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INTRODUCTION |
Glucosylceramide synthase
(GCS)1 transfers glucose from
UDP-glucose to ceramide and produces GC. GC serves as the core
structure for more than 300 glycolipids (1). Recently, it has been
shown that human GCS is a glycoprotein containing 394 amino acids
encoded from 1182 nucleotides, including a G+C-rich 5' untranslated
region of 290 nucleotides (2). A large body of literature shows that ceramide, the substrate of GCS, exerts an important role mediating myriad biological activities. Ceramide is a pleiotropic cellular activator capable of inducing two mutually exclusive cellular functions, cell proliferation and cell death. Ceramide is now recognized as a messenger of signaling events that originate from different cell surface receptors, including interferon-
, TNF-
, interferon-1
, CD95 (Fas/APO-1), nerve growth factor receptor, and
CD28 (3-5). Ceramide is also involved in the action of protein kinase
C
, vav protooncogene, 1
-25-dihydroxy vitamin
D3, dexamethasone, ionizing radiation, and chemotherapeutic
agents (3-5). There have been several lines of evidence suggesting
that loss of ceramide production is one cause of cellular resistance to
apoptosis induced by either ionizing radiation, TNF-
, or adriamycin
(6-13).
Glycolipids, in addition to being essential membrane structural
elements, are putatively involved in cell proliferation (14), differentiation (15-17), and oncogenic transformation (18, 19). GC has
recently been shown to be associated with resistance to chemotherapy
(12, 13, 20, 21). Accumulation of GC is a characteristic of some MDR
cancer cells and tumors derived from patients who are less responsive
to chemotherapy (20, 21). Further studies have shown that MDR
modulators of varying chemical structure inhibit the production of GC
(12, 21). Although these studies document the accumulation of GC in
multidrug-resistant cancer, little is known about the expression of GCS
in MDR cells and its relationship to drug resistance. In particular, it
is not known whether increased levels of GC are due to GCS gene
expression or to other drug resistance factors that play a role in
modulating GCS activation or GCS degradation.
The retroviral Tet-off/Tet-on vector is a highly regulatory, versatile
mammalian expression system (22-26). Expression of a target gene
inserted in multiple cloning sites under CMV promoter control can be
mediated simply by withdrawal of tetracycline or addition of
doxycycline. Utilizing a retroviral Tet-on system, we established the
MCF-7/GCS cell line from MCF-7 breast adenocarcinoma cells. The
MCF-7/GCS-transfected cells express high levels of GCS activity, and
they are resistant to cytotoxicity imparted by adriamycin and ceramide.
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EXPERIMENTAL PROCEDURES |
Materials--
[3H]UDP-glucose (40 Ci/mmol) was
purchased from American Radiolabeled Chemicals (St. Louis, MO). EcoLume
(liquid scintillation mixture) was from ICN (Costa Mesa, CA), and
[
-32P]dCTP (6,000 Ci/mmol) was from Amersham Pharmacia
Biotech. C6-Ceramide (N-hexanoylsphingosine) was
purchased from LC Laboratories (Woburn, MA). Sulfatides (ceramide
galactoside 3-sulfate) were from Matreya (Pleasant Gap, PA), and
phosphatidylcholine (1, 2-dioleoyl-sn-glycero-3-phosphocholine) was from Avanti
Polar Lipids (Alabaster, AL). C219, the monoclonal antibody against
human P-glycoprotein, was from Signet Laboratories (Dedham, MA), and
Bcl-2 (Ab-1) monoclonal antibody against human Bcl-2 was from Oncogene
Research Products (Cambridge, MA). Hygromycin B was purchased from
Boehringer Mannheim. Doxycycline hydrochloride, adriamycin (doxorubicin
hydrochloride), and other chemicals were purchased from Sigma. FBS was
purchased from HyClone (Logan, UT). RPMI 1640 medium and Dulbecco's
modified Eagle's medium (high glucose) were from Life Technologies,
Inc., and cultureware was from Corning-Costar (Cambridge, MA).
Cell Lines and Culture Conditions--
Human breast
adenocarcinoma cells, MCF-7 cells, and MCF-7 adriamycin-resistant cells
(MDR clone) were kindly provided by Drs. Kenneth Cowan and Merrill
Goldsmith (National Institutes of Health, NCI, Bethesda, MD). Cells
were maintained in RPMI 1640 medium containing 10% (v/v) FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 584 mg/liter
L-glutamine. Cells were cultured in a humidified, 5%
CO2 atmosphere tissue culture incubator and subcultured
weekly using a trypsin-EDTA (0.05%-0.53 mM) solution.
Transfected cells, MCF-7/GCS, were cultured in RPMI 1640 medium
containing 10% FBS and 200 µg/ml hygromycin in addition to the above components.
pTRE-GCS Expression Vector Construction and
Transfection--
pCG-2, a Bluescript II KS containing GlcT-1 (see
Ref. 2 for terminology for GCS) in the EcoRI site was kindly
provided by Drs. Shinichi Ichikawa and Yoshio Hirabayashi (Institute of Chemical and Physical Research, RIKEN, Saitama, Japan). The gene encoding human glucosylceramide synthase was immune-selected by monoclonal antibody M2590 from a human melanoma cell (SK-Mel-28) library (2). The full-length cDNA of human GCS was subcloned into
the EcoRI site in the pTRE, Tet-repressible expression
plasmid. The Tet-on gene expression system was purchased from
CLONTECH (Palo Alto, CA). This system contains
three vectors, pTet-on, pTRE, and pTK-Hyg. The pTet-on vector
(pUHD17-1neo) expresses a doxycycline-controlled rtTA that is a fusion
protein of a reverse Tet repressor and the C-terminal domain of protein
16 of herpes simplex virus, constitutively expressed under control of
human CMV promoter (22, 26). The pTRE vector (pUHD10-3) contains a
multiple cloning site to accept any cDNA to be expressed followed by an SV40 polyadenylation sequence (24). The promoter region upstream
from the multiple cloning site contains a minimal human CMV promoter
(PminCMV) with heptamerized tet operators. This
promoter is silent in the absence of binding of rtTA to the tet
operators. However, when the reverse Tet repressor of the rtTA binds to
the tet operators, the virion protein 16 domain of the rtTA can
activate PminCMV activity to a very high level
and switch on expression of the target gene, GCS. Binding of
doxycycline to the reverse Tet repressor domain of the rtTA can almost
completely activate rtTA binding to the promoter (22, 26).
Sense orientation of the GCS cDNA was analyzed with Vector NTI 4.0 and doubly checked by restriction enzyme digestion with HindIII and with XhoI plus NotI. The
pTK-Hyg vector, which has a hygromycin-resistant gene under control of
the mouse
-globin promoter, was used to select the stable
transfectants. When MCF-7 cells reached 20% confluence, pTet-on DNA
(10 µg/ml, 100-mm dish) was introduced by co-precipitation with
calcium phosphate (Mammalian Transfection Kit, Stratagene, La Jolla,
CA). The transfected cells were selected in RPMI 1640 medium containing
10% FBS and 400 µg/ml G418. Each G418-resistant clone was screened
by luciferase assay, after transient transfection with pTRE-Luc vector
containing the reporter gene, luciferase. pTK-Hyg (10 µg DNA) and
pTRE-GCS (10 µg DNA) were introduced into the selected MCF-7 Tet-on
cells by co-precipitation with calcium phosphate. The GCS-transfected
cells were primarily selected in RPMI medium containing 10% FBS and 200 µg/ml hygromycin. As a control for transfection, MCF-7 Tet-on cells were co-precipitated with pTK-Hyg and pTRE plasmid without GCS cDNA.
Transient Transfection and Luciferase Assay--
This procedure
was performed as described previously (22, 24, 25). After MCF-7 cell
transfection with pTet-on vector, each G418-resistant clone was grown
for 16 h in 6-well plates (4000 cells/well) in 10% FBS RPMI
medium, then shifted to 10% FBS Dulbecco's modified Eagle's medium.
After a 6-h incubation, pTRE-Luc (1.5 µg of DNA) was introduced by
co-precipitation with calcium phosphate. After culturing in 10% FBS
Dulbecco's modified Eagle's medium for 18 h and in 10% FBS RPMI
medium for 48 h, luciferase activity was measured using a
commercial luciferase assay system according to the instruction manual
(Promega, Madison, WI). Incubation for 48 h in 3.0 µg/ml
doxycycline was used to induce expression of rtTA protein. Cellular
extracts (100 µg of protein) from each clone were used. The activity
of luciferase was measured by scintillation spectroscopy 2 min after
the addition of substrate. MCF-7 cells transfected with pTRE-Luc were
used as controls.
Glucosylceramide Synthase Assay--
To determine the expression
of GCS in the hygromycin-resistant clones, a modified radioenzymatic
assay was utilized (12, 27). After incubation in the absence or
presence of doxycycline (3 µg/ml for 48 h), cells were
homogenized by sonication in lysis buffer (50 mM Tris-HCl,
pH 7.4, 1.0 µg/ml leupeptin, 10 µg/ml aprotinin, 25 µM phenylmethylsulfonyl fluoride). Microsomes were isolated by centrifugation (129,000 × g for 60 min).
The enzyme assay, containing 50 µg of microsomal protein, in a final
volume of 0.2 ml, was performed in a shaking water bath at 37 °C for 60 min. The reaction contained liposomal substrate composed of C6-ceramide (1.0 mM), phosphatidylcholine (3.6 mM; molecular weight, 786.15), and brain sulfatides (0.9 mM; molecular weight, 563). The liposomal substrate was
prepared by mixing the components, evaporating the solvents under a
stream of nitrogen, and sonicating in water over ice for 1 min using a
microtip at 50% output (Kontes, Micro Ultrasonic Cell Disrupter).
Other reaction components included sodium phosphate buffer (0.1 M), pH 7.8, EDTA (2.0 mM), MgCl2 (10 mM), dithiothreitol (1.0 mM),
-nicotinamide adenine dinucleotide (2.0 mM), and
[3H]UDP-glucose (0.5 mM). Radiolabeled and
unlabeled UDP-glucose were diluted to achieve the desired radiospecific
activity (4700 dpm/nmol). To terminate the reaction, tubes were placed
on ice, and 0.5 ml isopropanol and 0.4 ml
Na2SO4 were added. After brief vortex mixing, 3 ml t-butyl methyl ether was added, and the tubes were mixed
for 30 s. After centrifugation, 0.5 ml of the upper phase, which
contained GC, was withdrawn and mixed with 4.5 ml of EcoLume for
analysis of radioactivity by liquid scintillation spectroscopy.
Analysis of Ceramide and Glucosylceramide--
Analyses were
performed as described previously (12). Cellular lipids were
radiolabeled by incubating cells with [3H]palmitic acid
(2.5 µCi/ml culture medium) for 24 h. After removal of medium,
cells were rinsed twice with phosphate-buffered saline (pH 7.4), and
lipids were extracted (12). After nitrogen evaporation of solvents,
total lipids were resuspended in 100 µl of chloroform/methanol (1:1,
v/v), and aliquots were applied to TLC plates. Ceramide was resolved
using solvent system I, which contained chloroform/acetic acid (90:10,
v/v). GC was resolved using solvent system II, which contained
chloroform/methanol/ammonium hydroxide (70:20:4, v/v). Commercial lipid
standards were co-chromatographed. After development, lipids were
visualized by iodine vapor staining, and areas of interest were scraped
into 0.5 ml of water. EcoLume counting fluid (4.5 ml) was added, the
samples were mixed, and radioactivity was quantitated by liquid
scintillation spectrometry.
RNA Analysis--
RNA was extracted from cells using the
single-step method described by Chomczynski and Sacchi (28). Equal
amounts of total RNA (15 µg) were denatured in 59% formamide/2.2
M formaldehyde, size-separated by electrophoresis on 1%
agarose-formaldehyde, and then blotted onto NitroPure nitrocellulose
transfer membrane (29). GCS cDNA was prepared from pCG-2 plasmid,
digested with EcoRI, and HindIII (Stratagene).
The 1.1-kilobase pair fragment was then isolated by 1% low melt
agarose electrophoresis using a commercial agarose gel DNA extraction
kit (Boehringer Mannheim). Probing of 32P-GCS cDNA was
performed by nick translation according to the instruction manual
(Boehringer Mannheim). Nitrocellulose-plus membranes were hybridized
with the 32P-GCS probe at 68 °C for 18 h. The
filters were exposed at -70 °C for autoradiography. For even gel
loading, 28 S RNA was used.
Cytotoxicity Assay--
The assay was performed as described
previously (12). Briefly, after culture in the absence or presence of
3.0 µg/ml doxycycline for 48 h, cells were harvested and seeded
in 96-well plates (2,000 cells/well), in 0.1 ml of RPMI 1640 medium
containing 10% FBS in the absence or presence of 3.0 µg/ml
doxycycline. Cultures were incubated at 37 °C for 24 h before
addition of drug. Drugs were added in FBS-free medium (0.1 ml), and
cells were cultured at 37 °C for the indicated periods. Drug
cytotoxicity was determined using the Promega 96 Aqueous cell
proliferation assay kit. Absorbance at 490 nm was recorded using an
enzyme-linked immunosorbent assay reader (Molecular Devices, San Diego, CA).
Western Blot Analysis--
Western blots were performed using a
modified procedure (30, 31). Confluent cells were washed twice with
phosphate-buffered saline containing 1.0 mM
phenylmethylsulfonyl fluoride, and detached with trypsin-EDTA solution.
Cells were pelleted by centrifuging at 500 × g for 5 min. Cell pellets were solubilized in 1.0 ml of cold TNT buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin)
for 60 min with shaking. The insoluble debris was excluded by
centrifugation at 12,000 × g for 45 min at 4 °C.
The detergent soluble fraction was loaded in equal aliquots by protein
and resolved using 4-20% gradient SDS-polyacrylamide gel
electrophoresis. The transferred nitrocellulose blot was blocked with
3% fat-free milk powder in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) at room temperature for
1 h. The membrane was then immunoblotted with monoclonal
antibodies C219 (5 µg/ml) or Bcl-2 (Ab-1) (2.5 µg/ml) in
Tris-buffered saline containing 0.5% bovine serum albumin (10 mM Tris-HCl, pH 8.0, and 150 mM NaCl) at
4 °C for 18 h. Detection was performed using ECL (Amersham
Pharmacia Biotech).
Statistics--
All data represent the mean ± S.D.
Experiments were repeated two or three times. Student's t
test was used to compare mean values, and linear correlation between
variables was tested using Pearson's correlation coefficient.
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RESULTS |
Expression of Glucosylceramide Synthase--
MCF-7 cells were
transfected with pTet vector and co-transfected with pTRE-GCS and
pTK-Hyg. The stable, high expression clones were selected by screening
GCS activity using the cell-free enzyme assay and by Northern blot.
After transfection of pTet-on in MCF-7 cells, more than 30 G418-resistant clones were collected. Luciferase activity, which is a
measure of expression of rtTA in the G418-resistant clones, was
analyzed after 3 days of transient transfection with pTRE-luciferase
vector. After stimulation with doxycycline, maximal expression of
luciferase, 16,000-fold above that of MCF-7 cells, was found in clone
16. Luciferase activity in clone 16 in the absence of doxycycline was
15,000-fold higher than that of MCF-7 cells. Clone 1 demonstrated low
basal rtTA expression; however, clone 1 was highly responsive to
doxycycline, with induced luciferase activity that was 100-fold over
MCF-7 cells. Clones 1 and 16 were selected as the optimal MCF-7 Tet-on
clones for expression of rtTA.
After co-transfection of pTRE-GCS and pTK-Hyg into clone 1 and clone 16 of MCF-7 Tet-on cells, 65 hygromycin-resistant clones were selected.
Utilizing the [3H]UDP-glucose enzyme assay, we analyzed
GCS activity and identified three clones that exhibited 5-11-fold
increases in enzyme activity (Fig.
1A). Compared with a basal
level of 17.2 ± 0.1 pmol of GC in MCF-7 wt cells,
doxycycline-induced GCS activity in MCF-7/GCS12, MCF-7/GCS13, and MCF-7/GCS14 was 167.4 ± 17.2, 183.3 ± 12.4, and 90.2 ± 2.76 pmol of GC,
respectively (Fig. 1A). There were no differences in either
basal or doxycycline-induced GCS activity in transfection control cells
(TC) or in the basal level of GCS in MCF-7 wt cells (Fig.
1A). In MCF-7/GCS13 and MCF-7/GCS14,
the doxycycline-inducible GCS activities were 1.6- and 4.1-fold,
respectively, above untreated cells. The MCF-7/GCS14 clone
was designated MCF-7/GCS, and this clone was used in further
experiments.

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Fig. 1.
Glucosylceramide synthase activity and
GCS mRNA expression in MCF-7/GCS cells. Cells were incubated
without ( ) or with (+) doxycycline (3 µg/ml) for 72 h.
A, GCS activity. GCS activity was assayed as detailed under
"Experimental Procedures." WT, MCF-7 wild type;
TC, transfected control cells; GCS12,
GCS13, and GCS14, subclones of
MCF-7/GCS. *, p < 0.001 compared with MCF-7 cells.
Subclone GCS14 was designed as MCF-7/GCS and used in
further experiments. B, Northern blot analysis of GCS
mRNA expression. Total RNA (15 µg/lane) was subjected to
agarose-formaldehyde electrophoresis, transblotted to
nitrocellulose-plus membrane, and hybridized with GCS cDNA probe.
28 S RNA stained with ethidium bromide was used as control for even
loading. Dox, doxycycline; TC, transfected
control. C, ceramide and GC levels in MCF-7/GCS cells.
Lipids were radiolabeled by incubating cells with
[3H]palmitic acid. After lipid extraction, ceramide and
GC were resolved by TLC using solvent systems I and II,
respectively.
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Doxycycline-induced GCS mRNA was highly elevated in MCF-7/GCS cells
compared with doxycycline-naive MCF-7/GCS cells. A representative Northern blot is shown in Fig. 1B. Only traces of GCS
mRNA were observed in MCF-7 cells, TC, and MCF-7/GCS cells without
doxycycline (Fig. 1B). The levels of ceramide and GC in
MCF-7 and in MCF-7/GCS cells were assessed by steady-state
radiolabeling of cultured cells using [3H]palmitic acid.
As shown in Fig. 1C, transfection with GCS elicited only a
moderate decrease in ceramide, compared with MCF-7 cells. The decrease
was not statistically significant. GC in MCF-7/GCS compared with MCF-7
cells increased slightly and accounted for 1.8 and 1.5%, respectively,
of total cellular radiolabeled lipid.
Adriamycin and Ceramide Resistance in Transfected MCF-7/GCS
Cells--
Recent work has revealed that effects of therapeutic doses
of anthracyclines are closely related to the generation of ceramide, and elevated GC has been shown to be associated with adriamycin resistance in MDR cells (9, 11-13). Adriamycin was used to assess the
influence of GCS transfection on cellular response to anthracyclines. After pretreatment with doxycycline for 2 days, MCF-7/GCS cells were
incubated with increasing concentrations of adriamycin for 4 days. Fig.
2A shows that MCF-7/GCS cells,
compared with MCF-7 cells, are resistant to adriamycin toxicity. At
0.5, 1.0, 2.0, and 3.0 µM adriamycin, survival of
transfected MCF-7/GCS cells was significantly greater than that of
MCF-7 cells (p < 0.0005, Fig. 2A). As
presupposed, it was observed that MCF-7/GCS cells were also resistant
to ceramide toxicity. At 2.5 and 5.0 µM
C6-ceramide, MCF-7/GCS cell survival was significantly
higher than that of MCF-7 cells (p < 0.0005, Fig.
2B). The EC50 of adriamycin in MCF-7/GCS cells
was approximately 11 times greater than the EC50 observed in MCF-7 cells (4.01 ± 0.12 versus 0.37 ± 0.01 µM, p < 0.0005, Fig. 2C).
However, the EC50 in the TC group was nearly identical with
that of MCF-7 cells, and there was no statistical difference between
the two groups (Fig. 2C). The EC50 of
C6-ceramide in MCF-7/GCS cells was 4-fold greater than that
observed in MCF-7 cells (12.07 ± 1.50 versus 3.10 ± 0.50 µM, p < 0.0005, Fig.
2C), and survival of TC cells was not statistically
different from that of the parent cell line, MCF-7 (Fig.
2C).

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Fig. 2.
Adriamycin and ceramide toxicity in MCF-7 and
in GCS-transfected MCF-7/GCS cells. A, cytotoxicity of
adriamycin. After a 48-h incubation with doxycycline (3 µg/ml),
MCF-7/GCS cells were seeded into 96-well plates and treated the
following day with adriamycin at increasing concentrations in 5% FBS
RPMI 1640 medium. After a 96-h exposure, cell survival was determined.
MCF-7/GCS cells cultured with doxycycline and without adriamycin served
as control. The wild type MCF-7 cells were treated without doxycycline
and with the adriamycin concentrations shown. Data represent the
mean ± S.D. of six replicates from three independent experiments.
*, p < 0.001 compared with MCF-7 cells. B,
cytotoxicity of ceramide. The same conditions as cited above were
employed, and C6-ceramide was used in place of adriamycin.
*, p < 0.01; **, p < 0.001 compared
with MCF-7 cells. C, EC50 of adriamycin and
ceramide. * p < 0.001 compared with MCF-7 cells.
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Regulation of Ceramide Resistance in MCF-7/GCS Cells--
If
ceramide resistance is induced by GCS expression in MCF-7/GCS cells,
the resistance response should be tightly correlated with the level of
the inducer, doxycycline. We found that increasing doxycycline
concentrations correlated closely with increased expression of GCS,
which in turn correlated well with increased resistance of the cells to
C6-ceramide. After cells were exposed to increasing concentrations of doxycycline, higher expression of GCS mRNA was observed in MCF-7/GCS cells with 1.0 and 3.0 µg/ml doxycycline (Fig.
3A); the densities were 97 and
256, respectively (GCS band/28 S RNA × 100). In contrast, the
mRNA was scarcely detectable at 0 and 0.1 µg/ml doxycycline, with
densities measuring 16 and 18, respectively. Only traces of GCS
mRNA were found in MCF-7 cells treated with doxycycline (Fig.
3A). GCS activity in MCF-7/GCS cells exposed to 0.1, 1.0, and 3.0 µg/ml doxycycline was significantly higher than GCS activity
observed in MCF-7 cells (p < 0.001, Fig. 3B). The r of GCS activity to doxycycline in
MCF-7/GCS cells was 0.84. In contrast, increasing amounts of
doxycycline did not elevate GCS activity in MCF-7 cells, and the
r was 0.48. In concert with enhanced GCS activity, ceramide
cytotoxicity in MCF-7/GCS cells was reversed following target gene
expression by exposing cells to increasing concentrations of
doxycycline. Treatment with C6-ceramide (5 µM) in conjunction with doxycycline dose escalation
effected a dose-dependent increase in survival of MCF-7/GCS
cells, and the survival was significantly higher than that of MCF-7
cells (p < 0.001, Fig. 3C). MCF-7/GCS cell
survival upon exposure to ceramide was highly correlated with the
concentration of doxycycline in the pretreatment regimen
(r = 0.84 at 0.1-3.0 µg/ml doxycycline). In
comparing Fig. 3C with Fig. 3B, it is seen that
the increase in cell survival mirrored the induction of GCS activity.
The correlation coefficient for these biological parameters was 0.99, verifying that cell survival is closely associated with GCS
activity.

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Fig. 3.
Doxycycline-induced GCS activity and ceramide
resistance coincide with regulated expression of GCS mRNA.
A, regulated expression of GCS mRNA (Northern blot).
MCF-7 and MCF-7/GCS cells were incubated for 96 h with the
indicated concentrations of doxycycline (dox) (µg/ml).
Total RNA (15 µg/lane) was subjected to agarose-formaldehyde
electrophoresis. After transfer, filters were hybridized with GCS
cDNA probe. Ethidium bromide-stained RNA (28 S) was used as a
control for even loading. Densities of GCS/28 S RNA (×100) in
MCF-7/GCS cells were 16, 18, 97, and 256 at 0, 0.1, 1.0, and 3.0 µg/ml doxycycline, respectively. B, dose-response of GCS
activity to doxycycline. Cells were incubated for 96 h in medium
containing the indicated concentrations of doxycycline. GCS activity
was analyzed by radioenzymatic assay. *, p < 0.01; **,
p < 0.001 compared with MCF-7 cells. C,
doxycycline-induced resistance to ceramide. Cells were pretreated with
the indicated concentrations of doxycycline for 48 h, seeded in
96-well plates, and treated the following day with 5 µM
C6-ceramide in RPMI 1640 medium containing 5% FBS. Cell
viability was determined after 96 h. Data represent the mean ± S.D. of six replicates from two independent experiments. Control
cells were cultured in medium without C6-ceramide. *,
p < 0.001 compared with MCF-7 cells; **,
p < 0.001 compared with MCF-7 cells exposed to 1.0 µg/ml doxycycline.
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Adriamycin Induced Hyperglycosylation of Ceramide in MCF-7/GCS
Cells--
To further define the mechanism of drug resistance in
MCF-7/GCS cells, cells were challenged with adriamycin, and the
metabolism of ceramide was evaluated (Fig.
4). As illustrated in Fig. 4A, after 24- and 48-h exposures to adriamycin, ceramide levels in MCF-7
cells increased 3.4- and 3-fold, respectively; however, in
counterpoint, ceramide levels in response to adriamycin in MCF-7/GCS
cells increased 1.4- and 1.2- fold at 24 and 48 h, respectively. Examination of GC metabolism (Fig. 4B) shows that whereas
adriamycin had little impact on GC levels in MCF-7 cells, a
time-dependent increase in GC was observed in MCF-7/GCS
cells exposed to adriamycin. After 24 and 48 h with adriamycin, GC
levels in the GCS-transfected cells increased 1.4- and 2.1-fold,
respectively.

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Fig. 4.
Ceramide metabolism in MCF-7 and in MCF-7/GCS
cells in response to treatment with adriamycin. Cells were exposed
to adriamycin (1. 7 µM) for the times indicated and
radiolabeled with [3H]palmitic acid. Ceramide and GC were
resolved by TLC of the total cellular lipid extract, and quantitation
of tritium was by liquid scintillation spectroscopy. A,
influence of adriamycin on ceramide metabolism in MCF-7 and in
MCF-7/GCS cells. **, p < 0.01 compared with MCF-7/GCS
cells treated with adriamycin (Adr). B, influence
of adriamycin on GC metabolism in MCF-7 and MCF-7/GCS cells. *,
p < 0.05; **, p < 0.01 compared with
MCF-7 cells treated with adriamycin.
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To exclude the possibility that allied factors, such as P-glycoprotein
or Bcl-2, were responsible for conferring ceramide and adriamycin
resistance in the transfected cells, the expression of P-glycoprotein
and Bcl-2 was measured. Western blot analysis showed that
P-glycoprotein was not detected in either MCF-7/GCS or in MCF-7 cells
(Fig. 5A), regardless of the
absence or presence of doxycycline. Therefore, transfection and
inducible expression of GCS did not influence P-glycoprotein levels in
MCF-7/GCS cells. Western blot analysis also shows that the
phosphorylation/dephosphorylation of Bcl-2 was the same in MCF-7 and in
MCF-7/GCS cells, regardless of the absence or presence of doxycycline
(Fig. 5B).

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Fig. 5.
P-glycoprotein and Bcl-2 expression in MCF-7
and in MCF-7/GCS cells. After cell culture without ( ) or with
(+) the indicated concentrations of doxycycline for 72 h,
detergent-soluble cellular protein was isolated and subjected to
SDS-polyacrylamide gel electrophoresis (50 µg/lane). Proteins were
transferred to nitrocellulose, and immunoblots were incubated with
antibody. A, P-glycoprotein (P-gp) Western blot.
The immunoblots were incubated with C219, a monoclonal antibody against
human P-glycoprotein. When doxycycline (dox) was present,
the concentration was 3 µg/ml in the culture medium. MCF-7
adriamycin-resistant (AdrR) cells were used as
P-glycoprotein positive controls. B, Bcl-2 Western blot. The
immunoblot was incubated with Bcl-2 (Ab-1), a monoclonal antibody
against human Bcl-2. The phosphorylated Bcl-2 (top band) and
dephosphorylated Bcl-2 (bottom band) localized at ~25 kDa.
Doxycycline (dox) was present at the indicated concentration
(µg/ml).
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 |
DISCUSSION |
We have introduced GCS cDNA into MCF-7 cells and characterized
the resulting MCF-7/GCS cell line. MCF-7/GCS cells highly expressed GCS
mRNA (Fig. 1B) and demonstrated increased GCS enzymatic
activity (Fig. 1A). We show that GCS modulates drug
resistance in human breast cancer cells. Stable expression of GCS
induced adriamycin resistance in MCF-7/GCS cells. Ceramide resistance,
also a property of the transfected cells, was highly correlated with
induced expression of GCS. MCF-7/GCS cells represent a novel model to
study the role of GCS in cell differentiation, programmed cell death,
and resistance to anticancer agents that cause ceramide production (9,
11, 13).
Doxycycline-regulated gene expression is different from the
tetracycline-regulated systems in the parameters of control. In contrast with the Tet-off vector, target gene expression in the Tet-on
system is positively correlated with the amount of doxycycline in the
medium (22). MCF-7/GCS cells represent the first cell line that has
been developed from MCF-7 cells using the retroviral Tet-on expression
system. We found, at concentrations >2 µg/ml, that doxycycline was
slightly cytotoxic in MFC-7 cells but had no influence on MCF-7/GCS
cells. During transient transfection with pTRE-Luc and stable
transfection with pTRE-GCS, doxycycline exerted tight regulation on
target gene expression. We obtained one subclone of MCF-7 Tet-on cells
in which rtTA expression was increased 100-fold by doxycycline. In
MCF-7/GCS cells, maximum expression of the GCS gene occurred after
48 h with doxycycline (3 µg/ml).
Previous work from our laboratory has demonstrated that MDR is
associated with an accumulation of GC and that reversal of drug
resistance by MDR modulators is accompanied by a decrease in the
cellular GC component (12, 13, 20, 21). Based on these data, we
believed the GC pathway to be an alternative mechanism of drug
resistance and that increased expression of GCS would be a key step in
this biological event. MCF-7 cells are sensitive to anticancer drugs,
ionizing radiation, TNF-
, and CD95 (7, 11-13). After introduction
and expression of GCS, MCF-7/GCS cells were resistant to adriamycin and
ceramide. The EC50 for adriamycin and ceramide was elevated
11- and 5-fold, respectively. Moreover, ceramide resistance was closely
correlated with the induced expression of GCS mRNA and GCS enzyme
activity. Several studies have shown that the effects of therapeutic
doses of adriamycin and daunorubicin are related to the elevation of
cellular ceramide (9, 11-13). In this study, we confirmed that
adriamycin treatment elicits an increase in cellular ceramide and that
induced GCS activity catalyzes the removal of ceramide via
glycosylation (Fig. 4). Adriamycin resistance conferred by GCS
transfection and expression gives substantially direct evidence for the
relationship of ceramide to anthracycline action.
Among the causes of multidrug resistance, P-glycoprotein is the most
widely studied (32, 33). P-glycoprotein is an ATP-dependent transporter involved in removal of drug from the cells by efflux pumping (32, 33). It is highly expressed in MCF-7 adriamycin-resistant cells; however, we did not observe elevation of P-glycoprotein in
MCF-7/GCS. This implies that in MCF-7/GCS cells, adriamycin resistance
induced by GCS was independent of P-glycoprotein. In the ceramide
signal transduction pathway of apoptosis, increased Bcl-2, especially
dephosphorylated Bcl-2, has strong anti-apoptosis effects (31, 34-37).
As analyzed by Western blot, we did not find a significant alteration
in Bcl-2 expression and/or phosphorylation/dephosphorylation status in
MCF-7/GCS cells. Thus, drug resistance in MCF-7/GCS cells is also
divorced from Bcl-2 involvement.
Apoptosis can be induced by cytokines, ionizing radiation, and
anticancer agents (4, 5). Ceramide has been shown to mediate apoptosis,
acting a cellular second messenger in myriad signal transduction
cascades (3-5). Extracellular agents, such as calcitriol, TNF-
,
-interferon, and interleukin-1 promote ceramide production by
hydrolysis of sphingomyelin (4-6). Anthracycline anticancer drugs and
the cyclosporine MDR modulator SDZ PSC 833 also mediate cell killing
through enhancing the generation of cellular ceramide (9, 11-13, 38,
39). Cell-permeable ceramide analogs can directly induce apoptosis in
U937, P388, HL-60, MCF-7, and BL 30A Burkitt's lymphoma cells (6, 9,
11, 13, 40, 41). Ionizing radiation also elicits ceramide production
and initiates apoptosis (6, 8, 10, 42); however, loss of ceramide
production has been shown to confer resistance to radiation-induced apoptosis (6, 8, 10). Ceramide glycosylation catalyzed by GCS is an
effective mechanism for lowering elevations in ceramide caused by
exogenous agents. GCS activity contributes to lessening the potential
cytotoxic effects of high ceramide, as shown in the experiments in
which MCF-7/GCS cells were challenged with either adriamycin or
C6-ceramide.
In addition to accumulation in MDR cells, GC has been shown to exert a
regulatory role in cell proliferation (43-46). Administration of
conduritol B epoxide, a
-glucocerobrosidase inhibitor, causes an
elevation in cellular GC and tissue hyperplasia in mice (43, 44).
Intraperitoneal or intracutaneous injection of emulsified GC into mice
induces liver hypertrophy and epidermal proliferation (43, 44). These
data suggest that elevated GC stimulates hepatocyte proliferation and
epidermal mitogenesis through a relatively direct mechanism. Lowering
of GC by use of the GCS inhibitor,
D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, or by inhibiting GC hydrolysis results in proliferation of Madin-Darby canine kidney and other cell types (45). Depletion of endogenous GC
causes a cell cycle arrest (46). Only a slight increase in GC was
observed in MCF-7/GCS cells compared with MCF-7 cells. This suggests
that GC does not contribute to drug resistance; however, we do not know
the exact role of GC in adriamycin resistance exhibited in MCF-7/GCS
cells. With the direct evidence presented in this paper, we postulate
that GCS activity is one of the causes of cellular resistance to
adriamycin and resistance to ceramide. The glycosylation of ceramide,
in response to de novo generation of ceramide elicited by
adriamycin treatment, attenuated the cytotoxic response.