 p.1 are incubated with 20 pl View, CA). Red fluorescence, for DNA-content measurements, MRK-16 (50 pg/ml) for 60 min on ice. Cells are washed three times in PBS with 1%BSA and stained with 50 was detected through a 630 nm long pass filter and pl fluoresceinated rabbit anti-mouse IgG (FITC-RAM- photomultiplier pulses were amplified linearly. DNA-

638

GHEUENS ET AL.

found in DNR-content of these cell lines (DNA-index of 1.27 for mdr- vs. 0.96 for m d r + ) .

Dual Parameter Flow Cytometry With Cell Mixtures Figure 3 shows a contour plot of log membrane P-gp fluorescence vs. log intracellular DNR content for a 1 : l mixture of mdr- and m d r + cells after dual labeling with MRK-16 and DNR a s described above. Both cell populations are clearly separated from each other with this method. There is a n inverse relationship between both parameters: high P-gp expression is related with low DNR content. 1

4

Green fluorescence (FLl -H; log) FIG.1. P-gp expression in the human ovarian cancer cell lines mdr- and mdr + determined by flow cytometry, using MRK-16 as the marker for P-gp. The figure shows cell count on the ordinate and green fluorescence intensity (MRK-16IRAM-FITC) on the abscissa.

indices were obtained by calculating the ratio of mean fluorescence channels of G,/G, phase of the tumor cells over G,/G, of the normal lymphocytes.

Statistical Analysis Linear regression analysis was performed using the Complete Statistical Analysis System (CSS), release 2.1 (StatSoft, Inc., Tulsa, OK). RESULTS Flow Cytometric Measurement of P-Glycoprotein Expression With the Monoclonal Antibody MRK-16 Figure 1shows the frequency histograms of log membrane fluorescence for mdr- and mdr+ cells after incubation with the monoclonal antibody MRK-16. With this method multidrug resistant mdr + cells have approximately a 7.6 fold higher expression of P-glycoprotein than drug sensitive mdr- cells, when relative numeric values are calculated for fluorescence intensity (Table 1). Flow Cytometric Measurement of DNR Content Figure 2 shows the frequency histograms of log intracellular DNR content for mdr- and mdr + cells after a 1 h incubation with DNR as described above. In our hands multidrug resistant mdr+ cells have approximately a 14 fold lower DNR content than drug sensitive mdr- cells, when relative numeric values are calculated for fluorescence intensity (Table 1).Incubation in the presence of verapamil restores DNR accumulation, but the level of the mdr- cells, however, was never attained (Table 1). From the data of DNA-content measurements it could be concluded that the difference between DNA-content of mdr- and mdr + cells does not proportionally attribute to the differences

Discrimination of Viable and Non-Viable Cells Although the use of flow cytometry to quantify DNR levels in tissue culture cells is easily accomplished, the occurrence of a high number of dead cells in clinical specimens could be a problem with the proposed double labeling technique. To evaluate interference from nonviable cells we have set up a n experiment with a mixture of viable and artificially damaged cells (methanol fixation). A mixture of mdr + cells and mdr- cells was made in a 1 : l ratio. An aliquot of these cells was fixed with 70% methanol at -20°C to induce membrane damage. Viable and non-viable cells were mixed and subsequently labeled according to the method described here and analyzed on the flow cytometer. The resulting cytogram is depicted in Figure 4A. From this i t is clear that membrane damaged cells show up in the double positive region. However, these dead cells can easily be cleared out from this important region by staining with PI (Fig. 4B). MDR-Detection in Clinical Samples The assay was applied to a bone marrow sample of a patient with a malignant non-Hodgkin lymphoma containing 65% malignant cells (Fig. 5A,B). The sample contained only a small amount of dead cells which could clearly be discriminated using PI staining. Backgating analysis of the lymphoma-population demonstrated that i t consisted of three different subpopulations:

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-cells having a high DNR-content and not expressing p-gP -cells having a high DNR-content and expressing PgP

-cells having a low DNR-content and not expressing p-gP Incubation in the presence of VPL, in a concentration that is able to reverse MDR in the mdr + cell lines, had no effect on DNR-content in any of the subpopulations described (data not shown). We also applied the assay to a pleura-exudate obtained from a 63 year old male patient with a small cell lung carcinoma, who at the time of sampling had received eight courses of chemotherapy including two

FLOW CYTOMETRIC DOUBLE LABELING FOR MDR DETECTION

639

Table 1 Means and Standard Deviations for P-gp-Expressionand Intracellular DNR-Content (n= 3)" Parameters DNR-content in P-gp expression DNR-content presence of verapamil 240 C 27 618 65 590 465 C 42 322 k 51 500 "Incubation with DNR in the presence of verapamil (6.6 pg/ml) was performed in one experiment. Values shown for mean P-gp-expression and DNR-content are channel numbers. Logarithmic amplification was used in these studies. One log decade equals 256 channels. The relative difference between P-gp-expression or DNR-content of sensitive cells compared to resistant cells can be calculated from this by obtaining the antilogarithm of the value (mean channel number mdr- cells - mean channel number mdr + cellsY256 for DNR accumulation or the antilogarithm of the value (mean channel number mdr+ cells - mean channel number mdr- cells)/256 for P-gp-expression. This gives the value of DNR-content or P-gp-expression in sensitive cells relative to that of resistant cells, which is set at 1.0. Cell type mdr mdr +

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Orange fluorescence (FL2-H; log) FIG.2. MDR in the human ovarian cancer cell lines mdr- and mdr + determined by flow cytometry, using DNR as the marker for MDR. The figure shows cell count on the ordinate and orange fluorescence intensity (DNR) on the abscissa.

MDR-related cytotoxic compounds (doxorubicin and vincristine). Malignant cells were isolated by density gradient centrifugation and labeled according t o the method described here (Fig. 5C,D). Again dead cells could be discriminated by PI staining. The sample contained a large amount of cells with scatter parameters closely resembling the scatter profile of macrophages. Backgating analysis of the malignant population demonstrated the existence of two subpopulations:

1644

Relative P-gp expression FIG. 3. MDR in the human ovarian cancer cell lines mdr- and mdr+ determined by flow cytometry, using MRK-16 and DNR as the markers for MDR in a dual-parameter assay. The figure shows a contourplot of a 1:1 mixture of mdr- and mdr+ cells with orange fluorescence intensity (DNR) on the ordinate and green fluorescence intensity (MRK-16-RAM-FITC) on the abscissa.

Determination of MDR in Mixed Cell Populations In order to determine the selectivity of all assays described, mixtures of mdr + cells and mdr- cells were made in concentrations ranging from 50 to 0.1% mdr + -cells having a high DNR-content and not expressing cells in a population of mdr-. They were labeled subsequently according to the methods described here and p-gP -cells having a low DNR-content and not expressing analyzed on the flow cytometer. To determine the percentage of multidrug resistant cells, for all three methp-gP ods a window was drawn electronically around the Incubation in the presence of VPL, in a concentration multidrug resistant subpopulation on a biparametric that is able to reverse MDR in the mdr + cell lines, had plot of DNR-content vs. P-gp-expression from the 1:l no effect on DNR-content in any of the subpopulations mixture samples (Fig. 6). The percentage of mdr + cells was then defined in each sample as the percentage of described (data not shown).

640

GHEUENS ET AL.

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FIG. 4. MDR in the human ovarian cancer cell lines mdr- and mdr + determined by flow cytometry, using MRK-16 and DNR as the markers for MDR in a dual-parameter assay. Discrimination of viable and non-viable cells was performed using PI (10 pM)which was added to the samples a t room temperature a few minutes prior to flow cy-

tometric analysis. The figure shows a contourplot of a 1:l mixture of viable and non-viable mdr- and mdr+ cells with orange fluorescence intensity (DNR) on the ordinate and green fluorescence intensity (MRK-16-RAM-FITC)on the abscissa. A prior to PI staining; B: after PI staining.

events in this window. Linear regression analysis was done between expected percentages and the actual measured percentages. For all three methods a correlation was found beyond the 95% confidence interval (Fig. 7).

human ovarian cancer cell line A2780 (mdr-) and its multidrug resistant variant 2780AD (mdr + 1, which have to be treated with trypsin to make a cell suspension for flow cytometric analysis. The 2780ADcell line displays the typical MDR pattern with resistance to the selecting agent doxorubicin (adriamycin)(RF = 100-160) and cross resistance to vincristine (RF = 600-10,000) and actinomycin D (RF = 200N16, 27,31,38). Elevated md r l gene expression and increased P-glycoprotein levels have been demonstrated in this cell line (38). Tryptic digestion of monolayer cultures clearly did not destroy the antigenic determinant of MRK-16. We detected a 7.6 fold higher expression of P-gp in the md r+ cells. Function of P-gp was not impaired since a 14 fold lower accumulation of DNR in mdr + cells than in the mdr- parental cells was detected. Both techniques represent a totally different approach in the detection of multidrug resistant cells. Using a MoAb (MRK-16) to detect the presence of an antigen on the membrane of cancer cells depends on the presence and the conservation of the antigenic determinant for the specific MoAb and is therefore a structural assay. The information provided by this kind of assay is limited in the sense that it does not provide any insight into the function of the antigen. For MDR this is not without importance (6). Therefore the combination with a functional assay (measurement of DNR accumulation) offers interesting possibilities. We now not only have a tool to detect the presence of a molecular antigen related to MDR but also to detect simultaneously its functional implications for the accumula-

DISCUSSION To establish the clinical importance of MDR in vivo, screening of various malignancies for the presence of P-gp before, during, and after treatment is needed. A suitable detection assay should be sensitive, selective, simple, fast, and cheap. Because of its single-cell analytical capability, flow cytometry represents a potentially powerful means of detection for the presence of small subpopulations of cells. Flow cytometric detection of multidrug resistance or its consequences in clinical settings has been described already using daunorubicin (DNR) as a fluorescent marker for intracellular anthracyclin content or using monoclonal antibodies (C-219, MRK-16)(7,11,18,32,37,41). The application of flow cytometry in the field of solid tumors is complicated by the requirement for clean cell suspensions. Making a single-cell suspension of the tumor is necessary and this often involves a n enzymatic degradation step, usually with collagenase or trypsin. This hinders immunolabeling since antigenic determinants are often destroyed by this procedure. Recently Yoshimura et al. (42) have demonstrated that P-glycoprotein has a very rigid structure with a small number of protease sensitive sites and its global structure is not destroyed by tryptic cleavage. Therefore we tested our method on monolayer cultures of a well characterized

FLOW CYTOMETRIC DOUBLE LABELING FOR MDR DETECTION

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FIG.5. Application of the double labeling assay to clinical samples. The upper panels of the figure show the results for a malignant non-Hodgkin lymphoma, while the lower panels show the results for a small cell lung carcinoma. Panels A and C: The scatter profiles of the respective tumor samples. Panels B and D: A dot plot with orange

fluorescence intensity (DNR) on the ordinate and green fluorescence intensity (MRK-16-RAM-FITC) on the abscissa for the respective tumor samples. Notice the potentially interesting populations (arrows) with aberrant combinations of DNR-uptake/P-gp-expression.

tion of a n anticancer drug. An important advantage of the bi-parametric assay could be the possibility to discriminate between P-gp-mediated and non-P-gp-mediated mechanisms of resistance. Indeed the method can discriminate between cells with high P-gp-expression and low DNR-content (typical MDR cells) and cells with low P-gp-expression and high DNR-content (cells without MDR). Theoretically two other popula-

tions could be detected: a population of cells with high P-gp-expression and high DNR-content which would represent cells with a n immunologically detectable Pglycoprotein which in some way still accumulate DNR and a population of cells without P-gp-expression and low DNR-content which could represent cells with a resistance mechanism other than P-gp-expression resulting in low DNR-content or cells with a P-gp expres-

GHEUENS ET AL.

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FIG.6. (Left) Windows drawn electronically around the multidrug resistant population on a biparametric plot of DNR-content vs. Pgp-expression from the 1:l mixture samples. The upper panel shows the window for labeling with MRK-16; the middle panel shows the window for labeling with DNR, and the lower panel shows the window for the double labeling. FIG.7. (Above) Expected (X-axis) and measured (Y-axis) percentages of multidrug resistant mdr cells in a population of mdr-, with correlations and line equations. MRK-16: labeling for P-glycoprotein with the Moab MRK-16; DNR measurement of DNR-content; MRK16IDNR dual labeling technique.

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sion level not detectable by this flow cytometric assay but with significant effect on DNR accumulation. Analysis of the clinical samples presented in this paper indeed demonstrates the existence of such subpopulations which would not be detected using a single-parameter assay. In order to examine the selectivity of the present assay for MDR, mixed populations of mdr + and mdrcells in different ratios were analyzed (Fig. 7). Data of linear regression analysis demonstrated excellent linearity, whereas the dual-parameter assay proved discriminative power a t a level of 0.1% mdr + cells. This is assumed to be sufficient for monitoring the occurrence of MDR at least at the experimental level. Experiments are now ongoing using tumor biopsy material t o define selectivity and sensitivity of the assay described here. Since the cells used in this investigation differ considerably in their morphological aspects (mdr + cells are smaller than mdr- cells) the technique of cell volume normalization as described by Ross et al. (33) seems to have interesting possibilities to improve the

FLOW CYTOMETRIC DOUBLE LABELlNG FOR MDR DETECTION

assay. Cell volume normalization for P-glycoprotein expression would give a n idea of antigen density (assuming that the antigen is distributed evenly on the membrane), while for DNR-content this would represent DNR-concentration. The morphological differences between mdr + and mdr- cells have another implication with regard to DNR fluorescence. Since DNR is a cytotoxic agent which binds to the nucleus and other cellular components, DNR fluorescence is not a function of DNR accumulation only, but also reflects differences in ability to bind DNR. We found that, although a significant difference in DNA-content was found in our model, this was, however, not proportional to the differences in DNR fluorescence. Furthermore, fluorescence microscopy also demonstrated marked reduction of nuclear fluorescence in m d r+ cells compared to mdr- cells (data not shown). We conclude that flow cytometric analysis of P-gp overexpression and DNR-content is a rapid, selective, and economically interesting technique allowing large scale studies of multidrug resistance in human and animal cancer cell lines. It offers interesting research possibilities since the use of MRK-16 and DNR in human material can allow sorting of human multidrug resistant cells from a population of cancer cells. Furthermore the assay will allow studies on the clinical importance of MDR since analysis of the percentage of multidrug resistant cells in a newly diagnosed cancer can be correlated with the clinical response to chemotherapy. Hence it can be established if the presence of a number of multidrug resistant cells heralds clinical drug resistance. Eventually this can be helpful to the clinical oncologist to adjust to a n optimal, individualized treatment regimen.

ACKNOWLEDGMENTS We thank Dr. Tsuruo for his kind provision of the MoAb MRK-16 and we also wish to thank Dr. Borst who delivered the human ovarian cancer cell line with kind permission from Dr. Ozols.

LITERATURE CITED 1. Bell DR, Gerlach JH, Kartner N, Buick RN, Ling V: Detection of P-glycoprotein in ovarian cancer: a molecular marker associated with multidrug resistance. J Clin Oncol 3:311-315, 1985. 2. Broxterman HJ, Kuiper CM, Schuurhuis GJ, Tsuruo T, Pinedo HM, Lankelma J : Increase of daunorubicin and vincristine accumulation in multidrug resistant human ovarian carcinoma cells by a monoclonal antibody reacting with P-glycoprotein. Biochem Pharmacol 37:2389-2393, 1988. 3. Chen CJ, Chin J E , Ueda K, Clark DP, Pastan I, Gottesman MM, Roninson IB: Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein) gene from multidrug-resistant human cells. Cell 47:381-389, 1986. 4. Cornwell MM, Gottesman MM, Pastan IH: Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells. J Biol Chem 261:7921-7928, 1986. 5. Cornwell MM, Pastan I, Gottesman MM: Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein. J Biol Chem 262:2166-2170, 1987.

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6 Dalton WS, Grogan TM: Does P-glycoprotein predict response to chemotherapy, and if so, is there a reliable way to detect it? JNCI 83:80-81, 1991. 7. Epstein J , Barlogie B: Tumor resistance to chemotherapy associated with expression of the multidrug resistance phenotype. Cancer Bull 41:41-44, 1989. 8. Epstein J , Xiao H, Oba BK: P-glycoprotein expression in plasmacell myeloma is associated with resistance to VAD. Blood 74:913917, 1989. 9. Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesman MM, Pastan I: Expression of a multidrug-resistance gene in human tumors and tissues. Proc Natl Acad Sci USA 84:265-269, 1987. 10. Fuqua SAW, Moretti-Rajas IM, Schneider SL, McGuire W L Pglycoprotein expression in human breast cancer cells. Cancer Res 47:2103-2106, 1987. 11. Funato T, Bando Y, Kato H, Tokuhiro H, Shimoda Y, Ohtani H: Analysis of P-glycoprotein in patients with acute leukemias by flow cytometry. Rinsho Byori 37:899-904, 1989. 12. Gerlach J H , Bell DR, Karakousis C, Slocum HK, Kartner N, Rusturn YM, Ling V, Baker RM: P-glycoprotein in human sarcoma: evidence for multidrug resistance. J Clin Oncol 5:1452-1460, 1987. 13. Gerlach JH, Kartner N, Bell DR, Ling V: Multidrug resistance. Cancer Surv 5:25-46, 1986. 14. Goldstein LJ, Galski H, Fojo A, Willingham M, Lai SL, Gazdar A, Pirker R, Green A, Crist W, Brodeur GM, Lieber M, Cossman J, Gottesman MM, Pastan I: Expression of a multidrug resistance gene in human cancers. JNCI 81:116-124, 1989. 15. Gros P, Croop J, Housman D: Mammalian multidrug resistance gene: complete cDNA sequence indicates strong homology to bacterial transport proteins. Cell 47:371-380, 1986. 16. Hamada H, Okochi E, Watanabe M, Oh-Hara T, Sugimoto Y, Kawabata H, Tsuruo T: M, 85,000 membrane protein specifically expressed in adriamycin-resistant human tumor cells. Cancer Res 48:7082-7087, 1988. 17. Hamada H, Tsuruo T: Functional role for the 170- to 180-kDa glycoprotein specific to drug-resistant tumor cells a s revealed by monoclonal antibodies. Proc Natl Acad Sci USA 83:7785-7789, 1986. 18. Herweijer H, Sonneveld P, Baas F, Nooter K: Expression of mdrl and mdr3 multidrug resistance genes in human acute and chronic leukemias and association with stimulation of drug accumulation by cyclosporine. JNCI 82:1133-1140, 1990. 19. Herweijer H, Van den Engh G, Nooter K: A rapid and sensitive flow cytometric method for the detection of multidrug-resistant cells. Cytometry 10:463-468, 1989. 20. Juliana R, Ling V: A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim Biophys Acta 455:151-162, 1976. 21. Kakehi Y, Kanamuru H, Yoshida 0, Ohkubo H, Nakanishi S, Gottesman MM, Pastan I: Measurement of multidrug-resistance messenger RNA in urogenital cancers; elevated expression in renal cell carcinoma is associated with intrinsic drug resistance. J Urol 139:862-865, 1988. 22. Kanamura H, Kakehi Y, Yoshida 0, Nakanishi S, Pastan I, Gottesman MM: MDRl RNA levels in human renal cell carcinomas: correlation with grade and prediction of reversal of doxorubicin resistance by quinidine in tumor explants. JNCI 81:844849, 1989. 23 Kartner N, Evernden-Porelle D, Bradley G, Ling V: Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies. Nature 316:820-823, 1985. 24 Kartner N, Ling V: Multidrug resistance in cancer. Sci Am March:26-33, 1989. 25 Kartner N, Riordan JR, Ling V. CelI surface P-glycoprotein associated with multidrug resistance in mammalian cell lines. science 221:1285-1288, 1983. 26 Krishan A, Ganapathi R Laser flow cytometric studies on the intracellular fluorescence of anthracyclines. Cancer Res 40: 3895-3900. 1980. 0" ~ iLouie . KG,'Hamilton TC, Winker MA, Behrens BC, Tsuruo T, Klecker RW, McKoy WM, Grotzinger KR, Myers CE, Young R c ,

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29. 30. 31. 32.

33.

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