THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
Vol. 268, No. 35, Issue of December 15, pp. 2622646232,1993
Printed in U.S.A.
Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Selectivity of Ceramide-mediated Biology
LACK OF ACTIVITY OF erythro-DIHYDROCERAMIDE*
(Received for publication, March 22, 1993, and in revised form, July 14, 1993)
Alicia Bielawska, Heidi M. Crane#, DennisLiottal, Lina M. Obeid, and Yusuf A.
HannunQ
From the Department of Medicine, Duke University Medical Center, Durham, NorthCarolina 27710 and the $Department of
Chemistry, Emory University, Atlanta,Georgia 30322
Ceramide is emerging as a putative second messenger
cell death (apoptosis) (14). Thus, ceramide is emerging as an
mediating effects of extracellular agents on cell growth important regulator of cell growth, viability, and differentiaand differentiation (Okazaki, T., Bielawska, A., Bell, tion (15, 16). Recently, the ability of ceramide to inhibit
R. M., andHannun, Y. (1990) J. Biol. Chem. 266, growth of Saccharomyces cereuisiae was also demonstrated
16823-16831) and programed cell death (Obeid, L.
suggesting evolutionary conservation of this pathway of cell
M., Linardic, C. M., Karolak, L. A., and Hannun,Y.A. regulation (17).
(1993) Science 269, 1769-1771). In this study, the
To establish more conclusively a role for ceramide in cell
eight stereoisomers of Cz-ceramideanddihydrocerregulation, two important questions were addressed. First,
amide were synthesized, and their cellular
activity was what is the mechanism of action of ceramide, and what is the
investigated. The four stereoisomers of Cz-ceramide
were active in inhibition of cell growth and induction proximal molecular target for ceramide? Second, what is the
of apoptosiswith modest differences in potency. On the structural specificity of ceramide action? In an attempt to
other hand, with Cz-dihydroceramide only the threo identify the molecular target for ceramide action, the effects
compounds were active in these assays whereas the of ceramide on protein phosphorylation were examined. These
erythro compounds were totally inactive. Thus, of the studies led to the identification of a ceramide-activated protwo naturally occurring molecules, the analog of D- tein phosphatase (CAPP)’ as an in uitro target for ceramide
(12).
erythro-ceramide (with the 4-6 trans doublebond)
was active, whereas the analog of D-erythro-dihydro- In a previous study, the structural and stereospecific receramide was inactive. These results demonstrate the quirements for ceramide effects on HL-60 cells wereevaluated
using N-acylphenylamino alcohol analogs (18).These studies
specificity ofceramide action andsuggestthatthe
introduction of the double bond
is critical for imparting demonstrated that the amide-linked acyl chain was required
the biochemical and biological
activity of ceramide.
for activity, and these compounds demonstrated stereospecificity of action such that ~-erythro-2-(N-myristoyl-amino)-lSphingolipids are now well recognizedas playing important
roles in cell recognition, modulation of cell growth and differentiation, cell-cell contact, and othermembrane functions (13). A rolefor sphingolipids in signal transductionandas
precursors to bioactive intracellular mediators was initially
suggested by the discovery of inhibition of protein kinase C
by sphingosine (4) and lysosphingolipids (5). Investigation of
this hypothesis led to the discovery of a sphingomyelin cycle,
whereby a number of extracellular agents such as la,25dihydroxyvitamin D3(6), tumor necrosis factor cr (7, 8), yinterferon (7), and interleukin 1 (9, 10) activate a neutral
sphingomyelinase resulting in hydrolysis of sphingomyelin
and the formation of phosphorylcholine and ceramide.
A role for the generated ceramide in mediating some of the
activities of these extracellular agents was demonstrated with
the use of cell-permeable ceramides, which were found to
induce leukemia cell differentiation, inhibit cell growth, downregulate the c-myc protooncogene, and modulate cellular protein phosphorylation (7,ll-13). Recent studies demonstrated
the ability of Cz-ceramide to induce DNA fragmentation
suggesting a role for ceramide as a mediator of programmed
* This work wassupported in part by National Institutes of Health
Grant GM-43825. The costs of publication of this article were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “aduertisement” in accordance with 18
U.S.C. Section 1734 solelyto indicate this fact.
5 To whom correspondence should be addressed Duke University
Medical Center, Box 3355, Durham, NC 27710. Tel.: 919-684-2449;
Fax: 919-681-8253.
phenyl-1-propanol (D-e-MAPP) was active, whereas its enantiomer L-e-MAPP was inactive. These differences were not
related to differences in uptake of these compounds, suggesting that their cellular activity was specific.
In thisstudy we examined the effects of the eight stereoisomers of C2-ceramideand Cz-dihydroceramide (Fig. 1)on cell
growth and programmed cell death. The most critical finding
to emerge from these studies is the lack of activity of Derythro-Cz-dihydroceramideas compared with D-erythro-Czceramide.
EXPERIMENTAL PROCEDURES
Materials
DL-erythro-Dihydrosphingosine, DL-threo-dihydrosphingosine,
succimic anhydride, ( R ) ( + ) - or (S)(-)-1-phenylethylamine, L-glutamic acid, and acetic anhydride, were purchased from Sigma. [3Hl
Acetic anhydride was obtained from Amersham Corp. Cell lines used
in this study were from American Type Culture Collection.
Methods
Preparation of the Four Stereoisomers of DihydrosphingosineRacemic DL-erythro- and DL-threo-dihydrosphingosines
were resolved
using the method of Stoffel and Bister (19) in which the racemic
mixture is reacted with optically active acids resulting in the formation of the diastereomeric salts that are then separated by differential
crystallization. Liberating the dihydrosphingosines from the salts
The abbreviations used are: CAPP, ceramide-activated protein
phosphatase; D-e-MAPP, ~-erythro-2-(N-myristoyl-amino)-l-pheny~1-propanol; L-e-MAPP, ~-erythro-2-(N-myristoyl-amino)-l-phenyl1-propanol; MS, mass spectroscopy.
26226
Selectivity of Ceramide-mediated Biology
26227
3.83-3.86(1H,m,HC2).
results in optically active enantiomers. The threo forms were sepa- HC3);3.67-3.75(2H,m,H2C1);
(ZR,3S)-~-Dihydro-C~-ceramide-MS
and 'H NMR are identical
rated via their glutamates and theerythro forms via the saltsof (R)or (S)-N-(1-phenylethy1)-succinamicacid. (R)- and (S)-N-(1-phen- to D-erythro-dihydro-C2-ceramide.
(2S,3S)-~-threo-Dihydro-C~-cerarnide-MS
is m/z, 344 (M+). 'H
ylethy1)-succinamicacids were prepared from succimic anhydride and
R(+)- or S(-)-1-phenylethylamine. L-Glutamic acid was recrystal- NMR (CD30D) 6 is as follows: 1.0(3H,t,CH3); 1.28-1.48(26H,sbr,
CH,);1.50-1.56(2H,m,CH2);2.08(3H,s,COCH3); 3.58(1H,dd, HC1);
lized from 50% ethanol.
Resolution of DL-erythro-Dihydrosphingosine-Equimolar DL-ery- 3.66 (lH,dd,HCl); 3.78-3.81(1H,m,HC3); 3.90-3.93(1H,m,HC2).
(ZR,3R)-~-threo-Dihydro-C~-ceramide-MS
and 'H NMR are identhro-dihydrosphingosine and (R)-N-(1-phenylethy1)-succinamic
acid
were dissolved in boiling ethyl acetate then refluxed for 5 min. The tical to L-threo-dihydro-Cz-ceramide.
Synthesis and Uptake of rH]D-erythro-C2-ceramide, PHJD-eryreaction mixture was cooled down slowlyto about 50-60 "C (crystallization was initiated) and left at room temperature overnight to thro-C2-dihydroceramide, and fH]~-threo-Cz-dihydroceramideThis com- Compounds were synthesized by acetylation of the long chain bases
precipitate the R salt of D-erythro-dihydrosphingosine.
with [3H](CH3CO)20and purified by TLC (chloroform, methanol, 2
pound was then crystallized twice from boiling ethyl acetate (45 'C
was liberated from its organic N NH40H, 4:l:O.l). Specific activity of the obtained 3H-labeled Nfor 4 h). D-erythro-Dihydrosphingosine
acetyl derivatives was -1 X lo3 cpm/nmol, and the purity was close
salt by acidification with HC1, and organic acid wasremovedby
extraction with CH2C12.The aqueous phase was alkalinized with 2 N to 100%.
Uptake-HL-60 human myelocytic leukemia cells were grown as
KOH, and D-erythro-dihydrosphingosinewas precipitated overnight
a t 4 "C. L-erythro-Dihydrosphingosinewas obtained similarly from described and treated with [3H]C2-ceramidesat concentrations rangDL-erythro-dihydrosphingosine and (SI-N-(I-phenylethy1)-succi- ing from 1 to 10 p ~ At
. the indicated time points, pellets were
namic acid.
separated from media and 3H radioactivity was counted.
Resolution of DL-threo-Dihydrosphingosines-A boiling solution of
Metabolism Study-Lipids from cells treated with 2 p M [3H]C2DL-threo-dihydrosphingosinein 95% EtOH was added to a boiling ceramides were extracted (Bligh and Dyer method) and applied to
solution of equimolar L-glutamic acid in 50% EtOH andcooled down TLC, and radioactive spots of [3H]C2-ceramideswere scraped and
slowly to room temperature. Crystallization was initiated, and after counted (no metabolites were evident following autoradiography).
3 h the glutamate of o-threo-dihydrosphingosinewas separated and
DNA Fragmentation Analysis"U937 monoblastic leukemia cells
recrystallized from ethanol. D-threo-Dihydrosphingosinewas liber- were treated with ethanol vehicle, each of the stereoisomers ofCpated from its salt by alkalinization with 2 N Na2C03, extracted,and ceramide, and dihydroceramide. GenomicDNAwas
isolated and
crystallized from chloroform. The first two mother liquors from the fractionated on 1.8% agarose gels for detection of fragments as
above were concentrated and crystallized from boiling methanol. The described (14).
precipitated salt of L-threo-dihydrosphingosinewas decomposed with
Analysis of HL-60 Cell Proliferation-HL-60 human myelocytic
2 N NaZCO3,and the liberated L-threo-dihydrosphingosinewas ex- leukemia cells were used between passages 20 and 45. The cells were
tracted and crystallized from chloroform.
grown in RPMI 1640 medium containing 10% fetal calf serum at
(2S,3R)-D-erythro-,(ZR,BS)-~-erythro-,(2S,3S)-~-threo-,and 37 "C in a 5% COz incubator at a cell density of 2 X lo5 cells/ml.
(2R,3R)-~-threo-~phingosine
were prepared in stereoselective synthe- Before treatment thecells were washed once with phosphate-buffered
sis from either L- or D-serine (20). The purity of the obtained saline and resuspended in serum-free media containing insulin (5
sphingosines was >95% by TLC (chloroform, methanol, 2 N NHIOH, mg/liter) and transferin (5 mg/liter) for 2-3 h. Cells werethen treated
4:l:O.l). Detection by KMnO, or ninhydrin spray gave a single spot with ceramides (in fresh ethanol solution) or with ethanol vehicle.
of each with characteristic differences in RF value between erythro Ethanol concentration was less than 0.1%. Cells were counted using
and threo forms: sphingosine, RF erythro = 0.32 and RF threo = 0.29; a hemocytometer, and cell viability was evaluated by trypan blue dye
dihydrosphingosine, RF erythro = 0.20 and RF threo = 0.17. The exclusion.
optical purity of the obtained enantiomeric pairs of erythro and threo
forms of sphingosine and dihydrosphingosine was established by CD
RESULTS
spectra of the N-acetyl derivatives (see below).
Preparation of C2-ceramides and Dihydro-C2-ceramides-Compounds were synthesized by acetylation of the respective long chain
Growth Inhibition by Stereoisomers of Cz-ceramideand C2bases with acetic anhydride and crystallized from ethanol. (Purity dihydroceramide-Previous studies indicated that induction
was >95% by TLC, chloroform, methanol, 2 N NH40H, 4:1:0.1,
of differentiation and inhibition of cell growth required the
KMnO, spray). All structures were verified by'H NMR and mass
presence
of an amide-linked acyl chain (11) and showed
spectroscopy. The optical purity was evaluated by CD spectroscopy.
'H NMR spectra were obtained on a G.E. 500-MHz Omega spec- specificity for D-e-MAPP but not itsenantiomer, L-e-MAPP
trometer. Fig. 2.4 shows proton NMR spectra for areas covering (18). Therefore, the four stereoisomers of C2-ceramide (Fig.
carbons 1,2, and3 of the erythro and threo isomers of C2 and dihydro- 1)were prepared from the corresponding optically and chemCz-ceramides (for one representative of each enantiomeric pair). ically pure sphingosine isomers. The erythro and threo isomers
Chemical shifts (6) are indicated in parts/million relative to trime- showed different RF values on TLC, and the D- and L-enanthylsilane as internal standard. The C1,C2, and C3 protons were
tiomers showed opposite CD spectra (Fig. 2 B ) .
assigned by COSYHY method (data notshown).
The effects of the four stereoisomers of C2-ceramideon cell
CD spectra (Fig. 2B) were recorded on an ISA Inc. JOBIN YVON
Spectropolarimeter, from 400 to 200 nm (2-s averaging time, 1-nm proliferation were evaluated (Fig. 3 ) . Treatment of HL-60
stop size, 2-nm bandwidth, I-cm path length). Mass spectra were cells with D-erythro-Cz-ceramide(1-5 p M ) for 2 days resulted
obtained with Hewlett-Packard 5988GO/MS/DS system. Samples in a dose-dependent inhibition of cell growth (Fig. 3A). Its
were analyzed using chemical ionization mass spectrometry.
(ZS,3R)-~-erythro-C~-Ceramide-MS
is m/z, 342 (M+). 'H NMR enantiomer, L-erythro-Cz-ceramide, was equally potent. The
(CD3OD) d is as follows: 0.84(3H,t,CH3); 1.25(2 OH,sbr,CHz); 1.38- diastereomer, L-threo-Cp-ceramide,was more potent than Derythro-Cz-ceramide whereas D-threo-C2-ceramide was less
1.42(2H,m,CH2); 1.95(3H,s,COCH3); 1.98-2.08(2H,rn,CH2); 3.603.70(2H,m,H2C1); 3.80-3.90(1H,m,HC2); 4.02-4.08(1H,m,HC3); potent (Fig. 3 A ) . The ICso for these compounds ranged be5.42-5.00(1H,m,HC4); 5.62-5.72(1H,m,HC5).
tween 2.5 and 4.5 p ~ Fig.
. 3B shows a detailed time course of
(ZR,3S)-~-erythro-C~-Ceramide-MS
and 'H NMR are identical to inhibition of cell proliferation by the four stereoisomers of
D-erythro-Cz-ceramide.
(ZS,3S)-~-threo-C~-Ceramide"MS
is m/z, 342 (M+). 'H NMR C2-ceramide. These results again show that the four com(CD3OD) d is as follows: 0.85(3H,t,CH3); 1.23(20H,sbr,CH2); 1.35- pounds showed persistent inhibition of cell growth with con1.42(2H,m,CHz);1.98(3H,s,COCH3); 2.00-2.08(2H,m,CH2); 3.55(1H, sistent differences in potency.
dd,HC1); 3.66(1H,dd,HCl); 3.83-3.88(1H,m,HC2);4.20-4.25(1H,m,
The effects of Cp-dihydroceramideswere investigated next.
HC3); 5.40-5.48(1H,m,HC4); 5.65-5.74(1H,m,HC5).
Initially, the racemic mixtures of DL-erythro-Cp-dihydrocer(ZR,3R)-~-threo-C~-Ceramide-MSand 'H NMR are identical to
amide and DL-threo-Cz-dihydroceramidewere employed beL-threo-Cz-ceramide.
(2S,3R)-D-erythro-Dihydro-C2-ceramide-MS
is m/z, 344 (M+). 'H cause of the availability of the corresponding DL-dihydroNMR (CD30D) 6 is as follows: 0.92(3H,t,CH3); 1.28-1.44(26H,sbr, sphingosines. In these studies, DL-threo-Cz-dihydroceramide
CHZ);1.48-1.56(2H,m,CHz); 2.08(3H,s,COCH3); 3.60-3.63(1H,m, produced a dose-dependent inhibition of HL-60 growth. The
Selectivity of Ceramide-mediated Biology
26228
-
OH
H
O
OH
1
T
R
NHCOCH,
HO
R
NHCOCH~
D-e- (2SJR)
L-ei2RJS)
-
OH
OH
1
R= [ -HC=CH-(CH2),2CH3]
Ceramides :
Dihydrocerurnides : R= [-CH2-CH2-( CH,),,CH,]
FIG. 1. Structureof Cz- and dihydro-Cz-ceramide stereoisomers. Shown are the structures for the four stereoisomers of Czceramide and dihydro-C2-ceramide whereR is part of the sphingoid
backbone. In the case of ceramide stereoisomers, sphingosine constitutes the sphingoid backbone, which contains a C4-C5 double bond.
In the case of dihydroceramides, the sphingoid backbone is dihydrosphingosine, which lacks the double bond. The R/S nomenclature is
indicated, which is the preferred method of describing the stereochemical configuration at carbons 2 and 3 because the erythrolthreo
designation can sometimes result in ambiguities of assignment.
A -
-4
B,
1
250
son
350
Wavelength (nm)
400
FIG. 2. Spectroscopic analysis of Cz-ceramide and dihydroCp-ceramide stereoisomers.
A, proton NMR spectroscopy. Shown
here is the C1, C2, and C3 proton regions, which were assigned by
COSY. Chemical shifts are indicated in parts/million (pprn) relative
to trimethylsilane. Results are shown for only one of each enantiomeric pair of the eight different stereoisomers. B, CD spectroscopy for
D-erythro-C+eramide compared with L-erythro-C2-ceramideand Deythro-dihydro-C2-ceramidecompared with L-erythro-dihydro-Czceramide.
IC50 for DL-threo-C2-dihydroceramidewas approximately 2
PM (Fig. 4A), similar to thatobtained with C2-ceramides.On
was largely
the other hand, DL-erythro-Cz-dihydroceramide
inactive over the same concentration range extending up to
10 p~ (Fig. 4A).In a time course study, threo-C2-dihydrocer-
amide inhibited cell growth over the duration of the experiment and was equipotent with Cz-ceramide (Fig. 4B).However, erythro-C2-dihydroceramide was largely inactive (Fig.
4B).The results with DL-erythro-Cz-dihydroceramide,therefore, offered compelling evidence for specificity of action of
ceramide and itsanalogs.
Because the inactivity of DL-erythro-C2-dihydroceramide
could arise from either lack of activity of both enantiomers
or the effects of one enantiomer opposed by the other, it
became important to resolve the racemic mixture into its
individual enantiomers. To this end, D- and L-erythro-dihydrosphingosines were resolved by reacting the racemic mixture with D- or L-N-(1-phenylethy1)-succinamicacids thus
generating two diastereomeric salts, which were separated by
differential crystallization. Similarly, the two enantiomers of
DL-threo-dihydrosphingosinewere resolved by reacting the
racemic mixture with L-glutamic acid, and theresulting diastereomeric salts were separated by differential crystallization.
Thus, thefour individual stereoisomers of dihydrosphingosine
were obtained, and the corresponding Cn-dihydroceramides
(Fig. 1)were prepared. These stereoisomers were purified by
TLC, and structure and
purity were verified by NMR and fast
atom bombardment mass spectroscopy. CD spectroscopy was
used to demonstrate optical activity of the enantiomers (Fig.
2).
The addition of the individual enantiomers of threo-C2dihydroceramide to HL-60 cells resulted in a concentrationdependent inhibition of HL-60 growth. Both D-threo-C2-dihydroceramide and L-threo-C2-dihydroceramidedisplayed an
IC50of approximately 2 PM (Fig. 5A), whereas both enantiomers of erythro-C2-dihydroceramide
were largely inactive over
the concentration range of 1-10 pM (Fig. 5A). In a timecourse
experiment, both threo isomers caused inhibition of growth,
whereas both erythro isomers were inactive (Fig. 5B). Therefore, the inactivity of erythro-C2-dihydroceramideis shared
by both enantiomers and appears to be a property of the
erythro conformation of ceramides lacking the 4-5 double
bond.
UptarZe and Metabolism-Because of the potential physiologic importance of the differential activity of D-erythroceramide and D-erythro-dihydroceramide,it became important to investigate the uptake andmetabolism of these compounds by cells in tissue culture to rule out physical causes
for the differences in biologic activity (such as differences in
solubility or uptakeby cells). For these experiments,tritiumlabeled D-erythro-C2-ceramide,the racemic DL-erythro-dihydro-C2-ceramide, and the racemic DL-threo-dihydro-C2-ceramide were prepared. When different concentrationsof these
labeled compounds were added to HL-60 cells, there was very
early uptake within a few minutes that was concentrationdependent (Fig. 6A). D-erythro-Cz-Ceramidewas taken up by
cells to a greater extent than thetwo other compounds such
that at 1 PM, approximately 50% of D-erythro-Cz-ceramide
was taken up compared with 40% of DL-threo-dihydro-Cz(Fig.
ceramide and 32% of DL-erythro-dihydro-C2-ceramide
6A). With increasing concentrations of these compounds
there was an increase in the absolute amount of compounds
taken up by the cells, but the proportion taken UP became
progressively less with increasing concentrations such that at
15 PM only 10-15% of the compounds were taken up by cells
(Fig. 6A). Moreover, the proportion of compounds internalized by cells was similar asdetermined by albumin wash
studies. In these experimentsthe ceramide analogs were backextracted with 0.1% albumin to remove surface-bound compounds. The amount of compounds remaining in cells was
then determined. As shown in Table I, the amounts of the
26229
Selectivity of Ceramide-mediated Biology
A
120 1
80
60
40
FIG. 3. Effects of the four stereoisomers of C2-ceramide on HL-60
cell proliferation. A , dependence of
cell growth inhibition on concentration
of C2-ceramides. HL-60 cells were grown
in the presence of ethanol vehicle or the
indicated concentrations of the four stereoisomers of C2-ceramide(erythro is indicated by small e and threo indicated by
small t ) . At 2 days, cells were counted,
and the results are expressed as number
of cells at 2 days as percent of control.
These results are averages of three determinations and are representative of
four experiments. B, time dependence of
HL-60 growth on Ca-ceramides. HL-60
cells were treated with 3 PM of each of
the four stereoisomers or with ethanol
vehicle, and cells were counted a t 1, 2, 3,
and 5 days. Results are expressed as
absolute cell density. The results are representative of four experiments.
20
0
0
2
1
3
4
6
Concentration (pM)
25-
+ Control
B
“
r
.
?
”
Q
G
t
o
20-
15
-
10
-
0
“-0“
1
D-ta
2
3
4
5
1
6
Time (days)
compounds internalized (resistant to albumin washing) were dro-Cz-ceramide are analogs of naturally occurring ceramides,
similar for Cz-ceramide (8.1%f 0.9), DL-erythro-dihydro-Cz- it is conceivable that differences in their biologic activities
ceramide (11.6% -+ 2.7), and DL-threo-dihydro-Cz-ceramide may berelated to metabolism of one or the other. For example,
(10.3% A 1.0).
Cz-ceramide could be a substrate for (ceramide-metabolizing)
In a time course study, Cz-ceramide was taken up by cells enzymes that would generate bioactive products, or dihydroat theearliest time point examined (approximately 2-3 min) Cz-ceramide could be metabolized into inactive products thus
with no further uptakedetected over the next 24 h (Fig. 6B). explaining the differences in biologic activity between the two
In comparison, DL-threo-dihydro-Cz-ceramide
was taken up compounds. Therefore, the metabolism of D-erythro-Cz-certo a lesser degree initially but quickly reached levels equiva- amide, DL-erythro-dihydro-Cz-ceramide,
and DL-threo-dihylent to those of Cz-ceramide (Fig. 6B). DL-erythro-Dihydro- dro-C2-ceramide was examined next. HL-60 cells were incuCz-ceramide was taken up the least initially and with slower bated with 3 PM of tritium-labeled ceramides, and thefate of
kinetics, but at 24 h, cells contained equivalent amounts of the compounds taken up by cells was determined (Fig. 6C).
the threecompounds (Fig. 6B). While these results show that Similar to a previous result (ll),the Cz-ceramidetaken up by
there are some differences in initial and subsequent uptake cells was poorly metabolized with greater than 90% of the
of the three different compounds, these differences do not amount taken up remaining in cells as intact Cz-ceramide at
explain the different biologic activities of the three com- 4 h and approximately 80% present still at 24 h (Fig. 6C).
pounds. For example, the uptake of DL-threo-dihydro-Cz- Nearly identical results were obtained with both DL-erythroceramide is very similar to that of DL-erythro-dihydro-Cz- dihydro-Cz-ceramide and DL-threo-dihydro-C2-ceramideinceramide at 10 pM (Fig. 6 A ) , although at this concentration dicating that these compounds are poorly metabolized once
DL-erythro-dihydro-Cz-ceramide
is totally inactive in growth they are takenup by cells. No labeled products were observed
inhibition (Fig. 44).
to account for the small fraction of the compounds that was
Because both D-erythro-Cz-ceramide and D-erythro-dihy- metabolized. Because the label was in the acetate group, this
Selectivity of Ceramide-mediated Biology
26230
-.f- D L t D H O
"0- DM-DHO
204
A
\
CI
0
V
0
A
4
2
8
6
10
-
1
12
Concentration (pM)
Concentration (pM)
+ Control
-
25
B
Control
.
I
- 0
""*".
X
DGe-DHO
DM-DHQ
20
B
WDHO
LtDHQ
15
10
5
0
2
4
6
Time (days)
0
n
1
2
3
4
6
Time (days)
FIG. 4. Effects of the racemic dihydro-Cz-ceramides on HL60 growth. A, dependence of HL-60 growth on concentrationof DL-
FIG. 5. Effects of dihydro-Cz-ceramide stereoisomers on
HL-60 growth. A, dependence of HL-60 growth on concentration
erythro-dihydro-C2-ceramideand DL-threo-dihydro-Cz-ceramide.
B, of the four stereoisomers of dihydro-Cz-ceramide.HL-60 cells were
time dependence of HL-60 growth on DL-erythro-dihydro-Cz-ceram- treated with the indicated concentrations of the four stereoisomers
ide and DL-threo-dihydro-Cz-ceramide.
HL-60 cells were treated with of dihydro-Cz-ceramide (DHC2) and cells were counted at 2 days
3 FM of either Cz-ceramide (C2), DL-erythro-dihydro-Cz-ceramide
or following treatment. Results are expressed in cell number as percent
DL-threo-dihydro-C2-ceramide,
and cells were counted at the indi- of control and are averages of three determinations, which are representative of three individual experiments. B , time dependence of
cated time points.
HL-60 growth on dihydro-Cz-ceramide stereoisomers. HL-60 cells
were treated with 3 PM of each of the four stereoisomers of dihydromost probably indicates that thecompounds were hydrolyzed Cz-ceramide (DHC2), and cells were counted at the indicated time
to generate sphingosine or dihydrosphingosine (respectively), points.
which would then enter sphingolipid biosynthetic pathways
unlabeled. Thus, differences in metabolism do not appear to
explain the major differences in biologic activity between
erythro and dihydro ceramides and the otherceramides.
The Effects of Ceramide Isomers on Apoptosis-In a previous investigation of the role of ceramide as a mediator of
antiproliferative activities and in an attempt to understand
the cytotoxic action of ceramide, we found that ceramide is a
potent inducer of programed cell death in U937 cells as
evaluated by the ability of C2-ceramideto induce internucleosoma1DNA fragmentation (14). C2-ceramide was active in
concentrations ranging from 1-5 PM,and DNA fragmentation
was evident as early as 2-4 h following treatment of cells with
C2-ceramide.Dihydro-C2-ceramidewas not active in that assay (14).(HL-60 cells did not undergo internucleosomal DNA
fragmentation in response to either Cn-ceramide or tumor
necrosis factor, and therefore the cytotoxic mechanisms of
tumor necrosis factor and ceramide in these cells remain to
be determined.) Therefore, it became important to determine
whether the cytotoxic activities of ceramide also displayed
similar specificities as effects on c-myc down-regulation and
the antiproliferative effects. U937 cells were treated with 5
p~ of each of the four stereoisomers of C2-ceramideand with
either DL-erythro-dihdyro-C2-ceramideor DL-threo-dihydroC2-ceramide. As shown before (14),D~-erythro-dihydro-C2-
-
DtC2
A
3
0
‘
20
26231
Selectivity of Ceramide-mediatedBiology
TABLEI
Effects of albumin washing on retention of ceramide isomers by cells
3H-labeledcompounds were added to HL-60 cells as described
under “Experimental Procedures.” Thecells were washed twice with
media containing 0.1% albumin (w/v), and the amount of compounds
associating with the cell pellet was then determined. These results
are averagesof four determinations.
Cellular ceramide
-
Isomer
1st wash
2nd wash
% of total k S.D.
ceramide added
10.
04
5
10
D-e-Cz-ceramide
15
DL-e-dihydro-CZ-ceramide
DL-t-dihydro-CZ-ceramide
Concentration (pM)
B
8.1 -+ 0.9
11.6 f 2.7
10.3 -+ 1.0
DISCUSSION
whJ
I
6.0 f 0.4
13.5 f 0.7
9.5 f 2.8
The results with the stereoisomers
of Cz-ceramideshow
that
there
are
modest,
but
consistent,
differences
in theeffects
40
_“”””“”
of the stereoisomers of C2-ceramide on cell growth with the
rank order of potency as follows: L-threo-C2> D-erythro-C2 =
L-erythro-Cz > D-thFeO-cz. The lack of majordifferences
among these stereoisomers of ceramide was somewhat unexpected in lightof the results withD-e-MAPP and L-e-MAPP
where only the D enantiomer was active. This discrepancy
D-e-CZ
raised the possibility that D-e-MAPP mimicked the action of
ceramide indirectly or was active on a distinct pathway. In
ongoing studies, we find that D-e-MAPP is capable of elevat20
ing endogenous levels of ceramide, whereas L-e-MAPP isnot.
Time (h)
Moreover, neither D-e-MAPP nor L-e-MAPP is capable of
activatingCAPPin vitro. Theseresults suggest that D-eDsC2
C
MAPP may function indirectly throughelevation of intracel0 DL-e-DHC2
lular
ceramide levels perhaps by interacting with ceramideDL-CDHCZ
metabolizingenzymes specifically. Furtherstudiesarerela,
quired to evaluate thesepossibilities.
En
The most significantbiochemical result to emerge from
these studies pertains to the lack of activity of D-erythro60
dihydro-Cz-ceramide. The predominant naturally occurring
ceramides are D-erythro-ceramide and D-erythro-dihydrocer40
amide. In other studies investigating the effects of ceramide
20
on S. cerevisiae, we have also found thatceramide is capable
of inhibitingyeast
growth andactivatingyeastCAPP,
0
0.5
1
21
whereas dihydroceramide was inactive (17). Also, phyto-CzTime (h)
ceramide was active in those studies. These results suggest
that dihydroceramide with the saturatedsphingoid backbone
FIG.6. Uptake and metabolism of tritium-labeled ceramides. A , dependence of uptake on concentration of ceramides. HL-60 is inactive whereas introduction of either a double bond (at
cellswere treated with tritium-labeled D-erythro-C,-ceramide, DL- C4-C5) or a hydroxyl group (at C4) results in a biologically
erythro-dihydro-C,-ceramide,or DL-threo-dihydro-C,-cerarnide.
The active molecule.
percent of radioactivity recovered in the cell pellet is indicated as a
The lack of activity of dihydroceramide mayhave important
function of the concentration of these compounds. These results are physiological consequences. First, the lack of activity of dihaverages of three determinations and are representative of five exrole in biosynthesis of
periments. Uptake was determined at 10 min following addition of ydroceramide is consistent with its
compounds. B, time course for uptake of ceramide analogs. HL-60 sphingolipids because it is biologically inert (at least in the
cells were incubated in the presence of 3 PM of each of the three assays performed in this study), and the formation of dihytritium-labeled compounds, and percent of compounds recovered in droceramide would not be detrimental to cell viability and
the cell pellet was determined at the indicated time points.C, metab- regulation of growth. The introduction of the double bond
olism of ceramide analogs. HL-60 cells were incubated with 3 P M of
the above tritium-labeled compounds. Lipids werethen extracted at into sphingolipids occurs a t a step following the formation of
double
the indicated time points and fractionated on TLC. The percent of dihydroceramide (21,22).This delay in introducing the
the tritium-labeled compounds recovered as intact parent compound bond may serve to protect cells from these regulatory funcexogenous
is indicated in the figure. These results are averages of three deter- tions of ceramide. In this context, the finding that
minations and are representative of three experiments.
ceramides concentrate in the Golgi apparatus (23) also fits
this hypothesis whereby cells would tightly control the formation of bioactive ceramides by delaying the introductionof
ceramide was not active, but all four stereoisomers of Cz- the double bondandsequestering
excess ceramide in the
ceramide were active (data not shown), althougha quantita- Golgi. Second, the important biologic activities of ceramide
tive assessment could not be performed because of the quali- may be regulated not only by the formationof ceramide from
tativenature of the assay. Thesestudiesunderscorethe
sphingolipid breakdown as occurs in thesphingomyelin cycle
specificity of programed cell death in response to
ceramide.
but may occur as a consequence of changes in the relative
_”””“
-
26232
Selectivity of Ceramide-mediated Biology
proportion of inactive dihydroceramide and active ceramides
through regulation of introduction of the double bond. Dihydroceramide reductase is a poorly characterized enzyme activity. It is possible that modulation of the activity of dihydroceramide reductase may result in significant changes in ceramide levels with biological consequences. At the least, these
differences between ceramide and dihydroceramide underscore the need to evaluate and measure the levels of not only
total ceramide but also resolving ceramide from dihydroceramide.
Another issue raised by these studies concerns the mechanisms responsible for the lack of activity of dihydroceramide.
In thebiologic assays, the lack of activity of dihydroceramide
does not appear to be explained by the small differences in
uptake or metabolism of dihydroceramide compared with
ceramide (especially since erythro- and threo-dihydroceramides show similar uptake but different bioactivity). Moreover,
in other in vitro studies dihydroceramide was unable to activate CAPP? Thus, any explanation for the differences in
activity must account for the selective interaction of ceramide
and dihydroceramide with CAPP in vitro. These differences
do not appear to be a result of the introduction of the double
bond per se because threo-dihydroceramide is active in cells
and in vitro. Rather, this may be a consequence of the configuration of erythro-dihydroceramide compared with erythroceramide. An important result emerging from NMR spectroscopy shows that a major difference between erythro-dihydroceramide and the other ceramides (including threo-dihydroceramide) is in the chemical shift value of the carbon 3 proton,
which appears to be the least shifted in erythro-dihydro-C2ceramide, perhaps indicating intramolecular interactions involving carbon 3 in dihydroceramide that prevent interaction
with CAPP. These speculations suggest the need for further
structure function studies and evaluations of different substiR. T. Dobrowsky and Y. A. Hannun, unpublished observations.
tutions at carbon 3 in the context of ceramide and dihydroceramide.
In conclusion, these studies provide further evidence for a
specific intracellular signaling pathway involving ceramide
that may be involved in the regulation of cell growth and
apoptosis.
Acknowledgments-We thank Dr. Hanna Gracz for performingthe
proton NMR studies and for excellent advice and Marsha Haigood
for expert secretarial assistance.
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