Human T-cell lymphotropic virus type I-transformed T-cells have a partial defect in ceramide synthesis in response to N -(4-hydroxyphenyl)retinamide

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Biochem. J. (2005) 392, 231–239 (Printed in Great Britain) doi:10.1042/BJ20050578 231
Human T-cell lymphotropic virus type I-transformed T-cells have a partial
defect in ceramide synthesis in response to
N
-(4-hydroxyphenyl)retinamide
Nadine DARWICHE*1, Ghada ABOU-LTEIF*, Tarek NAJDI*, Lina KOZHAYA†, Ahmad ABOU TAYYOUN*, Ali BAZARBACHI‡
and Ghassan S. DBAIBO†§1
*Department of Biology, American University of Beirut, Beirut, Lebanon, †Department of Biochemistry, American University of Beirut, Beirut, Lebanon, ‡Department of Internal Medicine,
American University of Beirut, Beirut, Lebanon, and §Department of Pediatrics, American University of Beirut, Beirut, Lebanon
Treatment with the synthetic retinoid HPR [N-(4-hydroxyphenyl)-
retinamide] causes growth arrest and apoptosis in HTLV-I (human
T-cell lymphotropic virus type-I)-positive and HTLV-I-negative
malignant T-cells [8]. It was observed that HPR-mediated growth
inhibition was associated with ceramide accumulation only in
HTLV-I-negative cells. The aim of the present study was to in-
vestigate the mechanism by which HPR differentially regulates
ceramide metabolism in HTLV-I-negative and HTLV-I-positive
malignant T-cells. Clinically achievable concentrations of HPR
caused early dose-dependent increases in ceramide levels only in
HTLV-I-negative cells and preceded HPR-induced growth sup-
pression. HPR induced de novo synthesis of ceramide in HTLV-I-
negative, but not in HTLV-I-positive, cells. Blocking ceramide
glucosylation in HTLV-I-positive cells, which leads to accumul-
ation of endogenous ceramide, rendered these cells more sensitive
to HPR. Exogenous cell-permeant ceramides that function par-
tially by generating endogenous ceramide induced growth sup-
pression in all tested malignant lymphocytes, were consist-
ently found to be less effective in HTLV-I-positive cells confirm-
ing their defect in de novo ceramide synthesis. Owing to its
multipotent activities, the HTLV-I-encoded Tax protein was
suspected to inhibit ceramide synthesis. Tax-transfected Molt-4
and HELA cells were less sensitive to HPR and C6-ceramide me-
diated growth inhibition respectively and produced lower levels
of endogenous ceramide. Together, these results indicate that
HTLV-I-positive cells are defective in de novo synthesis of cer-
amide and that therapeutic modalities that bypass this defect are
more likely to be successful.
Key words: ceramide, human T-cell lymphotropic virus type-I
(HTLV-1), N-(4-hydroxyphenyl)retinamide (HPR), Tax protein.
INTRODUCTION
The retinoid HPR [N-(4-hydroxyphenyl)retinamide] inhibits
growth and induces apoptosis in many human cell lines, including
many that are all-trans-retinoic acid-resistant [1]. HPR’s cyto-
toxicity is mediated through retinoic acid receptor-dependent and
-independent mechanisms [2], may involve p53-independent path-
ways [3,4], is associated with the generation of ROS (reactive oxy-
gen species) [5], is coupled with JNK (c-Jun N-terminal kinase)
activation [6] and may require elevated levels of ceramide [7].
We previously reported that at clinically achievable concen-
trations, HPR is a potent and selective inducer of G1cell cycle
arrest and apoptosis in HTLV-I-positive and HTLV-I-negative
malignant T-cells with no effect on normal T-lymphocytes [8].
In HTLV-I-negative cells only, HPR-induced apoptosis was as-
sociated with ceramide accumulation, a sharp decrease in mito-
chondrial membrane potential, and activation of caspases 8, 9
and 3, and could be partially reverted by the caspase inhibitor
z-VAD. In contrast, HTLV-I-positive cells had a slower apoptotic
response and required higher HPR concentrations suggesting that
Tax protein, or other HTLV-I products, protected infected cells
from ceramide accumulation and caspase-mediated apoptosis [8].
Ceramide, a sphingolipid secondary messenger molecule, has
been proposed as a co-ordinator of eukaryotic stress responses [9].
Many inducers of stress responses result in ceramide accumul-
ation, usually as a result of sphingomyelin breakdown or by
de novo synthesis, and sometimes as a result of inhibition of cer-
amide clearance through sphingomyelin synthase or ceramidases
[10,11].
Ceramide exerts growth suppressive effects including those on
differentiation [12], cell cycle arrest by dephosphorylation of Rb
protein [13,14], senescence by inhibiting telomerase [15] and
most importantly promotes apoptosis in a variety of cell types
[16,17]. Ceramide may induce apoptosis through ROS generation
[18], activation of JNK/stress-activated protein kinase [19] and
through both caspase-dependent and -independent mechanisms
[20]. Inducers of ceramide accumulation include TNF-α(tumour
necrosis factor-α), Fas ligand, interleukin-1, γ-interferon, CD28
ligation, complement, serum deprivation, γ-irradiation, heat
shock, ultraviolet radiation, and most chemotherapeutic agents
examined [21]. Defects in ceramide production render the cells
more resistant to killing by these inducers [22–24], while elevation
of endogenous ceramide levels lowers the threshold for apoptosis
induction by these agents [25,26]. Cell permeant ceramide ana-
logues can also produce the growth suppressive effects of these
inducers [27].
Several studies described elevations in ceramide levels fol-
lowing HPR treatment and its role in apoptosis [28]. HL-60
leukaemic cells were first shown to exhibit a transient rise in cer-
amide levels following HPR treatment. The addition of FB1
(fumonisin B1), an inhibitor of de novo ceramide synthesis, in-
hibited HPR-induced apoptosis, while the addition of caspase
inhibitors had no effect on ceramide levels suggesting that
ceramide synthesis occurs upstream of caspase activation [29].
Abbreviations used: Dh-C6,dihydro-C
6-ceramide; DMEM, Dulbecco’s modified Eagle’s medium; DGK, diacylglycerol kinase; FBS, fetal bovine serum;
FB1, fumonisin B1; GCS, glucosylceramide synthase; HPR,
N
-(4-hydroxyphenyl)retinamide; HTLV-I, human T-cell lymphotropic virus type-I; JNK, c-Jun
N-terminal kinase; NF-κB, nuclear factor-κB; PDMP, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; ROS, reactive oxygen species; TNF-α,
tumour necrosis factor-α.
1Correspondence should be addressed to either author (email darwichn@aub.edu.lb and gdbaibo@aub.edu.lb).
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232 N. Darwiche and others
Neuroblastoma cells showed a sustained elevation in ceramide
levels after HPR treatment, at concentrations that induced both
necrosis and apoptosis, and again FB1inhibited apoptosis [4].
This HPR-induced ceramide accumulation in neuroblastoma cell
lines was due to the co-ordinated activation of serine palmitoyl-
transferase and ceramide synthase in the de novo synthesis path-
way [30]. As a consequence, combinations of HPR and some
modulators of ceramide metabolism may provide a novel chemo-
therapeutic approach [31]. In previous studies, cytotoxic concen-
trations of HPR have been shown to increase ceramide levels in
acute lymphoblastic leukaemia cell lines, and inhibitors of the
ceramide de novo pathway abrogated this ceramide accumulation
[32]. Furthermore, PC-3 prostate cancer cells, which are relatively
resistant to HPR, were rendered much more sensitive by blocking
the metabolism of ceramide to glucosylceramide using tamoxifen
[33]. All of these studies firmly establish ceramide as an important
player in apoptosis induction by HPR.
In this report, we demonstrate that HPR induces distinct cer-
amide responses in HTLV-I-negative and HTLV-I-positive malig-
nant T-cells. Clinically achievable concentrations of HPR caused
early dose-dependent increases in ceramide levels only in
HTLV-I-negative cells, which preceded HPR-induced growth
suppression. Furthermore, HPR specifically induced de novo syn-
thesis of ceramide in these latter cells. Exogenous cell-permeant
ceramides induced growth suppression in all malignant lympho-
cytes tested. Blocking ceramide glucosylation in HTLV-I-positive
cells enhanced HPR cytotoxicity in these cells. Tax protein-
transfected cells were less sensitive to HPR-mediated growth
inhibition and generated lower levels of ceramide.
EXPERIMENTAL
Cell lines and culture conditions
The HTLV-I-transformed CD4+T-cell lines HuT-102, MT-2 and
C8166, and the HTLV-I-negative CD4+T-cell lines CEM, Jurkat
and Molt-4 were grown as described previously [34]. HeLa cells
were grown in DMEM (Dulbecco’s modified Eagle’s medium).
Where indicated, Molt-4, Jurkat, or HeLa cells were transfected
with either pSG5-Tax or empty vector using Lipofectamine Plus®
(Gibco, Invitrogen) according to manufacturer’s recommenda-
tions.
HPR (Sigma) was prepared as stock solutions in DMSO at 1 ×
10−2M and stored in amber tubes at −80 ◦C. Before HPR ad-
dition, cells were cultured for 24 h at 1×105cells/ml in RPMI-
1640 medium containing 10 %(w/v) FBS (fetal bovine serum)
(Gibco, Invitrogen) and antibiotics. All experiments using HPR
were performed under yellow light (λ>500 nm) to prevent photo-
isomerization. PDMP (D,L-threo-1-phenyl-2-decanoylamino-3-
morpholino-1-propanol), purchased from Biomol (Plymouth
Meeting, PA, U.S.A.), was reconstituted in DMSO at a concen-
tration of 50 mM and was stored at −20 ◦C. PDMP was added to
cells, 2 h before HPR treatment, to a final concentration ranging
from 10 µMto30µM. The final concentration of DMSO never
exceeded 0.1 %and this concentration showed no effect on the
proliferation of all tested cell lines (results not shown). The short-
chain cell-permeant ceramides C2-andC
6-ceramide or C2-and
Dh-C6(dihydro-C6-ceramide) (Biomol) were reconstituted in
100 %pure ethanol at a concentration of 40 mM, stored at
−20 ◦C and used at final concentrations ranging from 1 µMto
20 µM. Before ceramide and dihydroceramide treatments, cells
were cultured in RPMI-1640 medium containing 2 %(w/v) FBS.
FB1 (Biomol) was reconstituted in 1 ×PBS at a concentration
of 20 mM, stored at −20 ◦C and added to cells at 50 µMfinal
concentration 1 h before HPR treatment.
Growth assays
Cell growth was assessed by cell counts using Trypan Blue dye
exclusion protocols and/or the use of the CellTiter 96®non-
radioactive cell proliferation assay kit (Promega Corp., Madison,
WI, U.S.A.) according to the manufacturer’s instructions. Cells
were grown in 96-well plates (Nunc, Naperville, IL, U.S.A.) and
at the initiation of cultures drugs were added at the concentrations
and time points indicated. Results are expressed as cell growth
relative to that in DMSO-treated controls and are derived from
the mean cell growth of quadruplicate wells. Results are represent-
ative of at least three independent experiments. Similar growth
trends were observed using the Trypan Blue dye exclusion and
the CellTiter 96®non-radioactive cell proliferation assays.
Ceramide measurement
Lipids were collected by the method of Bligh and Dyer [35].
Ceramide was measured with a modified DGK (diacylglycerol
kinase) assay using external ceramide standards as described
previously [36]. Briefly, 80 %of the lipid sample was dried under
N2. The dried lipid was solubilized in 20 µlofanoctylβ-D-
glucoside/dioleoyl phosphatidylglycerol micellar solution (7.5 %
octyl β-D-glucoside, 25 mM dioleoyl phosphatidylglycerol) by
several cycles of sonication in a bath sonicator followed by resting
at room temperature for 15–20 min. The reaction buffer was
prepared as a 2×solution, containing 100 mM imidazole HCl
(pH 6.6), 100 mM LiCl, 25 mM MgCl2and 2 mM EGTA. To the
lipid micelles, 50 µlof2×reaction buffer were added, 0.2 µlof
1M dithiothreitol, 5 µg of diglycerol kinase membranes and dilu-
tion buffer (10 mM imidazole, pH 6.6, 1 mM diethylenetriamine-
penta-acetic acid, pH 7) to a final volume of 90 µl. The reaction
was started by adding 10 µlof2.5mM[γ-32 P]ATP solution (spe-
cific activity of 75 000–200 000 c.p.m./nmol). The reaction was
allowed to proceed at 25◦C for 30 min. Bligh and Dyer lipid
extraction was performed and a 1.5 ml aliquot of the organic
phase was dried under N2. Lipids were then resuspended in
50 µl of methanol/chloroform (1:9, v/v) and 25 µl was spotted
on to a 20 cm silica gel TLC plate. Plates were developed using
chloroform/acetone/methanol/acetic acid/H2O (50:20:15:10:5, by
vol), air-dried and subjected to autoradiography. The radioactive
spots corresponding to phosphatidic acid and ceramide-phos-
phate, the phosphorylated products of diacylglycerol and cer-
amide respectively, were identified by comparison with known
standards. Spots were scraped into a scintillation vial containing
4 ml of scintillation fluid and counted on a scintillation counter.
Linear curves of phosphorylation were produced over a concen-
tration range of 0–960 pM of external standards (dioleoyl glycerol
and CIII-ceramide, Sigma). Ceramide levels were routinely
normalized to lipid phosphate levels. It is important to note that
under these conditions, there was a total conversion of ceramide
and diacylglycerol into their phosphorylated products, and there
was no change in the specific activity of the DGK enzyme.
De novo
ceramide synthesis
At initiation of treatment, [3H]palmitic acid (1 µCi/ml medium),
purchased from PerkinElmer (Boston, MA, U.S.A.) was added to
HPR-treated and control samples. Extracted lipids, dried under
N2, were resuspended in 1 ml of chloroform, 200 µlwasset
aside for the first phosphate measurement and 800 µl was used in
the base hydrolysis assay. In this assay, 200 µl of chloroform
and 800 µl of 0.125 M methanolic KOH were added to the
samples before incubation at 37 ◦C for 75 min. The base was
neutralized by adding 100 µl of 1 M methanolic HCl to each
sample. A 2 ml volume of chloroform and 600 µl of distilled H2O
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Tax protein-induced defect in ceramide synthesis 233
were next added to the samples at room temperature for 30 min.
Subsequently, lipids were extracted by the method of Bligh and
Dyer [35], dried under N2and resuspended in 75 µl of chloroform.
A50µl sample of hydrolysed lipids was subjected to separation
for 1 h on a 20 cm silica gel TLC plate and 25 µlwaskeptfor
a second phosphate assay. The solvent system employed in
TLC separation was chloroform/methanol/2 M NH4OH (4:1:0.1,
by vol). The TLC plates were then sprayed lightly with En3hance®
(PerkinElmer) to enhance tritium readings. The ceramide spots
were visualized by iodine vapour mark. Radioactivity was also
visualized by autoradiography after 48 and 96 h at 80◦Candthe
[3H]ceramide spots were scraped into scintillation vials containing
4 ml of scintillation fluid and counted on a Packard scintillation
counter. Ceramide was quantified using external standards and
was normalized to phosphate counts.
Treatment with 17C6-ceramide
Hela cells, seeded at 2 ×106cells/ml, were transfected with
either pSG5-Tax or empty vector using Lipofectamine Plus®
(Gibco) according to the manufacturer’s recommendations. At
24 h post-transfection, cells were treated with D-erythro-17C6-
ceramide (generously provided by Dr Alicia Bielawska from the
Lipidomics Core at the Medical University of South Carolina,
Charleston, South Carolina, U.S.A.) for a further 24 h. This
unnatural ceramide undergoes deacylation and reacylation shortly
after cell entry where the generated 17-sphingosine backbone is
recycled and the activity of different ceramide synthases results in
the formation of several species of endogenous 17-ceramide. Cell
pellets were spiked with internal standards, lipids were extracted
and the unnatural 17-ceramide molecular species reflecting the
activity of ceramide synthases were analysed by MS. Data were
normalized to total protein levels.
Microsome preparation
Microsomes were prepared by sonication of cell pellets from CEM
and HuT-102 cells in 25 mM Tris, pH 7.4, 5 mM EDTA, 1 mM
PMSF and 1×complete protease inhibitor (Roche, Mannheim,
Germany)-containing buffer. The preparation was centrifuged at
1000 g, and the resulting supernatant was then ultracentrifuged
at 100 000 g. The pellet was resuspended by homogenization
in Hepes buffer (20 mM Hepes, 2 mM KCl and 2 mM MgCl2)
at pH 7.4. Protein concentration was determined using the DC
protein assay (Bio-Rad Laboratories, Hercules, CA, U.S.A.).
In vitro
ceramide synthase activity assay
Ceramide synthase activity was determined using microsome
fractions isolated from HPR-treated and control cells. A 50 µg
sample of microsomal proteins was incubated in 25 mM potas-
sium phosphate, pH 7.4, containing 50 µM palmitoyl-CoA and
15 µMD-erythro-17-sphingosine (a kind gift from Dr Alicia
Bielawska) delivered using BSA as vehicle. The incubation was
performed for 15 min at 37 ◦C and was stopped by the addition of
the extraction solvent. Extracted lipids were used to measure the
levels of 17C16-ceramide by MS.
MS analysis of lipids
Samples were fortified with internal standards and lipids were
extracted with ethyl acetate/isopropanol/water (60:30:10 by vol),
evaporated to dryness and reconstituted in 100 µl of methanol.
Analysis was performed using electrospray ionization MS/MS
analysis on a Thermo Finnigan TSQ 7000 triple quadruple
mass spectrometer, operating in multiple-reactions-monitoring
positive-ionization mode, as described previously [37].
Statistical analysis
Three statistical tests were performed to validate the significance
of the observed results: the Dunnett test, the two-way analysis of
variance test, and the two-sample Students t-test.
RESULTS
HPR induces distinct ceramide responses in HTLV-I-negative and
HTLV-I-positive malignant T-cells
The effects of HPR treatment on ceramide levels was examined
in HTLV-I-negative cells. A time-dependent increase in ceramide
was observed in CEM (Figure 1A), Jurkat and Molt-4 cells (results
not shown) treated with HPR. This increase in ceramide levels
started after as little as 6 h in Jurkat cells and 12 h in CEM cells,
reaching a 2-fold increase after 18 h and increased to at least 3-
fold after 24 h in both cell lines. Ceramide generation preceded
the significant HPR-induced growth suppression and cell death
observed in these cell lines at the designated concentrations
(Figure 1B). A dose-dependent accumulation of ceramide was
noted in CEM cells treated for 24 h with increasing concentrations
of HPR, ranging from 0.1 µMto5µM (Figure 1C). In contrast,
no increase in ceramide levels was observed in HTLV-I-positive
HuT-102 (Figure 1D), C8166, and MT-2 cells (results not shown)
after 24 h of HPR treatment at 5 µM despite significant growth
suppression and cell death observed under these conditions [8].
Only late, modest increases in ceramide levels were observed in
HTLV-I-positive cells treated with 5 µMHPRfor48h(P<0.01,
t-test) (Figure 1D), after significant cell death had occurred [8].
Also, lower HPR doses had no effect (P<0.01, Dunnett test)
(Figure 1E).
HPR induces
de novo
synthesis of ceramide in HTLV-I-negative,
but not in HTLV-I-positive, malignant T-cells
Previous studies in different cell lines indicated that HPR in-
duces de novo synthesis of ceramide [30–32]. We examined the
role of de novo ceramide synthesis following HPR treatment
of HTLV-I-negative and HTLV-I-positive cell lines. CEM and
C8166 cells were treated with 1 and 5 µM HPR concentrations
respectively, based on their differential HPR sensitivity [8] at
indicated time points, and de novo synthesized ceramide was
measured by [3H]palmitate incorporation. HPR treatment caused
a time-dependent increase in [3H]ceramide, indicating de novo
synthesis in CEM cells, starting at 18 h post-treatment (P<0.01,
Dunnett test) (Figure 2). In contrast, C8166 cells showed no
change in [3H]ceramide after treatment using a 5-fold higher
HPR concentration (Figure 2). The activity of ceramide synthase
in CEM and HuT-102 cells in response to HPR treatment was
129.1 +
−5.4 and 89.3 +
−2.8 pmol of ceramide/min per mg of
protein respectively. There was no significant induction of this
activity when compared to the corresponding untreated cells.
These results suggest that de novo ceramide synthesis was im-
paired in HTLV-I-infected cells.
Inhibition of GCS (glucosylceramide synthase) increases
sensitivity to HPR-induced cell death in HTLV-I-positive cells
We next aimed to determine whether increasing the endogenous
levels of ceramide in HTLV-1-positive cells renders them more
sensitive to HPR. We therefore investigated the effect of PDMP, an
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234 N. Darwiche and others
Figure 1 HPR induces distinct ceramide responses in HTLV-I-negative and HTLV-I-positive malignant human T-cell lines
(A) Ceramide levels in the HTLV-I-negative CEM human T-cell line in response to HPR. CEM cells were seeded at a density of 3.5 ×105cells/ml and treated with 0.1% DMSO as a control or with
1µM HPR for the times indicated. Ceramide levels were determined using the DGK assay as described in the Experimental section and normalized to total cellular lipid phosphate levels. Data points
represent the mean +
−range (
n
=2). Results are representative of three independent experiments. (B) Effects of HPR treatment on the growth of CEM human T-cell line. Cells were seeded at a density
of 2 ×105cells/ml and treated with 0.1 % DMSO as a control or with 1 µM HPR for the times indicated. Viable cell counts, calculated from triplicate wells by Trypan Blue dye exclusion, are expressed
as a percentage of controls and are representative of two independent experiments. (C) Dose–response to HPR treatment in CEM human T-cell line. Cells were seeded at a density of 3.5×105
cells/ml and treated with 0.1% DMSO as a control, or the indicated concentrations of HPR, for 24 h. Ceramide levels were determined as in (A). Data points represent the mean+
−range (
n
=2).
Results are representative of three independent experiments. (D) HPR treatment induces late ceramide accumulation in HTLV-I-positive human T-cell line Hut-102. Cells were seeded at a density of
3.5 ×105cells/ml and treated with 0.1% DMSO as a control or 5 µM HPR for the times indicated. Ceramide levels were determined as in (A). Data points represent the mean+
−range (
n
=2).
Results are representative of two independent experiments. (E) Dose–response to HPR treatment in HTLV-I-positive human T-cell line C8166. Cells were seeded at a density of 3.5 ×105cells/ml
and treated with the indicated concentrations of HPR for 48 h. Ceramide levels were determined as in (A). Data points represent the mean +
−range (
n
=2). Results are representative of two independent
experiments.
Figure 2 HPR treatment induces
de novo
synthesis of ceramide in HTLV-I-
negative human T-cell lines only
CEM and C8166 cells were seeded at a density of 3.5 ×105cells/ml and treated with 0.1 % DMSO
as a control or with 1 and 5 µM HPR respectively.
De novo
ceramide levels were determined
using the [3H]palmitate incorporation method as described in the Experimental section and
normalized to total cellular lipid phosphate levels. Data points represent the mean +
−range
(
n
=2) and are percentages of treated over control cells. Results are representative of two
independent experiments.
inhibitor of GCS [38] and thus an inducer of ceramide accumul-
ation, on HPR-induced cytotoxicity. At 2 h before HPR ex-
posure, cells were pretreated with PDMP at non-cytotoxic con-
centrations: 20 µM in C8166 and HuT-102 and 30 µMinMT2
cells. These concentrations resulted in a modest elevation of
cellular ceramide levels (Figure 3A, and results not shown).
The combination of HPR and PDMP in HTLV-I-positive cells
resulted in a reduction in cell viability (P<0.01, t-test) exceeding
their additive effects (Figure 3B). Treatment with 1 µMHPR
and 20 µM PDMP for 24 h led to a 12 %and 16 %synergistic
reduction in cell viability in C8166 and HuT-102 cells respectively
(P<0.01, t-test) (Figure 3B). Because of their lower sensitivity
to both PDMP and HPR [8], MT2 cells were treated with
3µMHPRand30µM PDMP for 24 h, resulting in a significant
21 %synergistic reduction in cell viability (P<0.01, t-test)
(results not shown). Only additive effects of HPR and PDMP
treatments were observed in the HTLV-I-negative cell line CEM,
although ceramide levels increased by approx. 100%over
control in response to PDMP (results not shown). These results
indicate that increasing endogenous ceramide levels by PDMP
partially restores the sensitivity of HTLV-I-positive cells to HPR
treatment.
Exogenous ceramides induce cell death in HTLV-I-negative and
HTLV-I-positive malignant T-cells
The results above suggested that HTLV-I-positive cells may have
a defect in ceramide synthesis in response to HPR and that
elevation of endogenous ceramide by PDMP renders them more
sensitive to HPR. Therefore it became important to determine
the sensitivity of both HTLV-I-positive and HTLV-I-negative
cells to exogenous ceramide as this might bypass the defect.
Addition of cell-permeant C2-andC
6-ceramide analogues triggers
many of the biological responses of agonists in cells [22]. We
treated the HTLV-I-negative Jurkat cells and the HTLV-I-positive
HuT-102, MT2 and C8166 cells with increasing concentrations
of C2-ceramide, ranging from 1 to 20 µM for up to 3 days. In
all cells tested, C2-ceramide caused a reduction in their viability
in a time- and dose-dependent manner (Table 1). A 20 µMC
2-
ceramide concentration completely killed the majority of tested
cells by 24 h. When the effects of the same concentration of
C2-ceramide in both cell type groups were compared, HTLV-I-
positive cells were slightly more resistant than HTLV-I-negative
cells. Additionally, cell treatment with Dh-C6, the immediate
inactive precursor of ceramide, ranging from 1 to 20 µMfor
up to 3 days, showed no effect on cell viability in any cell line,
underscoring the specificity of ceramide action (Table 1).
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Tax protein-induced defect in ceramide synthesis 235
Figure 3 PDMP synergizes with HPR treatment to induce cell death in
HTLV-I-positive human T-cell lines
(A) Ceramide levels in the HTLV-I-positive C8166 and HuT-102 cell lines in response to
combined HPR and PDMP treatment. Cells were seeded at a density of 3.5 ×105cells/ml and
were pretreated with 20 µM PDMP, an inhibitor of GCS. PDMP pretreatment was performed
2 h before treatment with 1 µM HPR. Data points represent the mean +
−range (
n
=2). Results
are representative of two independent experiments. (B) Effect of combined HPR and PDMP
treatment on the growth of the HTLV-I-positive C8166 and HuT-102 cells. Cells were seeded
at a density of 2 ×105cells/ml and were p retreated with 20 µM PDMP, an inhibitor of GCS.
PDMP pretreatment was performed 2 h prior to treatment with 1 µM HPR. Viability was assayed
in 96-well plates at 24 h using the Cell Titer 96®non-radioactive cell proliferation kit. Data
points represent the mean +
−S.D. (
n
=3). Results are expressed as percentage of control cells
(0.1% DMSO) and represent the mean of three independent experiments.
Recently, C6-ceramides were shown to induce the generation
of endogenous long-chain ceramides [39]. Our previous results
demonstrated that the HTLV-I-positive cells are not capable of
HPR-induced de novo ceramide synthesis and therefore may be
relatively less sensitive to C6-ceramides than the HTLV-I-negative
cells. We treated several HTLV-I-positive HuT-102, MT2 and
C8166 cells and HTLV-I-negative Jurkat and CEM cells with C6-
ceramide, ranging from 1 to 20 µM for up to 3 days. C6-ceramide
caused a time- and dose-dependent reduction in the growth of
all cells tested (Table 1). Viability of HTLV-I-positive-C8166
cells was measured using the CellTiter 96®non-radioactive cell
proliferation assay owing to excessive clumping of cells observed
in this cell line. In response to 20 µMC
2-ceramide, viability
decreased to 49 %,2%and 0 %of control at 24, 48 and 72 h
after treatment respectively. In response to the same concentration
of C6-ceramide, viability decreased to 69 %,23%and 18 %at
the same respective time points. Interestingly, when the effects
of the same concentration of C6-ceramide were compared in both
cell type groups, the HTLV-I-negative cells were found to be
significantly more sensitive to C6-ceramide than HTLV-I-positive
cells (Table 1). These results suggested, that in response to C6-
ceramide, HTLV-I-positive cells might generate lower levels of
endogenous ceramides.
Response to C6-ceramide in HTLV-I-positive cells is blunted,
resulting from a defect in endogenous ceramide synthesis
In order to verify whether ceramide synthesis in response to
exogenous C6-ceramide is affected in HTLV-I-positive cells, we
treated HuT-102, MT2 and C8166 cells, and the control CEM,
Jurkat and Molt-4 cells with 20 µMC
6-ceramide. Ceramide levels
were measured using the DGK assay, where the phosphorylated
products of C6-ceramide and endogenous ceramide can be identi-
fied by their distinct migration. C6-ceramide induced significantly
lower endogenous ceramide accumulation after 12 (results not
shown) and 18 h (Figure 4A) in all HTLV-I-positive cells tested,
compared with HTLV-I-negative cells. Elevated endogenous cer-
amide levels in HTLV-I-negative cells correlated with higher
levels of growth suppression and cell death in these cells after
12 (results not shown) and 18 h (Figure 4B) as compared with
HTLV-I-positive cells (C8166 viability decreased to 74%of
Table 1 Exogenous ceramides induce cell death in HTLV-I-negative and HTLV-I -positive malignant human T-cell lines
Effects of synthetic ceramides C2and C6on the growth of the HTLV-I-negative (Jurkat) and the HTLV-I-positive (HuT-102 and MT2) human T-cell lines. Cells were seeded in RPMI 1640 medium
containing 2 % (w/v) FBS in 24-well plates at a density of 2 ×105cells/ml and were treated with the indicated concentrations of C2-ceramide, C6-ceramide and Dh-C6. Viability was assayed using the
Trypan Blue dye exclusion method. Data points represent the mean viability (
n
=3). Results are expressed as percentage of control cells (0.1 % ethanol) and represent the mean of three independent
experiments.
Treatment time (h)
24 48 72
Cells Ceramide (µM) C2C6Dh-C6C2C6Dh-C6C2C6Dh-C6
JURKAT 1 µM 90 90 100 62 80 100 83 100 99
5µM 879599 386598 247096
10 µM 408298 116197 35595
20 µM009500950094
HuT-102 1 µM 92 94 100 67 65 100 56 100 100
5µM 556098 385698 299296
10 µM 315197 104296 07894
20 µM 8 42 95 0 35 94 0 52 94
MT2 10 µM 819397 798095 916495
15 µM 327096 206495 135895
20 µM 12 65 96 1 39 94 0 25 94
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236 N. Darwiche and others
Figure 4 Response to exogenous C6-ceramide in HTLV-I-positive human T-cell lines is blunted because of a defect in endogenous ceramide synthesis
(A) Endogenous ceramide levels in the HTLV-I-positive(HuT-102, MT2 and C8166) and the HTLV-I-negative(CEM, Jurkat and Molt-4) human T-cell lines in response to C6-ceramide. Cells were seeded
in RPMI-1640 medium containing 2 % FBS at a density of 3.5×105cell/ml. Cells were treated with 20 µMC
6-ceramide for 18 h. Endogenous ceramide levels were determined as in Figure 1(A). The
phosphorylated product of C6-ceramide is easily distinguishable from that of endogenous ceramide on the TLC plate (results not shown). Data points represent the mean +
−range (
n
=2). Results are
representative of two independent experiments. (B) Effects of C6-ceramide on the growth of the HTLV-I-positive (HuT-102 and MT2) and the HTLV-I-negative (CEM, Jurkat and Molt-4) human T-cell
lines. Cells were seeded in RPMI-1640 medium containing 2 % FBS at a density of 2×105cells/ml. Cells were treated with 20 µMC
6-ceramide for 18 h. Viability was assayed using the Trypan Blue
dye exclusion method. Data points represent the mean +
−S.D. (
n
=3). Results are expressed as percentage of control cells (0.1% ethanol) and represent the mean of two independent experiments.
(C) Endogenous ceramide levels in the HTLV-I-positive HuT-102 and the HTLV-I-negative Jurkat human T-cell lines in response to C6-ceramide and FB1treatment. Cells were seeded in RPMI-1640
medium containing 2 % FBS at a density of 3.5×105cell/ml. Cells were treated with 20 µMC
6-ceramide, 50 µMFB
1, or combined C6-ceramide and FB1for 18 h. Endogenous ceramide levels were
determined as in Figure 1(A). Data points represent the mean +
−range (
n
=2). Results are representative of two independent experiments. (D) Endogenous ceramide levels in the HTLV-I-positive
HuT-102 and the HTLV-I-negative Jurkat human T-cell lines in response to exogenous C2-ceramide. Cells were seeded in RPMI-1640 medium containing 2 % FBS at a density of 3.5×105cell/ml.
Cells were treated with 20 µMC
2-ceramide for 18 h. Endogenous ceramide levels were determined as in Figure 1(A). Data points represent the mean +
−range (
n
=2). Results are representative of
two independent experiments.
controls as measured using the CellTiter 96®non-radioactive cell
proliferation assay kit). To investigate the role of the de novo
ceramide synthetic pathway upon exogenous C6-ceramide ad-
dition, HuT-102 and Jurkat cells were pretreated with FB1,an
inhibitor of ceramide synthase. The addition of FB1almost com-
pletely abrogated the endogenous ceramide levels that were gene-
rated at 12 and 18 h after C6-ceramide addition in both cell lines
(Figure 4C). The viability of HPR-treated Jurkat and HuT-102
cells for up to 12 h increased by 35 %and 14 %respectively, fol-
lowing FB1pre-treatment, suggesting that endogenous ceramide
accumulation contributes significantly to HPR-induced cell death
of HTLV-I-negative cells.
The more potent growth suppressive effects of C2-ceramide
compared to C6-ceramide in both HTLV-I-positive and HTLV-I-
negative cells (Table 1) led us to examine whether the activity
of this analogue was less dependent on endogenous ceramide
generation. We treated HuT-102 and Jurkat cells with 20 µMC
2-
ceramide to evaluate its ability to induce endogenous ceramide
synthesis. Indeed, lower levels of endogenous ceramide levels
accumulated in the HTLV-I-positive cells after C2-ceramide ad-
ministration versus C6-ceramide (compare Figure 4D with Fig-
ure 4C), suggesting an explanation for the improved activity of this
analogue in HTLV-I-positive cells. Taken together, these results
clearly demonstrate that the HTLV-I-positive cells are defective
in endogenous ceramide synthesis, not only in response to HPR,
but also in response to exogenous ceramides.
Tax protein represses the ceramide response in
HTLV-I-positive cells
In order to test whether, in HTLV-I-infected cells, the oncoprotein
Tax is responsible for inhibiting ceramide accumulation, we
treated Tax-transfected Molt-4 cells with HPR (5 and 10 µM)
and measured ceramide levels. The levels of ceramide generated
were significantly lower in Tax-transfected cells in response to
both HPR concentrations after 14 and 18 h (P<0.05, t-test)
(Figure 5A). However, 5 µMHPRtreatmentfor20and38hinthe
HTLV-I-negative CEM cells and HTLV-I-positive HuT-102 cells
did not significantly stimulate ceramide synthase activity (results
not shown). Furthermore, HeLa cells transiently transfected
with Tax protein had a significantly reduced accumulation of
endogenous ceramide after 18 h (Figure 5B) in response to
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Tax protein-induced defect in ceramide synthesis 237
Figure 5 Role of Tax protein in suppressing the ceramide response in the
HTLV-I-positive human T-cell lines
(A) Reduced ceramide levels in HPR-treated Tax-transfected Molt-4 cells. Molt-4 cells were
transiently transfected with pSG5-Taxor control vector and treated after 24 h with HPR (5 µMand
10 µM) for the times indicated. Ceramide levels were determined as in Figure 1(A). Data points
represent the mean +
−range (
n
=2). Results are representative of two independent experiments.
(B) Effects of exogenous C6-ceramide on endogenous ceramide levels in Tax-transfected HeLa
cells. pSG5-Tax or control cells were transfected for 24 h and then were treated with 20 µM
C6-ceramide in DMEM medium containing 2 % FBS for 18 h. Endogenous ceramide levels
were determined as in Figure 1(A). Data points represent the mean +
−range (
n
=2). Results
are representative of two independent experiments. (C) Ceramide synthase activity is reduced
in Tax-transfected Hela cells. pSG5-Tax or control cells were transfected for 24 h and then were
treated with 5 µM 17C6-ceramide in DMEM medium containing 2 % FBS for 24 h. Endogenous
17-ceramide species levels were determined as described in the Experimental section and
normalized to total cellular protein levels. Data points represent the mean +
−range (
n
=2).
C6-ceramide treatment (P<0.01, t-test). Moreover, we utilized
the synthetic ceramide 17C6-ceramide, that has an unnatural
17-sphingosine backbone, to indirectly determine the activity of
ceramide synthase(s). Measurement of the unnatural endogenous
17-ceramides that result from the deacylation/reacylation reaction
(Figure 5C) after 24 h showed that in Tax-transfected cells, the
production of most endogenous 17-ceramides was significantly
inhibited (P<0.01 for all except 17C24-ceramide, t-test). These
experiments when combined with the results above indicate that
Tax is responsible for suppression of de novo ceramide synthesis,
probably by inhibiting ceramide synthase.
DISCUSSION
We have determined that HPR induces distinct ceramide res-
ponses in HTLV-I-negative and HTLV-I-positive malignant T-
cells. This may be partly explained by an impaired de novo
ceramide synthetic pathway in the HTLV-I-positive cells owing
to expression of Tax protein. De novo synthesis of ceramide is
dependent on the activity of several enzymes, including several
ceramide synthases. Recently, ceramide synthase genes, identified
as Lass (longevity assurance-like) genes, were described but have
not yet been well characterized [40]. The results from this study
indicate that the activity of ceramide synthase(s) is suppressed in
HTLV-I-positive cells.
The HTLV-I-encoded oncoprotein, Tax, plays a key role in
the transformation of infected cells and their resistance to chemo-
therapy [8,41]. In the current study, it is shown that Tax expression
in cells not infected with HTLV-I is sufficient to inhibit the
generation of ceramide in response to HPR or C6-ceramide and to
suppress ceramide synthase activity as measured by the ability of
transfected cells to convert the 17C6-ceramide into endogenous
17-ceramides. A major biological function of Tax protein appears
to be its ability to induce a constitutive high level of activity
of the transcription factor NF-κB (nuclear factor-κB) [42].
Recent evidence suggests that the Tax oncoprotein represses
the transcriptional activity of the tumour suppressor protein p53
through the activation of the NF-κB pathway, but independent
of the CREB/ATF pathway, which is also disrupted by Tax [43].
Interestingly, p53 wasfound to regulate the generation of ceramide
in response to chemotherapeutic agents or γ-irradiation [44].
Therefore one future aim will be to examine whether NF-κBand
p53 mediate the inhibitory effects of Tax on de novo synthesis of
ceramide at the genetic level.
Cell permeant ceramide analogues C2 and C6 were shown to
reproduce the growth suppressive effects of chemotherapeutic
agents that induce ceramide accumulation [27]. These short-
chain ceramides have also been employed to support evidence for
apoptosis induction obtained after elevation of cellular ceramide
content in response to several inducers of ceramide accumulation
such as TNF-α, Fas ligand, interleukin-1 and other effectors
[45–47]. C6-ceramide was recently shown to induce de novo
ceramide synthesis in the A549 human lung adenocarcinoma
cell line where its sphingosine backbone becomes recycled after
deacylation/reacylation into endogenous long-chain ceramides
[39]. This revealed that the biological functioning of exogenous
ceramides was in part dependent on ceramide synthase activity
to produce endogenous ceramides. In the current study C2-as
well as C6-ceramides induced de novo ceramide synthesis in
both HTLV-I-positive and HTLV-I-negative cells. However, this
was markedly lower in HTLV-I-positive cells, and in both cell
types, was lower in response to the C2-ceramide analogue. Both
analogues induced growth suppression in both cell types in a time-
and dose-dependent manner. The lower sensitivity of HTLV-I-
positive cells to exogenous ceramides provided further support
for the suggestion that ceramide synthase is inhibited in these
cells. It was also noted, that under the same conditions, C2-
ceramide was more effective compared with C6-ceramide in
producing growth suppression. These differences could be due
to better uptake, slower metabolism, or an enhanced direct effect
for this analogue, as opposed to dependence on the generation of
endogenous ceramide.
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238 N. Darwiche and others
Several studies indicate that the co-administration of modul-
ators of ceramide metabolism and chemotherapeutic agents in-
creases the level of ceramide and further enhances cytotoxicity.
PDMP and related compounds are potent inhibitors of GCS
and consequently of ceramide clearance. These inhibitors have
been shown to sensitize a variety of drug-resistant cell lines to
chemotherapeutic reagents and cause their preferential killing
by such reagents [26,48,49]. Furthermore, D,L-threo-(1-phenyl-
2-hexadecanoylamino)-3-morpholino-1-propanol administration
produces a synergistic increase in cytotoxicity with HPR in
solid tumour cell lines [31] and in HPR-resistant PC-3 prostate
cancer cells [33]. In the present study HTLV-I-positive cells
were found to have a defect in ceramide synthesis and relative
resistance to HPR as compared with HTLV-I-negative cells. Re-
storation of elevated ceramide levels by PDMP during HPR treat-
ment led to synergistic cytotoxicity in HTLV-I-positive cell lines,
thus sensitizing these cells to HPR concentrations that are lower
than what is usually required to induce significant growth sup-
pression. This, together with results using exogenous ceramides,
indicated that HTLV-I-positive cells were sensitive to cera-
mide-induced cell killing once sufficient levels were generated.
Deregulation of ceramide metabolism may act as an indicator
of chemoresistance in tumour cells and could be a target in
cancer therapy [28,50]. The present study demonstrates that viral
oncogenes may target the ceramide pathway. In the case of Tax,
this occurs by inhibition of ceramide synthase activity and may
explain the resistance of HTLV-1-positive cells to treatment.
Therapeutic agents with the ability to bypass this defect are more
likely to be successful.
This work was supported by grants from the Lebanese National Council for Scientific
Research and from the American University of Beirut, University Research Board (N. D.)
and by the National Science Foundation (U.S.A.) Grant No. 0090859 (G.S.D.). We thank
Dr Jacek Bielawski for performing MS analysis of ceramides at the Lipidomics Core
Laboratory at the Medical University of South Carolina (M.U.S.C.) and Dr Alicia Bielawska
(M.U.S.C.) for providing the 17C6-ceramide and 17-sphingosine.
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Received 8 April 2005/29 July 2005; accepted 9 August 2005
Published as BJ Immediate Publication 9 August 2005, doi:10.1042/BJ20050578
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2005 Biochemical Society