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(CANCER RESEARCH 50. 6100-6106. September 15. 1990] Membrane Vesicle Formation Due to Acquired Mitoxantrone Resistance in Human Gastric Carcinoma Cell Line EPG85-2571 Manfred Dietel,2 Hartmut Arps, Hermann Lage, and Axel Niendorf Institut fur Pathologie, Christian-Albrechts-L'niversitat Republic of Germany zu Kiel, Kiel {M. D., H. L.] and Institut fürPathologie, UniversitätHamburg ¡H.A., A. N.], Hamhurg, Federal ABSTRACT A newly established gastric carcinoma cell line (EPG85-257P) exhib ited a high sensitivity to mitoxantrone (DHAD) as determined by a monolayer proliferation assay. The concentration to inhibit cell growth to 50% of controls (IG»)was 0.0022 «¿g/ml culture medium. The cells were continuously incubated for more than 4 months in the presence of stepwise increased concentrations of DHAD, and the ICS) was increased to 0.41 Mg/ml,i.e., 186.4-fold. This resistant variant was named EPG85257RNOV. The EPG85-257RNOV cells became cross-resistant to Adriamycin with enhanced K ..„ by 10.5-fold and to daunomycin with enhanced IC»by 3.9-fold. No distinct resistance was observed to vinblastine, vincristine, and colchicine. Verapamil (!()''. 4 x 10 " and Id \i) and cyclosporin A (10"*, 3 x 10"* and IO"5 M) did not reverse DHAD resistance. As shown by immunocytochemistry (monoclonal antibodies: C219 and JSB-1) and Northern blot analysis, DHAD resistance was not associated with the appearance of the multidrug resistance (MDR)associated (M, 170,000) P-glycoprotein or the overexpression of Pglycoprotein mRNA. The data indicate a chemoresistance pattern unlike typical MDR (often called "atypical" MDR). The phenotypes of parent and resistant EPG85-257 cells were com pared using interference contrast microscopy, electron microscopy, and immunocytochemistry. After DHAD application the following structural characteristics were found to be associated with emergence of resistance: (a) intensive formation of surface vesicles in the resistant variant. Such vesicles were almost absent in sensitive cells; (b) the vesicles contained the selecting DHAD which was visualized by its blue color; and (<•) in electron microscopy the vesicles were formed by an inner and an outer double membrane, presumably derived from the plasmalemma. These observations suggest a complex cellular mechanism responsible for DHAD resistance which includes formation of membrane vesicles, vesic ular drug binding, and drug compartmentalization. INTRODUCTION One limitation of cancer chemotherapy is the emergence of chemoresistant cell populations in malignant tumors. The mechanisms which enable tumor cells to survive and proliferate in the presence of relatively high concentrations of toxic sub stances are not fully understood. A great number of biochemical studies have described differences between parent (sensitive) cells and the resistant progeny, i.e., altered enzyme activity [e.g., 7-dihydrofolate reducÃ-ase, glutathione-S-transferase, protein kinase C, topoisomerase II) (1-5)], reduced intracellular drug toxification (6, 7), stimulated DNA repair (8-10), mdr gene amplification (11-18), overproduction of binding and transport proteins (e.g., M, 170,000 P-glycoprotein, M, 19,000 protein) (19-22) combined with reduced net drug accumulation (23Received 2/23/89; accepted 6/13/90. 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. 1This work was supported by the Deutsche Forschungsgemeinschaft. SFB 232 and Di 276/I-3, the Hamburger Stiftung zur Förderung der Krebsbekämpfung, the Hamburger Landesverband zur Krebsbekämpfung und Krebsforschung, and the E. und G. Roggenbuck-Stiftung. Presented in part at the 18th meeting of the University of California at Los Angeles. Keystone, CO, 1989. 2To whom requests for reprints should be addressed, at Institut für Pathologie. Christian-Albrechts-Universität. Michaelisstr. Il, D-2300 Kiel. Federal Republic of Germany. 25), and increased energy-dependent drug efflux (26, 27). Mor phological studies of phenotypic changes due to acquired che moresistance are relatively rare (28, 29). The experiments of Sehested et al. (30, 31) suggest that enhanced membrane traffic and vesicle formation play a key role in the emergence of drug resistance. The present study seeks to determine whether resistanceassociated phenotypic alterations of cellular and subcellular structures exist. This could be expressed, for example, by an altered membrane exchange, increased vesicular compartmen talization of drugs with augmented extrusion, or altered equip ment of subcellular organdÃ-es (e.g., lysosomes). To investigate these possibilities we used the human gastric carcinoma cell line EPG85-2571 (established in our laboratory), which was initially DHAD sensitive and became resistant to the drug by continuous incubation in the presence of stepwise increased concentrations. The phenotypes of sensitive (parent) and resist ant cells were compared by interference contrast microscopy, immunocytochemistry, and electron microscopy, supplemented by Northern blot analysis. MATERIALS AND METHODS Human Gastric Carcinoma Cell Line EPG85-257. A surgically re moved human gastric carcinoma was delivered to our laboratory in October 1985. The tumor was diagnosed histologically as adenocarcinoma of intestinal type. The procedure for establishing primary cell cultures is described elsewhere (32, 33). In brief, during transport the freshly explantcd specimen was kept at room temperature in serumfree medium. The tumor tissue was dissected mechanically by scraping and subsequently digested enzymatically for approximately 15-20 min with 0.1 unit collagenase/ml and 0.8 unit dispase/ml (Boehringer, Mannheim, FRG). After centrifugation (600 rpm at 25°C)the sediment was seeded in culture flasks (Falcon, Heidelberg, FRG). The culture medium was Leibovitz L 15 (Boehringer, Mannheim, FRG) supple mented per 500 ml with 5% fetal calf serum (Biochrom, Berlin, FRG), 500 ^g glutamine, 3.1 mg fetuin, 40 IE insulin. 1.25 mg transferrin, 0.56 g NaHCOi, 5 ml minimal essential medium vitamins, and 250 mg glucose (Sigma, Munich, FRG). After 2 weeks the cells were grown to confluence. To ensure that the cells in culture were derived from the carcinomatous human tumor they were characterized (a) by phase contrast microscopy showing atypical mitoses and dome formation, (b) by immunocytochemistry (see below) demonstrating keratin filaments as proof of epithelial origin (Fig. \A), and (c) by scanning cytophotometry (Leitz MPV 2) measuring nuclear DNA (33, 34). This disclosed 40% atypical cells with DNA values >4.5 n, indicating malignancy of the cultured cells (Fig. IA'). Drugs. The following cytostatic agents were used: DHAD (1,4dihydroxy-5,8-bis-l||2-[(2-hydroxyethyl)-amino]ethyl|amino||-9,10anthracenedione dihydrochloride: Cyanamid/Lederle, Wolfratshausen, FRG); Adriamycin and daunomycin (Farmitalia, Freiburg, FRG); col chicine (Sigma, Munich, FRG): actinomycin D (MSD Sharp & Dohme, Munich, FRG); VP-16 and cisplatin (Bristol-Myers, Neu-Isenburg, 'The abbreviations used are: EPG. Eppendorf Pathology Gastric; DHAD. mitoxantrone: P-glycoprotein, M, 170.000 glycoprotein; mab. monoclonal anti body; MDR. multidrug resistance: FRG. Federal Republic of Germany; ICso, drug concentration inhibiting cell growth to 50"'oof control; SSC, standard saline citrale: SDS. sodium dodecyl sulfate. 6100 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research. MEMBRANE VESICLE FORMATION AND DHAD RESISTANCE 1c 2c 3c 4c 5c 6c 7c 8c 9c 10c 12c DNA (rei. units) Fig. l . Phcnotypic characteristics of the human gastric carcinoma cell line EPG85-257. A, immunocytochemical visualization of the cytoskeletal protein keratin using the antibody KLI indicative of epithelial origin (x 250). B, DNA histogram with a highly atypical pattern of nDNA content proving malignancy of the cultured cells. DNA values (rei. units) are expressed in relation to cultured non-neoplastic fibroblasts (Fig. 2C, arrow). Table 1 Changes in the /C50 between parent anil resistant cells of the human gastric carcinoma cell line EPÕJ85-257 mediumMitoxantronAdriamycinDaunomycinColchicineVincristineVinblastine( 1C«," in jig/ml culture isplatinAclinomycin DVP-16EPG85-257P(parent)0.0022*0.001*0.013*0.00250.000050.000040.0210.0020.014EPG85-2 " Determined as described in Fig. 2. 'Significant differences. />< 0.001. 0,001 0,01 0,1 1 DHAD concentration in ug/ml EPG85-257RNOV EPG85-257P Fig. 2. Determination of the DHAD-induced ICM for parent and resistant EPG85-257 cells. Ordinate, cell growth during the 5-day control incubations without DHAD. Estimation of IC!0 was performed by dropping perpendicular lines (arrows) from the intercept of the dose-effect curves with the hori/ontal 50(V growth inhibition line (Glio). Points, mean cell counts of 6 cultures. SD does not exceed 5%. FRG); and vineristine and vinblastine (Lilly. Giessen, FRG). Verapamil was obtained from Sigma (Munich. FRG); cyclosporin A was a gift of Dr. Ryffel (Institute of Pathology, University of Basel and Sandoz Corp.. Basel, Switzerland). Selection of Resistance. The selecting drug was DHAD applied in stepwise increased concentrations for more than 120 days. The initial concentration was 0.001 ¿im/ml.After the cells were exposed to a particular concentration of DHAD the cultures were allowed to grow to near confluence. Subsequently, they were passaged and exposed to the same concentration for another 2 days, followed by a 2-fold en hancement of the concentration until they grew to near confluence again. During selection control cultures were kept without drug appli cation. At the beginning and the end of this procedure parent and resistant cells were cloned twice by establishing cultures from single cells according to the method described previously (24). Thus, cloned cell lines (parent = EPG85-257P and DHAD resistant = EPG85- 6101 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research. MEMBRANE VESICLE FORMATION AND DHAD RESISTANCE Fig. 3. Interference contrast microscopy of hematoxylin-cosin-stained parent and resist ant EPG85-257 cells after a 24-h incubation in 1.0 iig/ml of DHAD. A, sensitive EPG85257P cells showing almost complete cytolysis and condensed nuclei as sign of cell death: B, resistant EPG85-257RNOV cells without any alterations of the phenotype (x 250). 257NOV) of similar passage numbers were available for subsequent experiments. Monolayer Proliferation Assay to Assess ICW. The effects of growth modulating drugs were evaluated by counting living cells (as shown by trypan blue exclusion) with a hemocytometer or a coulter counter (model ZM plus Channelyzer 256). Test incubations were performed after the cultures reached the logarithmic growth phase estimated by microscopical observation. IC50 values were determined from doseresponse curves as detailed in Fig. 2 and Table 1. Thus, 0.5 x IO5 EPG85-257 cells/ml culture medium was seeded in 12-well cluster dishes (Costar, Cambridge, MA). The cells were incubated for 5 days either without drug (controls defined as 100%) or in the presence of increasing concentrations of the drug to be tested. All cultures were performed in triplicate. Statistical analyses for significance of differ ences were performed using the U test of Wilcoxon and Mann and Whitney. Immunocytochemistry. The cells were grown on glass slides, fixed in either methanol/acetone (1:2), 5% buffered formalin, or Bouin's solu cells still fixed to the slides. The Epon blocks including the cells were broken from the glass slide by repeatedly applying liquid nitrogen and warm water. From the blocks a I-mm discus containing the cells was dissected, cut along the diameter line, both parts were rotated for 90 degrees, and the parts were refixed, face to face, with a thin Epon film used as "glue." This particle was then remounted to an incompletely polymerized Epon block in such a way that the cells along the diameter line were located at the top. Next, the blocks were sectioned on an ultramicrotome (Reichert-Jung, Bielefeld, FRG) producing sections with two lines of cells cut from top to bottom (cf. Fig. 5). Northern Blot Analysis for P-glycoprotein mRNA. Total RNA was extracted from the resistant EPG85-257NOV cells using the guanidium thiocyanate procedure (39) with separation by ultracentrifugation through a dense cushion of cesium chloride. A total of 10 mg/ml of each RNA preparation was electrophoresed in a 1% agarose gel con taining 2.2 M formaldehyde and transferred to nitrocellulose mem branes (40). Cellular RNA was hybridized with the 0.6-kilobase pCHPl insert (41) (gratefully obtained from V. Ling) that had been multiprime labeled to specific activity (2.5 x 10' cpm/mg DNA) with ("PJdCTP (42). Hybridization was done at 42°Cin 50% formamide-5x SSC-Sx Denhardt'ssolution-50 mM sodium phosphate (pH 7.0)-15 mM EDTA0.1% SDS-250 mg/ml denatured salmon sperm DNA. After hybridi zation for 36 h the membranes were washed 3 times for 10 min with 2x SSC-0.1% SDS at room temperature followed by washing with 0.1% SSC-0.1% SDS at 66°Cfor l h and subsequent autoradiography. tion. They were processed by covering with the specific antibody, followed by an IgG (final concentration 1 ng/m\) directed against the first antibody and a compatible detection system, i.e., peroxidaseantiperoxidase or immunogold silver staining (Janssen. Ölen.Belgium) (for details see Refs. 33 and 35). The following mabs were used as purified IgG preparations: the anti-keratin KL-1 (final concentration 10 Mg/01') (Dakopatts, Hamburg, FRG) and the anti P-glycoprotein Sizes of RNA species were determined relative to the positions of 28S antibodies C219 (36) (provided by Dr. Victor Ling. The Ontario Cancer and 18S rRNA. As controls CHO-CH"C5 cells known to contain high Institute, Toronto, Ontario, Canada) and JSB-1 (given by H. J. Broxamounts of P-glycoprotein (mdr-I) mRNA were used. terman. Free University Hospital, Amsterdam, The Netherlands), both applied in a final concentration of 10 ng/ml. The mabs have been show n to be specific for the intracytoplasmic polypeptide domain of the Pglycoprotein (36, 37). Controls were performed (a) by replacing the RESULTS specific antisera with normal mouse serum or (b) with mouse IgG, (c) by replacing the anti-mouse IgG with normal serum or (d) phosphate Acquired DHAD Resistance of the Human Gastric Carcinoma buffered saline, and finally (e) by omitting separately each step of Cell Line EPG85-257. In the parent cells EPG85-257P, the detection. In order to check C219 and JSB-1 specificity and applicabil IC50 of DHAD was 0.0022 Mg/ml (Fig. 2). During the 120-day ity in immunocytochemistry, histológica! sections of a human adrenal gland known to contain considerable amounts of P-glycoprotein (38) DHAD exposure period the IC5(>was enhanced to 0.41 ng/m\ and P-glycoprotein (mdrl) mRNA (23) were covered with the mabs in the resistant variant EPG85-257NOV, i.e., by a factor of revealing intensive staining for P-glycoprotein in the cortex (not 186.4 (Table 1). The DHAD resistance remained preserved for shown). at least 90 days when cells were cultured without drug exposure. Electron Microscopy. Cells were grown on glass slides. To preserve Cross-resistance. Experiments on cross-resistance showed an structure and polarization the fixation was performed in situ, i.e., on increase of resistance to Adriamycin (10.5-fold) and daunomythe slides. For fixation a mixture of 0.35% glutaraldehyde, 3.5% cin (3.9-fold). However, EPG85-257NOV cells remained fully paraformaldehyde, 0.2% fannie acid, and 0.05% saponin diluted in 0.1 sensitive to colchicine, actinomycin D, VP-16, cisplatin, vinM phosphate-buffered saline (pH 7.0) was used. The cells were osmificated in 0.05% OsO4 buffered with 0.1 M phosphate-buffered saline blastine, and vincristine (Table 1). Control cultures never ex posed to DHAD did not exhibit a shift of chemosensitivity (pH 6.0). Dehydration and Epon embedding were performed with the 6102 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research. MEMBRANE VESICLE FORMATION AND DHAD RESISTANCE during the 120 days of parallel incubation. Reversibility of DHAD Resistance. Addition of verapamil alone for 4 days at concentrations ranging from 10~6, 4 x 10~6 to KT' M did not influence cell growth significantly, while concentrations higher than IO"5 M inhibited proliferation rate in a dose-dependent manner. In the noninhibiting range vera pamil did not reverse DHAD (0.2 Mg/ml) resistance of EPG85257NOV cells. Similar results were found with cyclosporin A when added at concentrations of 10~6, 3 x 10~6, and 10~5 M. Light Microscopy. If parent and resistant cells were incubated without drug addition, no morphological differences in size, shape, nuclear structure, etc. were observed. However, the ap plication of 1.0 ng/m\ DHAD for 24 h induced considerable changes in the phenotype of parent versus resistant EPG85257 cells. When stained with hematoxylin-eosin, the parent cells exhibited condensed and clotted nuclei as well as shrinkage and lysis of the cytoplasm (Fig. 3/1), whereas the resistant cells appeared unaltered (Fig. 3fi). In unstained cell preparations of the parent clones an intense blue staining of the nuclei was observed due to accumulation of the dark blue DHAD (Fig. 4A ). In contrast, the resistant cells showed completely unstained cell bodies and nuclei, which were both free of the DHADspecific blue staining (Fig. 4Ä).It is noteworthy that the resist ant cells formed cell surface-associated vesicles which contained DHAD, again shown by the blue staining (Fig. 4. B and C). The vesicles appeared to be of variable size. Ultrastructure. After 24 h incubation in 1.0 ng/m\ DHAD almost all cells of the parent population showed the signs of cell death, i.e., a dark condensed cytoplasm, enlarged mito chondria, a swollen endoplasmic rcticulum, and partially dis integrated cell membranes (Fig. 5A). As seen by light microscopy, the application of 1.0 /¿g/ml DHAD for 24 h to the resistant variant induced no changes in the cytoplasm, nuclei, or organelles (Fig. 5B) as compared with the untreated cells. However, the most important characteristic in the DHAD-incubated DHAD-resistant cells was the produc tion of multiple vesicles (Fig. 5, B and C). The vesicles were preferentially located near the outer cell surface and appeared to be expelled from the cells. They were formed by an inner and outer surface exhibiting a characteristic eccentric shape. Ultrastructurally, the layers resembled the cell membrane from which they obviously were derived. The membranous layers were separated from each other by an electron-lucent homoge neous material similar to the organelle-free cytoplasm. In this "vesiculoplasma" near the pole of the vesicles a condensed area was often present (Fig. 5C). Intracellular vesicles were observed only rarely. Immunocytochemistry and Northern Blot Analysis. Neither the sensitive EPG85-257P cells nor the DHAD-resistant EPG85-257RNOV cells exhibited P-glycoprotein immunoreactivity or contained P-glycoprotein-specific mRNA (Fig. 6). This finding was observed with and without drug exposure. DISCUSSION In the present study we report a cell line from a human gastric carcinoma (EPG85-257) which developed in vitro chemoresist- Fig. 4. Interference contrast microscopy of EPG85-257 cells in different stages of DHAD resistance. The cells have been fixed in buffered formalin: no artificial staining was performed: cultures after a 24-h incubation in 1.0 ,<L'.ml of the dark blue DHAD. A. parent DHAD-sensitive EPG85-257P cells. The nuclei are stained intensively blue indicating DHAD accumulation. The cytoplasm hasshrunk and disappeared nearly completely as a sign of cytolysis. B. interme diate stage of resistance development showing a mixture of parent and resistant cells. The resistant variant exhibits structurally intact noncolorcd cytoplasm and nuclei free of DHAD. At the surface multiple dark blue vesicles appear (arrows). The blue staining of the vesicles is due to DHAD accumulation. C, an almost exclusively DHAD resistant EPG85-257RNOV cell population, again showing DHAD in the surface vesicles but not in the cytoplasm and nuclei (x 400). 6103 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research. MEMBRANE VESICLE FORMATION AND DHAD RESISTANCE Fig. 5. Electron microscopy of EPG85257 cells. Cultured cells after a 24-h incubation in 1.0 Mg/ml DHAD. A. parent DHAD-sensitive EPG85-257P cells. The cytoplasm ap pears rather dark with multiple swollen mito chondria and dilated granules. The plasmalemma shows several breaks with discontinuity of the membrane layers due to cytolysis (pri mary magnification, x 4400; bar, 0.23 >im). B, DHAD-resistant EPG85-257RNOV cells. The cytoplasm, organelles, and the plasmalemma are well preserved. As demonstrated by light microscopy, multiple vesicles are at tached to the surface, presumably derived from the plasmalemma. The resistance-associated vesicles are seen almost only in the resistant variant (primary magnification, x 4.500; bar, 0.23 ¡im).C, vesicles exhibiting an outer and an inner membrane obviously derived from the plasmalemma. The membranes are separated by a homogeneous matrix similar to the organelle-free cytoplasm (primary magnification, x 85.000: bar. 0.012 »m). parent and resistant EPG85-257 cells were substantiated by ance for DHAD. We focus on phenotypic changes associated disturbed cellular integrity of the parent and unaltered mor with the in vitro acquired resistance to the selecting drug DHAD. Resistance was induced by culturing the cells in stepphology of the resistant variant. Furthermore, the resistant cells wise increased concentrations of DHAD, which resulted in an created DHAD-containing vesicles. The intensive vesicle for 186.4-fold increase in IC50. Moderate cross-resistance was mation and release was observed at the apical region of the found to the related anthracyclines, Adriamycin (10.5-fold) and resistant cells, indicating an outward transport mechanism. The daunomycin (3.9-fold), but not to other naturally occurring vesicles were derived from the plasmalemma, as shown by electron microscopy. It is noteworthy that these resistancecytostatic drugs, such as actinomycin D, VP-16, vinblastine, associated vesicles contained the selecting drug DHAD, as vincristine, and colchicine, or cisplatin. Furthermore, verapamil and cyclosporin A in various concentrations did not reverse evidenced by the dark blue staining of DHAD. Formation of DHAD resistance, as seen in a number of MDR cell lines. P- drug-containing vesicles appeared to represent a defense mech cells rapidly glycoprotein or the related mRNA could not be detected in anism by which the resistant EPG85-257RNOV EPG85-257RNOV using immunocytochemistry and Northern enclose DHAD and thus prevent intracellular drug accumula blot analysis. The data indicate that a typical MDR pattern (23, tion. Previously, Cornwell et al. (48) demonstrated biochemi cally the binding of vinblastine to membrane vesicles of MDR43-48) was not induced. A similar observation has been re ported for a leukemic cell line (49, 50), indicating that this resistant cells. This indicates that a vesicle-associated drug incomplete or "atypical" (49) form of MDR is not a unique transport is not a process exclusive to the EPG85-257NOV finding. Recently, another DHAD-resistant leukemic cell line cell line but may represent a general way of drug compartmen(HL-60/MX) and a colon carcinoma cell line (WiDr/R) were talization and extrusion. Accordingly, Sehested et al. (30) sug described (51-53) which, like the cell line EPG85-257RNOV, gested a vesicular transport of anthracyclines in resistant Ehr lack cross-resistance to Vinca alkaloids. lich ascites tumor cells. The vesicle-linked drug efflux appeared to function highly effectively in the resistant EPG85With DHAD application the phenotypic differences between 6104 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1990 American Association for Cancer Research. MEMBRANE VESICLE FORMATION 1 234567 28 S- 18SFig. 6. Northern blot analysis using the pC'HPI probe |0.6-fcilobase insert (41)]. The autoradiograph demonstrates lack of hybridization with total cellular RNA of EPG85-257RNOV cells (lanes 4 and f>). As control, the highly drugresistant CHO-CH*C9 cells were used and show a clear hybridization signal (lanes 2 and 7). The parental EPG85-257 cells are shown in lanes 3 and 5. as expected without hybridization signal. Lane I, molecular weight marker I from Boehringer. 257RNOV cells since cytoplasm and nuclei remained almost completely free of DHAD. Under identical conditions nuclei of parent cells stained dark blue by DHAD. cytoplasmic structures disintegrated almost completely, and vesicles did not appear. From the reported data a working hypothesis concerning the mechanism of DHAD resistance is proposed for the cell line investigated: When the drug approaches the resistant cell it is linked to a binding or receptor protein not present in the sensitive cells. The drug-receptor complex may induce vesicle formation as known for other receptor-mediated processes (5456). The complex is concentrated near the cell membrane and is rapidly released by exocytosis. This was demonstrated for the first time in the present study, since resistance-associated, drugloaded vesicles were predominantly located at the surface mem brane outside the cell. The following consideration gives further support: Several investigations, the present one included, report a decreased cellular drug accumulation and an increased drug efflux in resistant variants as compared to parent cells (23. 28, 57). With only one exception (30), none of these studies examined, or only speculated, what happens with the toxic drug when it is released. If resistant cells use a receptor protein to bind the drug and to pump it out of the cell without detoxification, the drug will attack the cell again. Thus, the resistant cell should at least in part inactivate the drug prior to release. One possible way to perform inactivation is to compartmentalize the drug in special vesicles, as shown in the present study. Whether the vesicles are provided with lysosomal activity, as described for intracellular vesicles in the MDR KB-C4 cells (28), remains to be shown. It has to be clarified whether the described mechanism can generally be attributed to cells resistant to naturally occurring antiproliferating substances. There are hints suggesting a ubiq uitous mechanism: (a) cells often exhibit resistance for several biochemically and functionally unrelated drugs (MDR), which requires a general defense mechanism, and (b) several authors described an increase of vesicle formation, drug trapping, and AND DIIAD RESISTANCE membrane traffic in resistant cells of different origin (30, 31, 47, 48, 58), again supporting the hypothesis that vesicular drug binding, processing, and extrusion might be a general mecha nism involved in chemoresistance. With regard to the in vivo development of drug-resistant tumors, it is still unclear whether the resistant cell clone is one of the heterogeneous cell clones present in each tumor (59) or whether these cells appear de novo, being genetically trans formed under the selection pressure. In the present study the parent cell culture, and presumably also the primary tumor, contained a small number of resistant cells as disclosed by their intact morphology following primary drug exposure. This ob servation, together with several reports (for review see Refs. 5 and 59), suggests that tumors may enclose resistant cells when they reach a clinically significant state. With chemotherapy the resistant mutants are selected by killing the sensitive popula tion. More knowledge is required concerning defense mecha nisms of the individual tumor cell and the distribution of resistant versus sensitive cells in a tumor to overcome drug resistance and to give chemotherapy a more permanent effec tiveness. 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