From the Center of Excellence on Neurodegenerative Diseases, Study
Center for the Biochemistry and Biotechnology of Glycolipids,
Department of Medical Chemistry, Biochemistry and Biotechnology,
University of Milan, 20090 Segrate, Italy and the
Department of Experimental Oncology, Istituto Nazionale
Tumori, 20133 Milan, Italy
Received for publication, July 19, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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In the present work, we studied the effects of
fenretinide (N-(4-hydroxyphenyl)retinamide (HPR)), a
hydroxyphenyl derivative of all-trans-retinoic acid, on
sphingolipid metabolism and expression in human ovarian carcinoma A2780
cells. A2780 cells, which are sensitive to a pharmacologically
achievable HPR concentration, become 10-fold more resistant after
exposure to increasing HPR concentrations. Our results showed that HPR
was able to induce a dose- and time-dependent increase in
cellular ceramide levels in sensitive but not in resistant cells. This
form of resistance in A2780 cells was not accompanied by the
overexpression of multidrug resistance-specific proteins MDR1
P-glycoprotein and multidrug resistance-associated protein,
whose mRNA levels did not differ in sensitive and resistant A2780
cells. HPR-resistant cells were characterized by an overall altered
sphingolipid metabolism. The overall content in glycosphingolipids was
similar in both cell types, but the expression of specific
glycosphingolipids was different. Specifically, our findings
indicated that glucosylceramide levels were similar in sensitive and
resistant cells, but resistant cells were characterized by a 6-fold
lower expression of lactosylceramide levels and by a 6-fold higher
expression of ganglioside levels than sensitive cells. The main
gangliosides from resistant A2780 cells were identified as GM3 and GM2.
The possible metabolic mechanisms leading to this difference were
investigated. Interestingly, the mRNA levels of glucosylceramide
and lactosylceramide synthases were similar in sensitive and resistant
cells, whereas GM3 synthase mRNA level and GM3 synthase activity
were remarkably higher in resistant cells.
Sphingolipid metabolism plays a pivotal role in the mechanism of
apoptosis induced in tumor cells. Ceramide, produced under physiological (tumor necrosis factor Interestingly, in tumor cell lines, resistance to chemotherapeutic
treatments is often associated with an increased ability of the cell to
glycosylate ceramide, as a consequence of a higher activity of
glucosylceramide synthase (3-12). High levels of
GlcCer1 (6-9, 11, 12) and of
glucosylceramide synthase activity (5, 8, 9) and/or expression (8, 9,
11, 12) were detected in a number of drug-resistant cancer cell lines and in specimens from cancer patients not responding to chemotherapy treatment (13). GlcCer accumulation results in the ability of drug-resistant cells to scavenge ceramide, thus preventing
ceramide-induced apoptotic death. Multidrug resistance (MDR), a
drug-resistant phenotype characterized by the ability of tumor cells to
become insensitive to a variety of chemically unrelated
chemotherapeutics after exposition to one single drug, is hallmarked by
the overexpression of energy-dependent drug efflux pump
proteins, such as MDR1 P-glycoprotein (MDR1) and multidrug
resistance-associated protein (MRP) (3, 14). Extensive studies by
Cabot's group lead to the conclusion that elevated GlcCer levels are a
specific marker for MDR phenotype in cancer cells and that modifying
ceramide metabolism might represent a winning strategy to overcome drug
resistance in tumor cells (4-11).
On the other hand, it has been shown (12, 15) that GlcCer accumulation
is not the only consequence of an altered sphingolipid metabolism in
MDR cancer cells. In MDR human ovarian carcinoma cells, SM and
galactosylceramide levels are also higher respect to parental sensitive
cells, whereas LacCer and all of the more complex GSLs are present in
lower amounts. These data can be at least in part interpreted in the
scenario described above. However, in addition to this it is necessary
to recall that sphingolipids and sphingolipid metabolites other than
ceramide have biological activities that could be responsible for the
acquisition of a drug resistance phenotype (16-18).
Retinoids are natural and synthetic derivatives of vitamin A that play
a critical role in different biological processes including morphogenesis in the embryo, cell proliferation, differentiation, and
apoptosis (19). Retinoids exert their effects by regulating gene
expression through two classes of nuclear receptors: retinoic acid (RA)
receptors 4-[3,7-Dimethyl-9-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,
6,8- nonatetraenamido]-1-hydroxybenzene, known as
N-(4-hydroxyphenyl)retinamide (HPR), pharmacologically known
as fenretinide, is a synthetic amide of all-trans-RA, which
has shown reduced toxicity relative to RA while maintaining a
significant biological activity (22). HPR is under investigation in
clinical trials as preventive and therapeutic agent and has already
shown a preventive effect for ovarian and breast cancers in
premenopausal women (23, 24) as well as chemopreventive and therapeutic
efficacy against different tumors in animal models (22). In
vitro studies have demonstrated that HPR has significant
antiproliferative activity associated with induction of apoptosis in
several tumor cell types (22). To date, the mechanism of action of HPR
is poorly understood. Some studies suggest that the effects of this
retinoid are mediated through RA receptor and retinoid X receptor
signaling (25-28). Other studies have shown that apoptosis in response
to HPR primarily occurs by a receptor-independent mechanism, which is
accompanied by generation of reactive oxygen species (29, 30) or
increases in ceramide (31, 32).
Discontinuation of retinoid treatment leads to recurrence of the
lesion. However, as occurs with chemotherapeutic agents, continuous
retinoid exposure might cause development of drug resistance. So far,
only few in vitro models had been developed to investigate mechanisms and molecular characteristics associated with retinoid resistance (33, 34). In a recent study, we hypothesized that continuous
HPR treatment might lead to resistance to the retinoid. We showed that
when A2780 human ovarian carcinoma cells, which are very sensitive to
HPR (35), were continuously exposed to the drug, they developed a
10-fold resistance to the drug (34). Differences in HPR uptake and
metabolism were observed between sensitive and resistant cells (34).
HPR intracellular peak levels were 2 times lower, and a polar
metabolite, not detected in sensitive cells, was found in cell extracts
from resistant cells. Moreover, the development of HPR resistance was
associated with changes in marker expression, suggestive of a more
differentiated status (34). The expression of RA receptor An increase in the cellular levels of ceramide upon HPR treatment was
reported in neuroblastoma (31, 37) and breast cancer cell lines (32).
In the present work, we studied the possible involvement of ceramide in
HPR-induced apoptosis in A2780 human ovarian carcinoma cells. Moreover,
we assessed whether resistance to HPR was associated with modifications
of sphingolipid patterns and metabolism in these cells.
Chemicals--
Commercial chemicals were the purest available,
common solvents were distilled before use, and water was doubly
distilled in a glass apparatus. HPR, obtained from Dr. J. A. Crowell, Division of Cancer Prevention, NCI, National Institutes of
Health (Bethesda, MD) was dissolved in Me2SO at a
concentration of 10 mM and stored at Lipids and Radioactive Lipids--
Sphingolipids and
glycerolipids to be used as standards were extracted from rat brain,
purified, and characterized (39). Gangliosides were extracted from
bovine brain and purified by partitioning (39).
GM12 was prepared from the
bovine brain ganglioside mixture by enzymatic treatment with C. perfringens sialidase (40). GM3 was prepared from GM1 using the
GM1-lactone hydrolysis procedure (41). Lactosylceramide was prepared
from GM3 by enzymatic treatment with Vibrio cholerae sialidase (42). Sphingosine was prepared from cerebroside (43). [1-3H]sphingosine was prepared by specific chemical
oxidation of the primary hydroxyl group of sphingosine followed by
reduction with sodium boro[3H]hydride (44) (radiochemical
purity over 98%; specific radioactivity 2.2 Ci/mmol).
N-Acetylsphingosine (C2Cer) and
N-palmitoylsphingosine (C16Cer) were prepared by
N-acylation of sphingosine using acetic anhydride and
hexadecanoic anhydride, respectively (45). The same procedure applied
to [1-3H]sphingosine was used to synthesize
tritium-labeled N-palmitoylsphingosine (radiochemical purity
over 99%; specific radioactivity 2.2 Ci/mmol). GM3 tritium labeled at
the position 3 of sphingosine,
[3-3H(sphingosine)]GM3, was prepared by the
dichlorodicyanobenzoquinone-sodium borohydride method (46), and the
natural erythro diastereoisomer was purified by reverse
phase HPLC as described (47) (radiochemical purity 98%, specific
radioactivity 1.2 Ci/mmol).
[3-3H(sphingosine)]LacCer was prepared from
[3-3H(sphingosine)]GM3 by enzymatic treatment
with V. cholerae sialidase and purified by silica gel column
chromatography (radiochemical purity over 99%, specific radioactivity
1.2 Ci/mmol). [3H]lipids used as chromatographic
standards were prepared from [1-3H]sphingosine-fed cell
cultures as previously described (48).
Cell Cultures--
An HPR-resistant cell line, A2780/HPR, was
developed from parental A2780 cells by in vitro incubation
of A2780 cells with increasing concentrations of HPR as previously
described (34). Briefly, cells surviving 60 rounds of selections in
HPR-containing medium (3 rounds at 1 µM, 11 rounds at 2 µM, 8 rounds at 3 µM, and 38 rounds at 5 µM) were cloned by limiting dilution. One clone (A2780/HPR) was expanded and when tested for HPR sensitivity
demonstrated a 10-fold resistance to HPR, which was slightly reversible
upon drug removal for five rounds (34). Therefore, A2780/HPR cells were
continuously maintained in 5 µM HPR and seeded without
the drug in all of the experiments. A2780 and A2780/HPR cells were grown in monolayer in RPMI 1640 medium (Biowhittaker) containing 10%
fetal bovine serum (Invitrogen) in 5% CO2 at
37 °C.
Effects of C2Cer on A2780 and A2780/HPR
Cells--
Cells were seeded at density of 10,000 cells/well in
96-well tissue culture plates, treated on the next day with different concentrations of C2Cer in the absence of fetal bovine serum, and
incubated for one additional day. Control cultures received the same
amount of ethanol as the treated cultures (0.1%). Cell number was
estimated by using the sulforhodamine B assay (49). Two analyses were
performed, and four replicate wells were used for each analysis.
Apoptotic cells were identified by the terminal dUTP nick-end labeling
(TUNEL) method using an in situ cell death detection kit
(Roche Diagnostics) according to the manufacturer's instructions.
Briefly, A2780 and A2780/HPR cells were treated 24 h after seeding
with 2.5 µM C2Cer in the absence of fetal bovine serum,
incubated for one additional day, harvested, and washed twice in
phosphate-buffered saline. After centrifugation, cells were fixed in
2% paraformaldehyde for 1 h at 26 °C and permeabilized with
0.1% Triton X-100 and 0.1% sodium citrate in phosphate-buffered saline. After washing, the cells were resuspended in TUNEL reaction mixture containing fluorescein isothiocyanate-dUTP and terminal deoxynucleotidyltransferase. Control cells were suspended in the TUNEL
reaction mixture containing fluorescein isothiocyanate-dUTP without
terminal deoxynucleotidyltransferase, and incubations were performed
for 1 h at 37 °C before washing the cells twice. The number of
TUNEL-positive cells, as detected by fluorescent microscopy, was
assessed on at least 100 cells in two different smears and referred to
the whole cell population.
Northern Analysis--
Total cellular RNA was isolated using the
TriReagent method (Molecular Research Center, Cincinnati, OH). Twenty
µg of total RNA were fractionated in formaldehyde/Mops-agarose gel
and blotted on Hybond N+ nylon membranes (Amersham Biosciences).
Filters were hybridized with cDNA probes for MDR1, MRP, GlcCer
synthase, LacCer synthase, and GM3 synthase, obtained by RT-PCR using
A2780/HPR cDNA as template. Briefly, single-stranded cDNA was
synthesized from 2 µg of RNA by reverse transcription and then
amplified by PCR. The primer pairs used for PCR amplification are the
following: MDR1, sense (5'-AAAAAGATCAACTCGTAGGAGTG-3') and antisense
(5'-GCACAAAATACACCAACAA-3') (161-bp amplified product); MRP, sense
(5'-CCACCTCCTCATTCGCATCCACCTTG-3') and antisense
(5'-GGAAACCATCCACGACCCTAATCCCT-3') (296-bp amplified product);
GlcCer synthase, sense (5'-TCTACACCCGATTACACCTC-3') and antisense
(5'-TGGGATAATCCAATTCAAAGAAT-3') (147-bp amplified product); LacCer
synthase, sense (5'-AAAAACAGTACGCTCAACGG-3') and antisense
(5'-ATCATCTTCTCCTCCCCATC-3') (676-bp amplified product); GM3 synthase,
sense (5'-AAAAATGCATTATGTGGACCC-3') and antisense (5'-GTCTTGGCTTTCAAGTGTTCA-3') (303-bp amplified product); human glyceraldehyde-3-phosphate dehydrogenase, sense
(5'-CCATGGAGAAGGCTGCGC-3') and antisense
(5'-CAAAGTTGTCATGGATGACC-3') (195-bp amplified product). The
cycling program was 94 °C for 30 s, 53 °C for 30 s, and
72 °C for 60 s for 35 cycles. PCR fragments were labeled with
[ Treatment of Cell Cultures with
[3H]Sphingosine--
24 h after seeding, cells were
incubated in the presence of 3 × 10 Treatment of [3H]Sphingosine-labeled Cell Cultures
with HPR--
After metabolic labeling of cell lipids with
[1-3H]sphingosine, at the end of a 24-h chase, the medium
was replaced with medium containing HPR at final concentrations of 5 and 10 µM for up to 72 h. Time-matched control cells
received the same amount of Me2SO as HPR-treated cells
(0.1%).
Lipid Extraction and Determination--
In the experiments
described above, at the end of the treatment periods, cells adherent to
the dishes were harvested in ice-cold water (2 ml) by scraping with a
rubber policeman. Cells floating in the culture medium were collected
by centrifugation. Adherent and floating cells were analyzed to
determine the content of radioactivity associated with lipids. Samples
were lyophilized, and lipids were extracted twice with
chloroform/methanol 2:1 by volume (first extraction, 1.5 ml; second
extraction, 0.25 ml) (51). The total lipid extracts were subjected to a
two-phase partitioning as previously described (52), resulting in the
separation of an aqueous phase containing gangliosides and in an
organic phase containing all other lipids. Aliquots of total lipid
extracts, aqueous and organic phases were analyzed by HPTLC as
described below, followed by radioactivity imaging for quantification
of radioactivity.
The identity of radioactive lipids separated by HPTLC (using HPTLC
Silica Gel 60 plates from Merck) was assessed by comigration with
standard lipids and confirmed by susceptibility of compounds to the
following enzymatic and chemical treatments (51). A sample of the
aqueous phase was treated at 37 °C for 2 h, in 50 µl of water, in the presence of 1 milliunit of V. cholerae
sialidase, to yield GM1. For the identification of SM, a sample of the
organic phase was treated at 37 °C overnight in 30 µl of 100 mM Tris-HCl, pH 7.4, 0.5 mM MgCl2,
0.05% sodium deoxycholate in the presence of 11 milliunits of
Staphylococcus aureus sphingomyelinase to yield ceramide; PE
contained in the organic phase was characterized following its
degradation under alkaline conditions. The enzymatic or chemical
reaction mixtures were separated by HPTLC, and the reaction products
were identified by chromatographic comparison with standard lipids.
The total lipid extract from about 50 mg of cell proteins was analyzed
for mass lipid content. The phospholipid content was determined in the
organic phase as phosphate after perchloric acid digestion by the
method of Bartlett (53). Gangliosides from the aqueous phase and
phospholipids and cholesterol from the organic phase were separated by
HPTLC as described below. Identification of lipids after separation was
assessed by comigration with standard lipids and confirmed by their
susceptibility to enzymatic and chemical treatments as previously
described (51). After chromatographic separation, compounds were
chemically detected, and their amount was determined by densitometry as
described below. Gangliosides from the aqueous phase were characterized
by HPLC-ESI-MS (46) (see below).
GM3 Synthase Assay--
Cells cultured in 100-mm dishes as
described above were harvested using a plastic scraper and washed two
times with phosphate-buffered saline. GM3 synthase activity was assayed
as previously described (54, 55) with some modifications. Cells were
resuspended in 150 mM sodium cacodylate-HCl buffer, pH 6.6 (20 mg of cell protein/ml) with protease inhibitors (2 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 0.0016 mM
aprotinin, 0.044 mM leupeptin, 0.08 mM
bestatin, 0.03 mM pepstatin A, 0.028 mM E-64)
(Sigma) and homogenized with a Dounce homogenizer (10 strokes, tight).
In each reaction tube, 10 µl of Triton CF-54 1.5% (v/v) in
chloroform/methanol (2:1) were mixed with 0.5-50 nmol of
[3-3H(sphingosine)]LacCer (corresponding to 45 nCi) from a stock solution in chloroform/methanol (2:1) and dried under
N2. To this mixture, 8 µl of 750 mM sodium
cacodylate-HCl buffer, pH 6.6, 4 µl of 125 mM
MgCl2, 4 µl of 125 mM 2-mercaptoethanol, 10 µl of 5 mM CMP-NeuAc, and 10 µl of cell homogenate
(containing 200 µg of protein) were added in a total reaction volume
of 50 µl. Negative controls were performed using heat-inactivated
cell homogenates (100 °C for 3 min). The incubation was performed at
37 °C for 3 h with continuous shaking. The reaction was stopped
by adding 1.5 ml of chloroform/methanol (2:1). The reaction mixture was
analyzed by HPTLC using the solvent system chloroform/methanol/water
(55:20:3 by volume). Radioactive lipids were detected and quantified by
radioactivity imaging as described below.
Ceramidase Assay--
Cell homogenates were prepared
as described for the GM3 synthase assay, using 20 mM
Tris-HCl, pH 7.4, as homogenization buffer. The activity of acidic,
neutral, and alkaline ceramidase was assayed as previously described
(56, 57) with some modifications. In each reaction tube, 25 µl of
0.1% Triton-X 100 (v/v) in chloroform/methanol (2:1) and 25 µl of
0.2% sodium cholate (w/v) in chloroform/methanol (2:1) were mixed with
2-100 pmol of [1-3H(sphingosine)]C16Cer
(corresponding to 4.5 nCi) from a stock solution in chloroform/methanol
(2:1) and dried under N2. To this mixture, 7.5 µl of
water was added, and the tubes were sonicated for 3 min, heated for
5 s at 80 °C, and then put on ice. Ceramidase activities were
assayed by adding 12.5 µl of 400 mM sodium acetate buffer, pH 4.5 (for the acidic enzyme), 12.5 µl of 400 mM
Tris-HCl, pH 7.4 (for the neutral enzyme), or 12.5 µl of 400 mM glycine-NaOH, pH 9.0 (for the alkaline enzyme), 5 µl
50 mM MgCl2, and 10 µl of cell homogenate
(containing 200 µg of protein) in a total reaction volume of
50 µl. Negative controls were performed using heat-inactivated cell
homogenates (100 °C for 3 min). The incubation was performed at
37 °C for 1 h with continuous shaking. The reaction was stopped by adding 1.5 ml of chloroform/methanol (2:1). The reaction mixture was
analyzed as described for GM3 synthase assay.
Analytical Procedures--
Lipids were separated by
monodimensional and two-dimensional HPTLC carried out with the
following solvent systems: chloroform/methanol/0.2% aqueous
CaCl2 (55:45:10 by volume) to separate ceramide from
sphingosine; chloroform/methanol/0.2% aqueous CaCl2
(50:42:11 or 55:45:10 by volume) and chloroform/methanol/0.2% aqueous
CaCl2/32% NH3 (60:50:9:1 by volume) to analyze
total lipids and gangliosides; chloroform/methanol/water (55:20:3 by
volume) to analyze total lipids and lipids from the organic phase and
to separate GM3 from LacCer; and 1-butanol/acetic acid/water
(3:1:1 by volume) and hexane/chloroform/acetone/acetic acid (10:35:10:1
by volume) to analyze ceramide. Endogenous phospholipids from organic
phase were separated by mono- or bidimensional HPTLC using the
following solvent systems and conditions (51):
chloroform/methanol/acetic acid/water (30:20:2:1 by volume) to analyze
glycerophospholipids and SM; chloroform/methanol/acetic acid/water
(30:20:2:1 by volume) first run and second run with intermediate
exposure to HCl vapors for 15 min to analyze plasmalogens of
glycerophospholipids. The solvent system chloroform/methanol/water
(55:40:3 by volume) was used to analyze neutral GSLs and SM from
organic phase after alkaline treatment. Hexane/diethylether/acetic acid
(80:20:1 by volume) and hexane/ethyl acetate (3:2 by volume) were used
to analyze cholesterol.
Ganglioside, neutral GSL, and phospholipid species were quantified
after separation on HPTLC followed by specific detection with a
p-dimethylaminobenzaldehyde reagent (58), an
aniline/diphenylamine reagent (59), or a molybdate reagent (60),
respectively. The relative amounts of each ganglioside, neutral GSL, or
phospholipid were determined by densitometry. The mass content of each
phospholipid was calculated on the basis of the percentage distribution
and total phospholipid content, determined as described above. The quantity of each ganglioside, neutral GSL, or SM was determined by
densitometry and comparison with known amounts of standard compounds
using the Molecular Analyst program (Bio-Rad). Cholesterol was
quantified by visualization with 15% concentrated sulfuric acid in
1-butanol (48). The quantity of cholesterol was determined by
densitometry and comparison with 0.1-2 µg of standard compounds.
Radioactive lipids were detected and quantified by radioactivity
imaging performed with a Beta-Imager 2000 instrument (Biospace, Paris,
France) using an acquisition time of about 48 h. The radioactivity associated with individual lipids was determined with the specific
The radioactivity associated with cells, lipids, lipid extracts, and
aqueous or organic phases was determined by liquid scintillation counting.
Structural characterization of gangliosides has been carried out using
a ThermoQuest Finnigan LCQDeca mass spectrometer (FINNIGAN MAT,
San Jose, CA) equipped with an ESI ion source and a Xcalibur data
system and connected to an HPLC TSP P4000 quaternary pump and an
automatic solvent degasser. Optimum conditions included sheath gas flow
of 70 arbitrary units, auxiliary gas flow of 10 arbitrary units, spray
voltage of 4 kV, capillary voltage of
HPLC separation of gangliosides dissolved in CH3CN/5
mM ammonium acetate buffer, pH 7 (64:36 by volume), was
carried out on a 5-µm LiChrospher C8 reverse-phase column (250 × 4 mm) (Merck) at a flow rate of 0.5 ml/min using a gradient formed
by the solvent system A, consisting of CH3CN, 5 mM ammonium acetate buffer, pH 7 (15:85 by volume), and
solvent system B containing CH3CN/H2O (85:15 by
volume). The gradient was linear from 30:70 to 20:80, by volume, of A:B
over 25 min and then to 100 of B in 5 min.
The protein content was determined according to Lowry (61), using
bovine serum albumin as the reference standard.
Effect of HPR on Ceramide Levels in A2780 Cells--
The possible
involvement of ceramide in the effect of HPR in A2780 human ovarian
carcinoma cells was investigated. To this purpose, A2780 and A2780/HPR
cells were incubated in the presence of [1-3H]sphingosine
for 2 h, followed by a 24-h chase. [1-3H]sphingosine
is efficiently incorporated by cultured cells and converted into
ceramide, which is further utilized for the synthesis of more complex
sphingolipids. This metabolic radiolabeling approach was successfully
used in the past as a valuable tool to quantitatively and sensitively
determine changes in cellular ceramide levels elicited by different
treatments, including RA (62) and HPR (32). Cells were then treated
with 5 and 10 µM HPR for 48 and 72 h. As previously
reported (35), treatment of A2780 cells with HPR under these
experimental conditions was able to induce apoptosis, and a relevant
number of cells (25.0 and 60.6% of total cells after treatment with 5 µM HPR for 48 and 72 h, respectively) were detached
from the culture dishes and floating in the culture medium. The same
treatment had only weak effect (3.8 and 5.5% of total cells were
floating after treatment with 5 µM HPR for 48 and 72 h, respectively) on A2780/HPR cells (34). Cell lipids were extracted
from both cell types, separated by HPTLC and analyzed by digital
autoradiography. The values for radioactivity incorporation into
ceramide in control and HPR-treated cells are reported in Fig.
1. Under these experimental conditions,
treatment of A2780 cells with HPR increased the incorporation of
radioactivity from [1-3H]sphingosine into ceramide in
floating cells respect to untreated cells or adherent cells (Fig. 1,
A and B). On the other hand, in the case of
A2780/HPR cells, the incorporation of [1-3H]sphingosine
into ceramide was very similar in adherent and floating cells, treated
with HPR or not (Fig. 1, C and D). HPR treatment of A2780/HPR cells is thus not able to determine a rise in the production of radioactive ceramide.
Effects of C2Cer on A2780 and A2780/HPR Cells--
To
further examine the involvement of ceramide in the HPR effect in A2780
cells, we investigated whether cells resistant to HPR were also
resistant to ceramide. To this aim, the antiproliferative and apoptotic
effect of C2Cer was evaluated in parental and HPR-resistant cells.
After 24 h of exposure to the drug, the sensitivity of the two
cell lines to C2Cer was different (Fig.
2A); doses of C2Cer ranging
from 1 to 5 µM caused ~60% growth inhibition in A2780,
whereas the same doses were ineffective in A2780/HPR cells. No
difference in sensitivity to C2Cer between A2780 and A2780/HPR cells
was observed at doses higher than 7.5 µM, which caused a strong dose-dependent growth inhibition in both cell lines.
TUNEL analysis performed in cells treated with 2.5 µM
C2Cer showed an increase in the percentage of apoptotic cells, compared
with untreated cells, in A2780 cells, whereas no increase was observed
in A2780/HPR cells (Fig. 2B).
Expression Levels of MDR1 and MRP--
In several tumor cell
lines, the loss of ability to respond to anticancer drugs with the rise
of cellular ceramide levels hallmarks the acquisition of the MDR
phenotype. To understand whether HPR resistance in A2780 cells is
accompanied by the overexpression of multidrug resistance-specific
proteins, MDR1 and MRP mRNA expression levels were analyzed in
parental and resistant cells (Fig. 3). No
differences were observed in gene expression between sensitive and
resistant cells; MDR1 mRNA was not detected in A2780 and A2780/HPR cells, whereas MRP1 was expressed in both cell lines at similar levels.
Sphingolipid Metabolism in A2780 and A2780/HPR
Cells--
To study possible differences in sphingolipid patterns
and metabolism, A2780 cells and A2780/HPR cells were incubated in the
presence of [1-3H]sphingosine for 2 h, followed by
different chase times (up to 4 days). As reported for other cell types
(48, 51), under these conditions, all sphingolipids (including
ceramide, SM, neutral glycolipids, and gangliosides) were
efficiently metabolically radiolabeled, reaching a steady-state
distribution of radioactivity after 48 h (data not shown). The use
of [1-3H]sphingosine as metabolic precursor allows the
quantitative analysis of cell sphingolipids with very high sensitivity.
Moreover, during the degradation of the exogenous
[1-3H]sphingosine taken up by the cells or of
[1-3H]sphingosine-containing sphingolipids, radioactive
ethanolamine is formed, which is recycled for the synthesis of PE. The
formation of [3H]PE is thus a very useful and sensitive
tool to quantify sphingolipid catabolism.
Under these experimental conditions, at all investigated chase times,
A2780 and A2780/HPR cells incorporated comparable amounts of
radioactivity (Table I). Cell lipids were
extracted, separated by HPTLC, and analyzed by digital autoradiography.
Fig. 4 shows the patterns of radioactive
lipids extracted from A2780 and A2780/HPR cells after HPTLC separation.
In both cell lines, radioactive bands co-migrating with standard
ceramide, glucosylceramide, PE, lactosylceramide, and SM were
detectable. In the case of A2780/HPR cells, at least two intense
radioactive bands migrating below SM were also present. To allow a
better resolution of these lipid profiles, the total lipid extracts
were subjected to a two-phase partitioning, resulting in the separation
of an aqueous phase containing gangliosides and an organic phase
containing all less polar lipids (including radioactive ceramide, PE,
neutral glycosphingolipids, and SM) that were further analyzed. As
reported in Table I, most radioactive lipids were associated with the
organic phase in both A2780 and A2780/HPR cells. Remarkably,
independently from the chase time, the radioactivity associated with
the aqueous phase in the case of resistant A2780/HPR cells was 3 times
higher than in sensitive A2780 cells.
Fig. 5 shows the patterns of radioactive
lipids present in aqueous (Fig. 5A) and organic phases (Fig.
5B) obtained from A2780 and A2780/HPR cell lipid extracts
after HPTLC separation. The major radioactive bands present in the
aqueous phase from both cell types showed a similar chromatographic
behavior. On the basis of their chromatographic migration and
sensitivity to V. cholerae sialidase treatment, the two
upper bands were identified as GM3, and the two lower bands, accounting
for about 60% of the aqueous phase radioactivity in the case of
A2780/HPR cells, were identified as GM2. In the organic phase from both
cell types (Fig. 5B), ceramide, glucosylceramide,
lactosylceramide, PE, and SM were identified. The quantitative data,
summarized in Table II as radioactivity incorporation into different lipids, indicate that the metabolic use of
[1-3H]sphingosine is different in A2780 and A2780/HPR
cells, thus resulting in quantitatively different
[3H]lipid patterns. In both cell types, a significant
portion of [1-3H]sphingosine-containing lipids underwent
complete degradation, as indicated by the formation of high levels of
radioactive PE (particularly in the case of A2780/HPR cells). In both
cell types, more than 50% of radioactivity from
[1-3H]sphingosine was associated with complex
sphingolipids, including SM and glycosphingolipids. Although the total
radioactivity associated with glycosphingolipids was similar in A2780
and A2780/HPR cells, its distribution among different
glycosphingolipids was radically different in these two cell lines. In
particular, A2780 cells were characterized by high levels of LacCer
(about 6-fold higher than in A2780/HPR), whereas A2780/HPR express
remarkably more elevated (6-fold higher) ganglioside levels than the
parental cell line.
Lipid Composition of A2780 and A2780/HPR Cells--
The
data obtained using metabolic labeling with
[1-3H]sphingosine strongly indicate that, in A2780 cells,
resistance to HPR is linked to an alteration in sphingolipid metabolism
leading to an increased expression of gangliosides. To evaluate
possible alterations in the metabolism of other lipid classes, we
analyzed the mass content of cholesterol and phospholipids in A2780 and A2780/HPR cells. Cholesterol content was 45.20 ± 3.12 and
45.20 ± 2.97 nmol/mg of cell protein in A2780 and A2780/HPR
cells, respectively (Fig. 6A).
Phospholipid phosphorous content was 130.60 ± 5.21 and
118.30 ± 6.13 nmol/mg of cell protein in A2780 and A2780/HPR cells, respectively, and the phospholipid pattern was very similar in
both cell types (Fig. 6B). Thus, A2780 and A2780/HPR cells were almost identical in their bulk lipid composition.
In the case of sphingolipids, colorimetric chemical detection did not
allow an accurate quantification, due to their low cellular content.
However, the qualitative comparison of endogenous neutral glycosphingolipid and ganglioside patterns confirmed the radiolabeling data. Mass spectrometry analyses showed that GM2 and GM3 were the main
components of the ganglioside mixture extracted from A2780/HPR cells.
Both gangliosides were present as molecular species containing
d18:1-16:0, d18:1-18:0, d18:1-24:1, and d18:1-24:0 ceramide. As
shown in Fig. 7, the GM2 and GM3 species
were characterized by the deprotonated molecular ions
[M Expression Levels of GlcCer Synthase, LacCer Synthase, and GM3
Synthase in A2780 and A2780/HPR Cells--
To investigate
potential mechanisms underlying the observed differences in
sphingolipid levels in A2780 and A2780/HPR cells, the mRNA
expression of the enzymes GlcCer synthase, LacCer synthase, and GM3
synthase was evaluated in parental and resistant cells. A2780/HPR cells
showed GlcCer synthase and LacCer synthase mRNA levels similar to
those of A2780 cells, whereas GM3 synthase mRNA was markedly
increased (Fig. 8). In A2780 cells, the
mRNA levels of GM3 synthase were below the detection limit of our
Northern blot analysis. Using RT-PCR, we were able to detect in A2780
cells a faint band relative to GM3 synthase.
To assess whether observed changes in the mRNA levels of GM3
synthase were paralleled by differences in the enzyme levels, we
measured the activity of GM3 synthase in A2780 and A2780/HPR cells by
means of a cell-free assay using radioactive LacCer as substrate. As
indicated in Fig. 9, the in
vitro activity of GM3 synthase was 20-fold higher in resistant
A2780/HPR cells than in sensitive A2780 cells.
Ceramidase Activities in A2780 and A2780/HPR
Cells--
Among the metabolic mechanisms that contribute to the
regulation of cellular ceramide levels, a possible role of ceramidases (enzymes that cleave the N-acyl bond of ceramide) was
recently highlighted (reviewed in Ref. 63). To determine whether
differences in the activity of ceramidases were associated with the
resistance to HPR in human ovarian carcinoma cells, we measured the
in vitro activity of ceramidase at acidic, neutral, and
alkaline pH in A2780 and A2780/HPR cells using
[1-3H(sphingosine)]C16Cer as substrate. As
reported in Table III, the in
vitro activities of acid, neutral and alkaline ceramidases were
similar in sensitive and resistant cells.
Conclusions--
The synthetic retinoid HPR exerts an
apoptotic effect in several tumor cell lines, including A2780
(35). In the case of neuroblastoma (37), breast carcinoma (32), and
acute lymphoblastic leukemia cell lines (64), HPR-induced
apoptosis is accompanied by the elevation of ceramide cellular
levels. In the present paper, we show that this is the case also for
human ovarian carcinoma A2780 cells. The administration of proapoptotic
concentrations of HPR to A2780 cells resulted in an up to 10-fold
increase of cellular ceramide levels. The rise of cellular ceramide
levels was not observed when A2780/HPR cells, resistant to HPR-induced apoptosis, were treated with HPR under the same experimental
conditions. These data indicate that (a) ceramide is a
possible mediator in HPR-induced apoptosis in parental A2780 cells and
(b) the different sensitivity of parental A2780 and
A2780/HPR cells to HPR might be linked to their different ability to
respond with an increase in the cellular levels of the proapoptotic
sphingolipid ceramide. Interestingly, a parallel between ceramide
generation in response to HPR treatment and sensitivity to HPR was
observed in cells of lymphoid origin (64). Different acute
lymphoblastic leukemia cell lines respond to cytotoxic HPR treatment
with an increase in ceramide. On the other hand, HPR is not cytotoxic
and has no effect on ceramide levels in nonmalignant lymphoid cell
types (64).
In the well studied case of multidrug resistance (3-12,
14), MDR cells are able to escape the drug-induced apoptotic death elicited in their sensitive counterparts by a rise in the cellular ceramide levels through two distinctive features. MDR cells express high levels of efflux pump proteins belonging to the ATP-binding cassette superfamily of membrane transport proteins, such as MDR1 and
MRP. The overexpression of energy-dependent drug efflux
pump proteins may lead to lowering intracellular drug levels to
sublethal concentrations. MDR cells show increased levels of GlcCer and GlcCer synthase expression and/or activity with respect to their sensitive counterparts. The ability to scavenge ceramide by converting it to GlcCer could represent an efficient way to reduce the drug effects at the intracellular level. In addition, in 2780AD cells (an
MDR cell line deriving from the parental A2780 line), in addition to enhanced levels of glucosylceramide, more complex
alteration of sphingolipid metabolism with respect to the parental
A2780 cells was reported (12). In these cells, a reduced synthesis of
LacCer was observed, which resulted in lower levels of LacCer, GM3, and
GM2 and higher levels of galactosylceramide and SM.
According to this finding, the resistance to a retinoid in A2780 cells
herein reported does not seem to be related to MDR. In fact, our data
indicate that levels of MDR1 mRNA and MRP mRNA as well as those
of GlcCer and GlcCer synthase mRNA were substantially unchanged in
A2780/HPR cells with respect to parental A2780 cells.
Instead, striking differences were observed between A2780 and A2780/HPR
cells in the metabolism of more complex glycosphingolipids. In
A2780/HPR cells, the sphingolipid metabolism is markedly oriented toward the synthesis of gangliosides. We characterized the main gangliosides of A2780/HPR cells as GM3 and GM2, and their levels were
6-fold higher than in parental cells, whereas LacCer, the direct
neutral glycolipid precursor of gangliosides, was proportionally lower.
Finally, differences were observed in the mRNA levels of the enzyme
GM3 synthase, which is more expressed in A2780/HPR than in sensitive
A2780 cells. This difference was reflected by the in vitro
activity of GM3 synthase, which was 20-fold higher in resistant
A2780/HPR cells than in sensitive A2780 cells.
Thus, the data presented in this paper indicate that all of the
biosynthetic pathway downstream of ceramide is more active in A2780/HPR
cells than in the parental sensitive cell line, leading to the
expression of higher levels of GM3 and GM2 gangliosides. This
particular alteration in sphingolipid metabolism associated with the
development of resistance to an apoptosis inducing drug is reported
here for the first time.
It could be argued that an overall increased flow through the
ganglioside biosynthetic pathway is also a mechanism, even if quite
elaborate, to scavenge ceramide, thus counteracting the apoptotic
effect of HPR, in analogy with what is observed in MDR cells
synthesizing high GlcCer levels. However, the role acknowledged for
complex sphingolipids in modulating the properties of tumor cells (such
as adhesion, motility, invasiveness, and proliferation) is well
documented (reviewed in Ref. 16) even if less codified that the role of
ceramide in tumor cell apoptosis. In bladder cancer, a specific role
for GM3 ganglioside as a negative modulator of malignant potential of
this tumor was hypothesized. In fact, superficial bladder tumors
express higher levels of GM3 and GM3 synthase compared with invasive
tumors (65), and exogenous GM3 treatment (65) or GM3 synthase
overexpression (66) reduced invasion potential and induced apoptosis in
bladder tumor cell lines. Even in the case of MDR cells (where the link
between sensitivity to drug and generation of ceramide as described
above seems extremely well established), a possible role for GlcCer as
substrate or modulator of the pump activity of MDR-related proteins
(that are interestingly localized at the level of plasma membrane in
sphingolipid-rich environments) was suggested (3). As presented in this
paper, the evidence we obtained using A2780/HPR cells suggests that, also in the case of retinoids, differences in sphingolipid metabolism are related to differences in the sensitivity of these cells to the
drug, as previously reported for chemotherapeutic drugs. However, in
our case, these differences might be not simply related to the ability
of cells to neutralize the proapoptotic action of ceramide. In
particular, our data suggest that changes in the expression of
gangliosides could contribute to the onset of a resistant phenotype in
tumor cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
-interferon, and
interleukins) and pharmacological (anticancer drugs, including
daunorubicin, vincristine,
1-
-D-arabinofuranosylcytosine, and retinoids) stimuli by
sphingomyelin hydrolysis or by de novo biosynthesis, is a
mediator of apoptosis and an inhibitor of cell proliferation in a
variety of tumor cell lines (reviewed in Refs. 1 and 2).
,
, and
and retinoid X receptors
,
, and
.
RA receptors and retinoid X receptors activate gene transcription by
binding as homo- or heterodimers to specific DNA sequences, the
retinoic acid-responsive elements, and the retinoid X-responsive elements, usually found in the 5'-flanking regions of responsive genes
(20). Retinoids have been shown to have differentiating and antitumor
activities in several experimental models, and their effectiveness in
the treatment and prevention of human cancer has already been
established (21).
, a
putative tumor suppressor (36), was markedly increased, whereas the
expression of cell surface molecules associated with tumor progression
including HER-2 laminin receptor and
1 integrin
was markedly reduced.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
80 °C under
N2 in the dark. Sodium boro[3H]hydride was
from Amersham Biosciences (specific radioactivity 12.0 Ci/mmol).
Clostridium perfringens sialidase was from Roche Molecular
Biochemicals, and Vibrio cholerae sialidase was from Sigma.
-32P]dCTP (3,000 Ci/nmol; ICN, Milan, Italy) using a
multiprime DNA labeling system (Amersham Biosciences) to a specific
activity of ~2 × 109 cpm/µg of cDNA.
Following hybridization for 3-12 h at 66 °C in Rapid-Hyb buffer
(Amersham Biosciences), the blots were washed twice with 2× SSC, 0.1%
SDS at room temperature for 10 min. The final washes were done with
0.1× SSC, 0.1% SDS at 65 °C for 20 min at least two times.
Membranes were exposed to x-ray film (Hyperfilm-MP; Amersham
Biosciences) using double intensifying screens. RNA from human colon
cancer LOVO-DX cells (kindly provided by Monica Binaschi) was used as
positive control for MDR1 (50).
8 M
[1-3H]sphingosine (5 ml/dish) in culture medium for
2 h (pulse). After the pulse, the medium was replaced with fresh
medium without radioactive sphingosine, and cells were further
incubated for up to 4 days (chase). Under these conditions, all
sphingolipids (including ceramide, SM, neutral glycolipids, and
gangliosides) and phosphatidylethanolamine (PE) (obtained by recycling
of radioactive ethanolamine formed in the catabolism of
[1-3H]sphingosine) were metabolically radiolabeled (32,
48, 51).
-Vision software provided by Biospace.
42 V, capillary temperature of
260 °C, fragmentor voltage (used for collision induced dissociation)
of 40-80%. Mass spectra were acquired over an
m/z range 200-2000.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (12K):
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Fig. 1.
Ceramide detection in A2780 and A2780/HPR
cells. After cell lipid metabolic labeling with
[1-3H]sphingosine as described under "Experimental
Procedures," A2780 (A and B) and A2780/HPR
(C and D) cells were treated with vehicle
(white) or 5 µM (gray) and 10 µM HPR (black) for 48 or 72 h.
A, A2780, adherent cells; B, A2780, floating
cells; C, A2780/HPR, adherent cells; D,
A2780/HPR, floating cells. Radioactive lipids were extracted, separated
by HPTLC, and detected by digital autoradiography (250 dpm applied on a
3-mm line). Time of acquisition was 48 h, and radioactivity
associated with ceramide was quantitatively determined. Data are
expressed as nCi/mg of cell protein and are the means of three
different experiments, with the S.D. never exceeding 10% of the mean
values.
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[in a new window]
Fig. 2.
C2Cer effects on A2780 and A2780/HPR
cells. A, C2Cer antiproliferative activity on A2780
(open circle) and A2780/HPR (solid circle) cells.
Cells were treated 24 h after seeding, and surviving cell number
was evaluated 1 day later. Data are expressed as percentage of control
untreated cells. Results are the mean ± S.D. of two independent
experiments. B, detection of DNA fragmentation induced by
C2Cer. A2780 (lane 1) and A2780/HPR (lane 2)
cells were treated with vehicle (white) or 2.5 µM C2Cer (gray) for 24 h 1 day after
seeding. Cells were then harvested, processed for TUNEL reaction, and
observed using a fluorescence microscope. The results are in
reference to the whole population and represent the mean ± S.D. of two independent experiments.
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Fig. 3.
Constitutive expression of MDR1 and MRP
mRNA in A2780 and A2780/HPR cells. Northern blot analysis was
performed as described under "Experimental Procedures". Each lane
contains 20 µg of total RNA. Filters were hybridized with cDNA
probes for MDR1 (A) and MRP (B), and as a control
for loading, the filters were stripped and rehybridized with
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(lower part of each panel). Lane 1,
A2780 cells; lane 2, A2780/HPR cells;
lane 3, RNA from LOVO-DX cells was used as a
positive control for MDR1.
Radioactivity incorporation into total lipid extracts and aqueous and
organic phases in A2780 and A2780/HPR cells after a 2-h
pulse with [1-3H]sphingosine followed by 48, 72, and
96 h of chase
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[in a new window]
Fig. 4.
Radioactive lipid patterns in total lipid
extracts from A2780 and A2780/HPR cells. After metabolic labeling
with [1-3H]sphingosine for a 2-h pulse followed by 48-h
chase, lipids were extracted from A2780 (lane 1) and
A2780/HPR (lane 2) cells and separated by HPTLC in the
solvent system chloroform/methanol/water (55:20:3 by volume).
Radioactive lipids were detected by digital autoradiography (250 dpm
applied on a 3-mm line). Time of acquisition was 48 h. The
position of pure standard lipids is indicated on the left.
The pattern is representative of that obtained in three different
experiments.
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[in a new window]
Fig. 5.
Radioactive lipid patterns in aqueous and
organic phases obtained after phase separation of total lipid extracts
from A2780 and A2780/HPR cells. After metabolic labeling with
[1-3H]sphingosine for a 2-h pulse followed by 48-h chase,
lipids were extracted from A2780 (lanes 1 and 3)
and A2780/HPR (lanes 2 and 4) cells. Total lipid
extracts were subjected to phase separation as described under
"Experimental Procedures." Lipids from the aqueous (A)
and organic (B) phases were separated by HPTLC using solvent
systems chloroform/methanol/0.2% aqueous CaCl2 (50:42:11
by volume) and chloroform/methanol/water (55:20:3 by volume),
respectively. To selectively remove PE and allow the quantitative
detection of LacCer, aliquots of the organic phases were subjected to
chemical treatment under alkaline conditions (lanes 3 and
4). Aliquots of aqueous and organic phases corresponding to
identical amounts of cell proteins were analyzed. Radioactive lipids
were detected by digital autoradiography (250-1000 dpm applied on a
3-mm line). Time of acquisition was 48 h. The position of pure
standard lipids is indicated on the left. The pattern is
representative of that obtained in three different experiments.
Radioactivity incorporation into different lipids after metabolic
labeling of A2780 and A2780/HPR cells with
[1-3H]sphingosine (2-h pulse followed by 48 h of chase)
View larger version (29K):
[in a new window]
Fig. 6.
Patterns of endogenous lipids from A2780 and
A2780/HPR cells. Cholesterol (Chol) and phospholipids
from A2780 (lane 1) and A2780/HPR cells (lane 2)
were analyzed by HPTLC followed by chemical detection as described
under "Experimental Procedures." Aliquots of samples corresponding
to identical amounts of cell proteins were analyzed for both cell
types. A, cholesterol was separated from organic phases
(corresponding to 0.4 mg of cell proteins). The solvent system was
hexane/ethyl acetate (3:2 by volume), and visualization was by charring
with 15% concentrated sulfuric acid in 1-butanol. B,
phospholipids were separated from organic phases (corresponding to 0.6 mg of cell proteins). The solvent system was chloroform/methanol/acetic
acid/water (30:20:2:1 by volume), and visualization was by detection
with a molybdate reagent. PC, phosphatidylcholine;
PS, phosphatidylserine; PI,
phosphatidylinositol.
H]
at m/z 1354, 1382, 1464, and 1466 and at m/z 1151, 1179, 1261, and 1263, respectively. Fig. 7 shows as an example the characterization of
GM2(d18:1-16:0) and GM3(d18:1-16:0). MS2 of molecular ion
[M-H]
gave a typical fragmentation pattern due to the
sequential loss of sugar units, providing information about the
carbohydrate structure and the total ceramide mass; GM3 and GM2
molecular species contained N-acetylneuraminic acid, whereas
species containing N-glycolylneuraminic acid were
undetectable.
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Fig. 7.
Mass spectra of main gangliosides from A2780
and A2780/HPR cells. Lipids were extracted from A2780 and
A2780/HPR cells, total lipid extracts were subjected to phase
separation as described under "Experimental Procedures," and
gangliosides present in the aqueous phases were analyzed by
HPLC-ESI-MS. A, total negative ESI-MS spectrum of the
gangliosides from HPR-resistant cells, with indication of the main GM3
and GM2 molecular species differing in the acyl structure.
B, HPLC-ESI-MS mass spectrum of the GM3(d18:1-16:0)
molecular species together with the MS2 spectrum derived from the ion
at m/z 1151 and the MS3 spectrum derived from the
ion at m/z 536. C, HPLC-ESI-MS mass
spectrum of the GM2(d18:1-16:0) molecular species together with the
MS2 spectrum derived from the ion at m/z 1354 and
the MS3 spectrum derived from the ion at m/z
536.
View larger version (32K):
[in a new window]
Fig. 8.
Constitutive expression for GlcCer synthase,
LacCer synthase, and GM3 synthase mRNA in A2780 and A2780/HPR
cells. Northern blot analysis was performed as described under
"Experimental Procedures." Each lane contains 20 µg of
total RNA. Filters were hybridized with cDNA probes for GlcCer
synthase (A), LacCer synthase (B), and GM3
synthase (C), and as a control for loading, the filters were
stripped and rehybridized with glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (lower part of each panel).
Lane 1, A2780 cells; lane
2, A2780/HPR cells.
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Fig. 9.
GM3 synthase activity in A2780 and A2780/HPR
cells. GM3 synthase activity was assayed on cell homogenates from
A2780 (1) and A2780/HPR cells (2) as described
under "Experimental Procedures" in the presence of 10 µM (dark gray) or 100 µM
(light gray)
[3-3H(sphingosine)]LacCer (corresponding to 45 nCi/assay). Aliquots of cell homogenates containing the same amount of
proteins (200 µg) were added in a total reaction volume of 50 µl.
The incubation was performed at 37 °C for 3 h. Negative
controls were performed using heat-inactivated cell homogenates. The
reaction mixture was analyzed by HPTLC using the solvent system
chloroform/methanol/water (55:20:3 by volume). Radioactive lipids were
detected and quantified by radioactivity imaging as described in the
legend to Fig. 1. Data are expressed as pmol of formed GM3/h/mg of cell
protein and are the means of three different experiments, with the S.D.
never exceeding 10% of the mean values.
Activity of acid, alkaline, and neutral ceramidase in A2780 and
A2780/HPR cells
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FOOTNOTES |
---|
* This work was supported by COFIN-PRIN Grants 2000 and 2001, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy, and Mizutani Foundation for Glycoscience Grant 2002 (to S. S.), and by Associazione Italiana per la Ricerca sul Cancro (to F. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Via Fratelli Cervi 93, 20090 Segrate, Italy. Fax: 39-0250330365; E-mail: Sandro.Sonnino@unimi.it.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M207269200
2 Ganglioside and glycosphingolipid nomenclature is in accordance with Svennerholm (67) and the IUPAC-IUBMB recommendations (38, 68).
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ABBREVIATIONS |
---|
The abbreviations used are:
GlcCer, -Glc-(1-1)-Cer;
LacCer,
-Gal-(1-4)-
-Glc-(1-1)-Cer;
Cer, ceramide, N-acyl-sphingosine;
C2Cer, N-acetylsphingosine;
C16Cer, N-palmitoylsphingosine;
sphingosine, (2S,3R,4E)-2-amino-1,3-dihydroxyoctadecene;
PE, phosphatidylethanolamine;
SM, sphingomyelin;
ESI, electrospray
ionization;
MS, mass spectrometry;
GSL(s), glycosphingolipid(s);
HPR, N-(4-hydroxyphenyl)retinamide;
HPTLC, high performance
thin layer chromatography;
HPLC, high performance liquid
chromatography;
MDR, multidrug resistance;
MDR1, MDR1 P-glycoprotein;
MRP, multidrug resistance-associated protein;
RA, retinoic acid;
RT, reverse transcription;
SSC, standard sodium citrate;
TUNEL, terminal
dUTP nick-end labeling;
Mops, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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