Download Membrane Vesicle Formation Due to Acquired

(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.
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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-
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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
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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).
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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
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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.
ACKNOWLEDGMENTS
We gratefully acknowledge the cooperation of Prof. Dr. H. W.
Schreiber (Director of the Chirurgische Klinik. Universitätskranken
haus Eppendorf) who provided us with the tumor material. We are
thankful for the assistance of Gisela Broers and James K. Hcywood in
preparing the manuscript.
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Membrane Vesicle Formation Due to Acquired Mitoxantrone
Resistance in Human Gastric Carcinoma Cell Line EPG85-257
Manfred Dietel, Hartmut Arps, Hermann Lage, et al.
Cancer Res 1990;50:6100-6106.
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