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Int. J. Cancer: 66,358-366 (1996)
0 1996 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publicationde I'Union Internationale Contre le Cancer
SPHINGOSINE AND ITS METHYLATED DERIVATIVE
N,N-DIMETHYLSPHINGOSINE (DMS) INDUCE APOPTOSIS
IN A VARIETY OF HUMAN CANCER CELL LINES
Elizabeth A. SWEENEY',~,
Chouhei SAKAKURA',
Tsutomu SHIRAHAMA',
Atsushi MASAMUNE~,
Hideki OHTA~,
Sen-itiroh H A K O M O Rand
I ~ . Yasuyuki
~
IGARASH11,2,3
The Biomembrane Institute and Department of Pathobiology, University of Washington, Seattle, WA, USA.
In the study of apoptosis initiated by various signals including induce apoptosis (Jarvis et al., 1994; Obeid et al., 1993).
ligands binding to cell membrane receptors such as Fas and Previously, we have reported that human neutrophils treated
TNFRl , the sphingomyelin pathway and its resulting metabo- with TNF-a (Ohta et al., 1995) and HL60 cells treated with
lites, the sphingolipids, have been suggested t o be involved in phorbol myristate acetate (PMA Ohta et al., 1994) exhibited
the signaling pathway. In earlier studies we presented data an increase in both Cer and Sph. Furthermore, we have shown
which indicated that sphingosine (Sph) itself was increased
that Sph exogenously added to neutrophils or HL60 cells
during apoptosis induced by phorbol myristate acetate (PMA)
in HL60 cells and tumor necrosis factor (TNF) in neutrophils, induces apoptosis in as little as 2 hr, whereas no effect was seen
and when added exogenously was able t o induce apoptosis. We at that time point with similar concentrations of the cellpermeable analogue of Cer, Cs-ceramide (CsCer), or with
report here that Sph and its methylated derivative N,N,dimethylsphingosine (DMS) are able t o induce apoptosis in Sph-1-phosphate (S-1-P; Ohta et al., 1995).
cancer cells of both hematopoietic and carcinoma origin. In
The current study examines the effect of Sph and its
human leukemic cell lines CMK-7, HL6O and U937, treatment
derivatives on apoptosis in a variety of human cancer cell lines
with 20 pM Sph for 6 hr caused apoptosis in up t o 90% of cells.
of both hernatopoietic and carcinoma origin. Derivatives
Human colonic carcinoma cells HT29, HRTI8, MKN74 and
COLO205 were shown t o be more susceptible to apoptosis upon examined included S-1-P, N,N,-dimethylsphingosine (DMS)
addition of DMS (>SO%) than of Sph (<SO%),
yet were weakly and N,N,N,-trimethylsphingosine (TMS). Unlike Sph, these
or not sensitive t o N,N,N-trimethylysphingosine (TMS). Under methylated derivatives cannot be acylated to Cer or become
phosphorylated to S-1-P. Our results were compared with
the same conditions, in the presence of serum, neither Sph- I
phosphate nor ceramide analogues Cz-, C6- or C8-ceramide effects of exogenously added analogues of Cer, which have
were able t o induce apoptosis in any cell lines. However, in the previously been reported to cause apoptosis (Jarvis et al., 1994;
absence of serum, ceramide analogues induced apoptosis in Obeid et al., 1993).
leukemia cell lines after 18 hr, yet much less so than Sph or
DMS. Furthermore, apoptosis inducedby Sph or DMS could not
MATERIAL AND METHODS
be inhibited by the ceramide synthase inhibitor fumonisin B I.
Apoptosis was not induced by sphingolipids in primary culture Culture of human cells
cells, such as HUVEC or rat mesangial cells, but was apparent in
Cancer and transformed cell lines used for these experitransformed rat mesangial cells. Additionally, apoptosis induced
by Sph, DMS or CzCerwas inhibited by protease inhibitors. Our ments were purchased from the ATCC (Rockville, MD), with
data further support the evidence that the catabolic pathway of the exception of the CMK-7 cell line, which was kindly
sphingomyelin involving Sph and other metabolites is an inte- provided by Dr. T. Sato (Sato et al., 1989), and HUVECs,
gral part of the apoptosis pathway.
which were purchased from Cell Systems (Kirland, WA).
-
o 1996 Wiley-Liss,Inc.
Apoptosis is a form of programmed cell death, the term
given to cellular death in which the cell itself is involved in its
own demise in responsc to specific external or internal signals.
Its hallmarks include nuclear compaction followed by internucleosomal degradation, cytoplasmic blebbing and condensation and, finally, disintegration into dense particles called
apoptotic bodies, which are then phagocytosed by macrophages or neighboring cells (for review, see Martin et al.,
1994). Apoptosis is known to be active during embryogenesis,
morphogenesis, cell selection and at the end of the cell's
pre-programmed life span, as in terminal differentiation. It can
also be activated in cells whose DNA has been damaged
beyond the cell's ability to repair it.
One mechanistic pathway proposed to be involved in signaling apoptosis is the sphingomyelin pathway (Hannun, 1995;
Hakomori and Igarashi, 1995). In this model, the enzyme
sphingomyelinase (SMase) is activated to cleave sphingomyelin (SM) into ceramide (Cer) and phosphocholine (PC). The
ceramide can be phosphorylated into Cer-1-phosphate or
deacylated to sphingosine (Sph). Both Cer and Sph have been
implicated as possible second-messengcr molecules in intracellular signaling. Cer has been suggested to be involved in
signaling apoptosis induced by the addition of extracellular
agents such tumor necrosis factor (TNF)-a (Dbaibo et al.,
1993; Kolesnick and Golde, 1994) or anti-Fas antibody (Cifone
et al., 1994), and analogues of Cer have been reported to
Suspension cell lines CMK-7, HL60 and U937 were grown in
RPMI 1640 media, and all others were grown in DMEM
supplemented with sodium pyruvate, sodium glutamate, penicillin-streptomycin and, unless noted otherwise, 10% heatinactivated (50°C for 30 min) FBS (Hyclone, Logan, UT).
For experiments performed in the absence of serum, hematopoietic cells were adapted to HL-1 serum-free media (Hycor,
Irvine, CA) supplemented with sodium glutamate by slowly
decreasing the amount of serum-containing RPMI and increasing the amount of HL-1 media over 1 week, until cells were
growing well in HL-1 media alone.
Isolation of glomeruli and culture of mesangial cells were
performed and the specificity of cell type confirmed as reported by Mackazy et al. (1988).
Treatment of cells with agents
DMS, TMS, S-1-P and CsCer were prepared in house
(Igarashi et al., 1989; Ruan et al., 1992; Vunnam and Radir,
1979). All other chemicals were purchased from Sigma (St.
Louis, MO) unless otherwise indicated. All lipids were dis7To whom correspondence and reprint requests should be sent, at
The Biomembrane Institute, 201 Elliott Ave. W., Suite 305, Seattle,
WA 98119, USA. Fax: (206) 281-9899.
Received: September 20, 1995 and in revised form December 12,
1995.
SPHINGOSINE AND DMS CAUSE APOPTOSIS
solved in ethanol/water (50/50) mixture as a stock solution,
then added directly to cell culture media. Cell cultures were
diluted with fresh media and incubated overnight to insure
that cells were in log phase before treatment.
Protease inhibitors were prepared in stock solutions as
follows: leupeptin 5 m M in distilled water, TLCK 20 mM and
ALLnM 10 mM in DMSO and PMSF 10 mM and TPCK 20
mM and 1 mM in ethanol. Fumonisin B1was diluted in PBS to
1mM. Final dilutions were added directly to cell cultures prior
359
to treatment with 20 p M Sph or DMS for 5-6 h r or, in cells
grown in HL-1 media, with 10 K M CzCer for 18 hr.
Analysis of DNA distribution by flow cytomety
The flow cytometric analysis of Nicoletti et al. (1991) was
modified for use in this study. Briefly, suspension cultures were
centrifuged, washed in PBS and resuspended in lysis buffer
(100 mM Na citrate with 0.1% Triton XlOO) containing 50
Kg/ml propidium iodide, then stored overnight at 4°C in the
dark. For adherent cell lines, detached cells in the culture
supernatant, washes and adherent cells which were detached
using trypsin/EDTA were collected by centrifugation, washed
DNA Content
FIGURE1 - Flow cytometry of sphingolipid-induced apoptosis in
hematopoietic cells. CMK-7, HL60 and U937 cells, grown in the
presence of 10% FCS, were untreated (Cont; U F C ) or treated with
20 pM of DMS (d-f, sphingosine (SPH; g i ) or Cz-ceramide
(CzCer;j-Z) for 6 hr. Sub-diploid peaks representative of apoptotic
DNA were apparent in cells treated with sphingosine or DMS but
not in ceramide analogues or untreated cells. Bar 1 measures
hypodiploid cells; bar 2, cells in G,/G1 phase of the cell cycle; bar
3, cells in G2/Mphase; and bar 4, total cells analyzed.
FIGURE2 - Morphology of sphingolipid-induced apoptosis in
hematopoietic cells stained with GiemsaIWright. CMK-7, HL60
and U937 cells, grown in the presence of 10% FCS, were untreated
(CONT) or treated with 20 p.M sphingosine (SPN) or DMS for 4
hr. Untreated cells exhibited a normal nucleus to cytoplasm ratio,
but condensed and fragmented DNA, apoptotic bodies and
membrane blebbing were observed in treated cells.
TABLE I - PERCENTAGE OF APOPTOTIC CELLS IN LEUKEMIC CELL LINES TREATED FOR 6 OR 18 HR
WITH 20 pM SPHINGOLIPIDS IN THE PRESENCE OR ABSENCE OF SERUM
Cell line
6 hr
CMK-7
CMK-7(SF)
HL60
HL60 (SF)
U937
U937 (SF)
18 hr
CMK-7
CMK-7(SF)
HL60
HL60(SF)
u937
U937 (SF)
Sphingolipids
s-1-P
-
+
++
-
SPN
DMS
+++++
+++++
++++
++++
++++
++++
+++++
++++
++
+
++++
++++
++++
++++
+++
+
++
ND
-I+
++
+
++
+++++
+++++
+++
+++
-/+
-
+
++
+
Data expressed using the following scale: -/+, <S%;
TMS
-
++
+++
+-
ND
-I+
ND
CzCer
CnCer
CsCer
-
-
-I+
-
-
-I+
-
-
++
+
-
-/+
-
-
-
-
-
+++
++
+
++++
-I+
++++ ++ - / +
-/+
ND
+++
++ ND
+, 10-25%; ++, 2545%; +++, 45-65%;
+ + + +, 6545%; + + + + +, > 85%. ND, not donc; SF, cells grown in serum-free media.
SWEENEY E T A L
360
with PBS and processed as above. Cells were analyzed using
the EPICS profile (Coulter, Hialeah, FL).
Analysis of DNA fragmentation by electrophoresis
To examine DNA, cells were treated and collected as above,
then resuspended in a TTE buffer (10 mM Tris HC1, 10 mM
EDTA, 0.1% Triton Xl00) containing 100 pg/ml proteinase K
and incubated for 18 hr at 50°C. RNAase was then added at
100 yg/ml and the mixture incubated for 1 hr at 37°C. DNA of
adherent cells was further processed by extraction with phenol/
chloroform/isoamyl alcohol followed by ethanol precipitation
and resuspended in TE buffer. DNA was then separated by
electrophoresis in agarose gel containing 1 pg/ml ethidium
bromide. Gels were examined and photographed under UV
light.
Examination of cellular morphology
To examine cellular morphology, cells were treated and
collected as above. Slides were prepared using a Cytospin
(Shandon, Pittsburgh, PA) and stained with Giemsa-Wright.
Cells were viewed and photographed using a Nikon inverted
microscope fit with a Nikon camera.
RESULTS
FIGURE
3 - DNA fragmentation in sphingolipid-treated hematopoietic cells. CMK-7, HL60 and U937 cells were treated for 4 or 6
hr in the presence of 10% FCS, and DNA samples were prepared
as described in “Material and Methods.” Treatments are marked
as untreated (C), sphingosine 20 I*.M(S20), DMS 20 I*.M(D20)
and DMS 10 pM (D10). No fragmented DNA was apparent in the
controls, but nucleosomal fragmentation associated with apoptosis
is clearly discernible in all 3 cell lines treated with either
sphingosine or DMS.
Sph and DMS induce apoptosis in hematopoietic cells
Hematopoietic cell populations routinely undergo apoptosis
during cell differentiation and selection. Consequently, leukemia cell lines, such as HL60 and U937, are commonly used in
the study of apoptosis. We examined the ability of sphingolipids and their derivatives to induce apoptosis in 3 human
leukemia cell lines, the promyelocytic cell line HL60, the
histiocytic lymphoma cell line U937 and the megakaryoblastic
cell line CMK-7. Initial studies employed the staining of
cellular DNA and distribution analysis by flow cytometry. Cells
were treated with Sph, its methylated derivatives DMS and
TMS, S-1-P or ceramide analogues C2Cer, C6Cer or C8Cer,
then stained and analyzed. In the resulting histograms, shown
in Figure 1, cells are gated for normal size in untreated
populations. Necrotic cells are much larger and are thus
excluded from analysis. Apoptotic cells appear as sub-diploid
peaks (represented here by reference line 1). In human
leukemic cell lines CMK-7, HL60 and U937, treatment with 20
p M Sph for 6 hr caused apoptosis in 85%, 75% and 36%,
respectively. DMS under the same conditions caused less
apoptosis, 65%, in CMK-7 cells but more apoptosis in HL60
(85%) and U937 (50%) cells. These responses were dose- and
time-dependent (not shown). Treatment with TMS at 20 pM
for 6 hr, however, showed apoptosis in only HL60 cells (60%),
not in the other 2 lines in the presence of FCS (though
apoptosis was induced in a low percentage of CMK-7 cells
grown in the absence of serum). Similarly, no apoptosis was
apparent after treatment for 6 hr with any of the ceramide
TABLE I1 - PERCENTAGE OF APOPTOTIC CELLS IN CARCINOMA, PRIMARY CULTURED AND TRANSFORMED
CELL LINES TREATED WITH 20 pM SPHINGOLIPIDS FOR 6 HR IN THE PRESENCE OF SERUM
Cell line
A431
MKN74
HT29
HRTl8
COL0205
HUVEC
Mesengial
SV40Mes13
Sphingolipid
s-I-P
SPN
DMS
ND
++
++
+++
+++
++
+++
++
-
ND
-!+
-
-
-I+
+
+++
-I+
-
+++
Data presented according to the scale used in Table I.
TMS
-I+
-
-!+
-!+
ND
ND
CzCer
ChCer
C&er
SPHINGOSINE AND DMS CAUSE APOPTOSIS
A431
MKN74
COL0205
361
HT29
HRTl8
E
4
DNA Conknt
FIGURE
4 - Flow cytometry of sphingolipid-induced apoptosis in human carcinoma cell lines. A431, MKN74, COLO205, HT29 and
HRTl8 cells, grown in the presence of 10% FCS, were untreated (Cont; a-e) or treated with 20 p M of DMS (f-j),sphingosine (SPN;
k+j or C2 -ceramide (C2Cer; pt) for 6 hr. Sub-diploid peaks representative of apoptotic DNA were apparent in cells treated with
sphingosine or DMS but not in those treated with ceramide analogues or untreated. Phases of the cell cycle are labeled as in Figure 1.
FIGURE5 - Morphology of sphingolipid-induced apoptosis in human carcinoma cell lines stained with Giemsa-Wright. MKN74,
HRT18, Col0205, A431 and HT29 cells were untreated (CONT) or treated for 6 hr in the presence of 10% FCS with 20 pM sphingosine
(SPN) or DMS. Untreated cells appeared normal, but DNA condensation, mebrane blebbing and apoptotic bodies were observed in all
treated cell lines.
362
SWEENEY ETAL.
plasm, thus illustrating a range of apoptotic stages. Apoptotic
bodies were apparent and increased in number with longer
treatment. Membrane blebbing was also apparent.
To observe if nucleosomal DNA fragmentation was also
induced by the lipids, hematopoietic cells were treated with
10-20 FM Sph or DMS for 4 or 6 hr, as indicated in Figure 3.
DNA was then extracted and separated on a 1.5% agarose gel.
No fragmentation was seen in controls for all 3 cell lines,
whereas in Sph- or DMS-treated cells the DNA ladder
indicating internucleosomal fragmentation typical of apoptosis
was quite clear at 4 hr, and by 6 hr nearly all DNA had
fragmented.
Apoptosis induction in serum-free cultures
Most published experiments to date on Cer-induced apoptosis were conducted in the absence of serum. For comparison,
then, hematopoietic cells were adapted to grow in HL-1
serum-free media and tested with agents as above. In comparison with those seen in Figure 1, responses to Sph and DMS
remained the same in the absence of serum after 6 hr of
treatment, but CzCer showed a small increase in the percentage of apoptosis (<20%) in HL60 and CMK-7 cells and
slightly higher in U937 cells (> 2S%), as summarized in Table
I. Cells were further treated for 18 hr and analyzed. As shown
in Table I, little change is seen from 6 to 18 hr of treatment in
the presence of serum. However, in the absence of serum,
apoptosis in CMK-7 or HL60 cells treated with C2Cer increased from less than 20% at 6 hr to 50% for CMK-7 cells and
more than 60% in HL60 at 18 hr. After 6 hr of treatment with
CdCer or C8Cer, no apoptosis was seen in these 2 cell lines. At
18 hr, apoptosis in cells treated with C&er increased to above
25% and a small percentage of apoptosis was seen with C8Cer.
In U937 cells treated for 6 hr with CfCer in the absence of
serum, apoptosis was observed in 25% of cells and, for C6Cer,
in 20%. These values also increased slightly after 18 hr.
Sph and DMS induce apoptosis in human carcinoma cell lines
As stated earlier, apoptosis is most widely tested in leukemia
cell lines. Apoptosis, however, is known to occur in most cell
types. Therefore, human colonic carcinoma cell lines HT29,
HRT18 and COLOZOS; stomach carcinoma cell line MKN74
and epidermoid carcinoma cell line A431 were also tested for
sensitivity to Sph and its derivatives. A summary of the flow
cytometry data on apoptosis induced by sphingolipids in cell
lines derived from solid tumors is presented in Table I1
FIGURE6 - DNA fragmentation in human tumor cell lines together with that of primary and transformed cell lines. All
grown in the presence of 10% FCS and treated with sphingolipids. cancer cell lines tested, except Colo205, proved to be more
Fragmentation is not observed in untreated (C) MKN74 cells, but susceptible to apoptosis upon addition of DMS ( > 50%) than
in those treated for 6 hr with 20 p M sphingosine or DMS a ladder Sph (25-50%), yet were weakly or not sensitive to TMS or
pattern indicative of inter-nucleosomalfragmentation is observed. S-1-P (Fig. 4). All results were dose- and time-dependent (data
Untreated A431 cells (C) and those treated with 5 pM of not shown).
C2-ceramide(Cer) for 12 hr also showed no fragmentation, but
We also tested the 3 Cer analogues under the same
cells treated with 5 pM sphingosine (SPN) or DMS for 12 hr
conditions as Sph and DMS (in 10% FCS), but in all cell lines
showed definite inter-nucleosomalfragmentation.
only a very weak response was seen after treatment with 20 pM
for 6 hr (Table 11). HT29 and HRT18 responded very slightly
analogues tested at concentrations from 2 to 20 KM in the to C2Cer (Fig. 4p-t) and ChCer. MKN74 and COL0205
presence of serum (Fig. l),nor with S-1-P in CMK-7 or HL60 responded just slightly to C&er only. No apoptosis was seen
cells (Table I).
with CsCer in any cell line. This response did not increase after
To examine cells for changes in morphology associated with incubation for 18 hr (data not shown).
apoptosis, treated and untreated hematopoietic cells were
To confirm the apoptosis observed by flow cytometry, cells
stained with Giemsa-Wright and observed under magnifica- were treated for 6 hr with Sph or DMS and stained with
tion. Figure 2 shows rcprcscntativc photographs of CMK-7, Giemsa-Wright. Untreated cells showed normal nuclei to
HL60 and U937 cells untreated or treated with 20 pM Sph or cytoplasm ratios and occasional cell division. Cells treated for
DMS for 4 hr. In untreated cultures, the majority of cells were 6 hr with 20 pM Sph or DMS showed darkly stained,
in GI phase with normal DNA to cytoplasm ratios. Occasional condensed nuclei corresponding to apoptosis with varying
cells in various stages of mitosis were evident. After treatment amounts of cytoplasm still associated (Fig. 5). Membrane
with Sph or DMS, cell nuclei had become condensed and blebbing was apparent in cells in early apoptosis, and apoptotic
fragmented and in some cases had lost all associated cyto- bodies were abundant in most cultures.
SPHINGOSINE AND DMS CAUSE APOPTOSIS
363
8
A
HUVEC
Mesangial
SV40 Mes 13
4
za
v)
DNA Content
FIGURE
7 - Effect of sphingolipidson apoptosis in primary cultured and transformed cell lines grown in the presence of 10% FCS. (a)
No apoptosis was apparent in HUVECs untreated (Cont) or treated for 6 hr with 20 pM sphingosine (SPN) or DMS. (6) Apoptosis is not
seen in normal rat mesangial cells, untreated (Cont) or treated for 6 hr with 20 p M sphingosine (SPN) or DMS, as indicated, but is
apparent in sphingosine- or DMS-treated SV40 Mes 13-transformed rat mesangial cells. (c) No inter-nucleosomal fragmentation is
apparent in DNA samples prepared from normal rat mesangial cells (lanes 5-7) or in SV40 Mes 13-transformed rat mesangial cells
untreated (lane 1) or treated with 20 pM C,-ceramide (lane 2). SV40 Mes 13 cells treated with 20 pM sphingosine (lane 3) or DMS (lane
4) exhibited ladder formation indicative of inter-nucleosomal fragmentation associated with apoptosis.
DNA samples from A431 and MKN74 cells were further
examined for the internucleosomal DNA fragmentation pattern often accompanying apoptosis. Both cell lines demonstrated some ladder-type DNA fragmentation after treatment
with DMS. Additionally, A431 cells showed similar fragmentation after treatment with 5 pM Sph for 12 hr but none after
CzCer (Fig. 6). MKN74 cells exhibited some fragmentation in
control DNA; however, no ladder appearance indicative of
nucleosomal fragmentation was observed and the control
culture appeared quite healthy, so this was determined to be
degradation after processing.
transformed rat mesangial cell line, SV40 Mes 13, Sph caused
apoptosis in SO% of cells and DMS in 40% (Fig. 7b). These
results were confirmed by fragmentation studies of DNA
samples prepared from primary cultured and transformed rat
mesangial cells untreated or treated with 20 pM Sph, DMS or
CzCer for 6 hr. No fragmentation was seen in control or
treated normal cells (lanes 5-7). Control transformed cells
(lane 1), as well as those treated with C2Cer (lane 2), also
exhibited no fragmentation. However, ladder formation is
apparent in transformed cells treated with Sph or DMS (lanes
3 and 4, respectively).
Apoptosis induction in primary cultures
and transformed cell lines
To examine the effect of Sph on normal cell lines, HUVEC
cultures at passages 5-7 were treated with sphingolipids and
observed for evidence of apoptosis. Almost no apoptosis was
seen after 6 hr treatment with any sphingolipid (Fig. 7a). After
18 hr, however, some apoptosis was apparent in DMS-treated
cells (data not shown).
Primary cultures of rat mesangial cells were also examined
for sensitivity to sphingolipids. Cells were isolated and cultured as indicated in “Material and Methods” and examined
morphologically and histochemically to confirm cell type
(Mackazy et al., 1988). Cells were examined for evidence of
apoptosis after treatment for 6 hr with 20 pM sphingolipids.
Similar to results seen with HUVECs, no apoptosis was
apparent in any of the treated cells (Fig. 7b). However, in a
Inhibition of sphingolipid-induced apoptosis
by protease inhibitors
It is accepted that the apoptotic process involves a protease
similar to those of the family known as ICE-like proteases.
Inhibitors of these enzymes inhibit apoptosis (Kumar, 1995).
We tested HL60 cells treated with 20 pM Sph or 20 pM DMS
for 5.5 hr or 10 pM CzCer for 18 hr in the presence of protease
inhibitors and analyzed them by flow cytometry. Figure 8a
summarizes the results of experiments using 5 different protease inhibitors, tosyl-L-lysine chloromethyl ketone (TLCK),
tosyl-L-phenylalanine chloromethyl ketone (TPCK), leupeptin
(Leu), phenylmethyl fluoro phosphate (PMSF) and acetylleucyl-leycyl-normethionel (ALLnM). The cysteine protease
inhibitor Leu at 25 pM inhibited apoptosis induced by the
sphingolipids by a minimum of 20%. Concentrations of up to
100 pM for the less specific cysteine protease inhibitors
364
SWEENEY ETAL.
ALLnM and PMSF failed to inhibit apoptosis. The serine
protease inhibitor TPCK greatly inhibited apoptosis induced
by all 3 agents, though DMS-induced apoptosis was only
partially inhibited. TLCK, also a serine protease inhibitor,
inhibited Sph-induced apoptosis completely and DMSinduced apoptosis by up to 70%, but at much higher concentrations. Figure 8b illustrates the ability of the 3 inhibitors to
block apoptosis in a dose-dependent manner. C2Cer-induced
apoptosis was the most sensitive to inhibition by the cysteine
inhibitor Leu, with 50 PM blocking 69% of the apoptosis. The
A
1
0SPN 20 uM
loo
e
G
n
80 -
DMS 20 uM
=
c
C, Cer 10pM
0
60m
._
40 -
c
Q
2
20-
ALLnM
100gM
TLCK
TPCK
ImM
100pM
Serine Protease
Inhibitors
Leu
25pM
PMSF
100pM
Cysteine Protease
Inhibitors
B
Leupeptin
1oc
80
60
40
20
0
I
I
I
I
0
10
20
30
I
40
I
same concentration was able to inhibit DMS-induced apoptosis only by 53% and that of Sph-induced by 40%. However,
TPCK was able to completely inhibit apoptosis induced by Sph
at the lowest concentration tested, 25 yM, yet DMS was
inhibited by 45% at o r above 50 p M ( p < 0.05). At 50 FM,
TPCK inhibited C2Cer by 85% ( p < 0.01); this dropped to
60% at 25 yM. At its effective concentrations, TLCK inhibited
DMS-induced apoptosis more effectively than TPCK, by up to
6 5 7 0 % ( p < 0.05). Sph-induced apoptosis was also inhibited
by more than 90% ( p < 0.01). These results support the
conclusion that these sphingolipids do indeed induce apoptosis
and are possible messcngers in apoptotic signaling. Furthermore, their apoptotic signal is apparently up-stream from the
protease(s) involved in apoptosis. The fact that CzCer is more
sensitive to the cysteine protease inhibitor and Sph and DMS
to the serine protease inhibitors also suggests that these 2
compounds are not affecting the pathway at the same point or
in the same manner. DMS and Sph, however, responded
similarly to serine protease inhibitors, yet DMS-induced apoptosis was never completely blocked. This, together with the
results that DMS is nearly always more effective in inducing
apoptosis, implies that DMS is the more potent inducer.
Sphingolipid-induced apoptosis in the presence of fumonisin BI
The conversion of Sph to Cer requires acylation of the
compound by the enzyme Cer synthase. The mycotoxin fumonisin B1 has been shown to completely inhibit this synthase
(Merrill et al., 1993), thereby blocking the formation of Cer. To
confirm that Sph was not causing apoptosis by being converted
to Cer, the induction of apoptosis by Sph was examined in the
presence of fumonisin B1. HL60 cells were treated with 20 p M
Sph or DMS in the presence of 25 y M fumonisin B1 for 5 hr
and analyzed for apoptosis by flow cytometry. In Figure 9, the
DNA distributions clearly demonstrate that apoptosis induced
by Sph was not affected by the fumonisin B,. Fumonisin B1
itself had no effect on the cells, and the apoptosis induced by
DMS, which cannot be acylated, was not affected by the
mycotoxin. Thcse results indicate that Sph is not being
converted to Cer prior to inducing apoptosis but is acting as
the inducer itself, further supporting the role of Sph or DMS
as messengers in apoptosis.
DISCUSSION
50
In the study of apoptosis through receptors such as Fas and
TNF, the sphingomyelin pathway has been implicated to be
involved in signal transduction. The data presented here
100
80
60
40
20
0
50
0
100
150
200
100 1.
TLCK
80
/
60
40
20
0
J
0
I
I
I
I
I
200 400 600 800 I000
Concentration (uM)
FIGURE 8 - Inhibition of sphingolipid-inducedapoptosis by protease inhibitors. (a) HL60 cells were treated for 5.5 hr with 20 FM
sphingosine (open bar) or DMS (hatched bar) or for 18 hr with 10
FM C2-ceramide(solid bar) in the presence of protease inhibitors
as indicated and analyzed by flow cytometry. The cysteine protease
inhibitor leupeptin inhibited sphingolipid-inducedapoptosis, especially C2-ceramide,but the less specific inhibitors ALLnM and
PMSF did not. TPCK and TLCK, serine protease inhibitors, were
able to completely block apoptosis induced by sphingosine and to
partially block that induced by DMS. (b) HL60 cells were treated
for 5.5 hr with 20 yM sphingosine (0)or DMS (W) or for 18 hr
with 10 yM C2-ceramide (A)in the presence of the indicated
concentrations of leupeptin, TPCK or TLCK, then analyzed by
flow cytometry. Leupeptin inhibited C2-ceramide-inducedapoptosis in a dose-dependent manner but weakly inhibited apoptosis
induced by sphingosine or DMS. Sphingosine- and C2-ceramideinduced apoptosis was completely inhibited by TPCK, yet DMSinduced apoptosis was only partially inhibited. TLCK effectively
inhibited both sphingosine- and DMS-induced apoptosis. Treated
cells in the absence of inhibitors were used as control, and the total
apoptosis for each treatment was assigned a 100% value (*p < 0.01,
+*p < 0.05).
SPHINGOSINE AND DMS CAUSE APOPTOSIS
Media
SPh
365
DMS
-
m
LL
I
z
m'
LL
+
DNA Content
FIGURE
9 - Sphingolipid-inducedapoptosis in HL60 cells in the presence of fumonisin B1. HL60 cells grown in 10% FCS were treated
for 5 hr with 20 pM sphingosine (b,e) or DMS (c,f) in the absence (a-c) or presence (d-f) of the ceramide synthase inhibitor fumonisin
B1(25 p,M) and analyzed by flow cytometry. No inhibition of apoptosis was seen in the presence of the inhibitor.
further support the evidence that this pathway, which is known
to involve precursors and catabolites of Sph, plays an integral
role in the mechanism of apoptosis. These results clearly show
that Sph and DMS, in a dose- and time-dependent manner,
cause apoptosis in some percentage of cells in all cancer cell
lines tested under conditions in which similar concentrations
of 3 Cer analogues, CzCer, C&er and C8Cer, were not able to
cause apoptosis. In comparison, Cer analogues did cause
apoptosis after 18 hr in hematopoietic cell lines grown in the
absence of serum. Sphinganine, which differs from Sph only in
the lack of the double bond, also caused apoptosis in the cells
tested (data not shown). However, S-1-P, which is a next step
in the metabolic pathway of Sph, did not cause apoptosis in
most cells tested. Furthermore, the methylated derivative
DMS, which is not further catabolized within the cell to either
Cer or S-1-P, induced apoptosis in all cancer cells tested. It
appeared to be a stronger inducer than Sph as it induced more
apoptosis and was less susceptible to inhibition by protease
inhibitors. Finally, the Cer synthase inhibitor fumonisin B1was
not able to inhibit apoptosis induced by Sph. These data
suggest that Sph and/or DMS may be a messenger in the
apoptotic pathway distinct from, and in addition to, Cer.
Our studies of Sph and DMS are performed in the presence
of 10% FCS, the normal growth conditions for cell culture, and
with the cells in logarithmic phase. Nearly all other studies
published on exogenously added ceramides use cells abruptly
removed from serum. In our experience, abrupt removal of
serum and the growth factors it contains can cause cell death,
even apoptosis, in a matter of hours, and it has even been
reported to increase cellular Cer levels (Jayadev et al., 1995).
Furthermore, Smith and Merrill (1995) have reported that
adding fresh media (without serum) to cells causes significant
changes in the levels of sphingolipids. To compare our results
with those published, the hematopoietic cells used in this study
were gradually adapted to grow in HL-1 medium. This medium
contains essential growth factors but no serum and therefore
eliminates some lipid-binding serum proteins. Overall, Sph
and DMS studies did not differ greatly in cells grown in serum
vs. those grown in HL-1, though the cells in the absence of
serum are slightly more fragile. However, abrupt withdrawal of
serum caused significant changes in all 3 cell lines, both
morphological and in the cell cycle, within 4 hr (data not
shown). Apoptosis assays are treated for a minimum of 2 hr,
and 6 hr or more is the usual treatment time. As incubations
for signal transduction assays can be as short as a minute, this
dilemma might not always apply, yet for observing cell responses in culture for apoptosis, differentiation or prolifera-
tion, this could be a crucial detail. In some cells, for example
HUVECs, removal from serum for 6 hr causes arrest in Go or
GI, with some apoptosis. It is probable, then, that the studies
done on serum-starved cells represent mixed effects of treatment and withdrawal of growth factors. It is, in the least, an
additional variable to be considered.
The effect of FCS on Cer-induced apoptosis may simply
reflect the presence of lipid-binding proteins on Cer analogues
or their ability to cross the cell membrane since significantly
higher concentrations of Cer can induce apoptosis even in the
presence of serum (though longer treatment times do not
increase apoptosis in the presence of serum). Yet, it is
conceivable that removal of serum and growth factors changes
the susceptibility of the cell, thus lowering some threshold for
the cell to suicide. The idea of a threshold for apoptosis is
considered by Fisher (1994), who discusses the concept that
different apoptosis thresholds exist for different cell types, as
in normal vs. malignant cells. Agents which cause apoptosis in
malignant cells cause only reversible cell cycle arrest in normal
cells, a fact exploited in many conventional anti-cancer therapies. Paradoxically, it appears that the same oncogenes that
transform cells actually sensitize them to apoptosis induced by
an exogenous agent, 2 of these genes being bcl-2 and c-myc,
which have been implicated as targets for the pathways
involving the SM pathway. Perhaps a disturbance in the
natural cell damage inspection and repair-or-apoptosis pathway allows uncontrolled proliferation but confers greater
susceptibility to apoptosis once triggered. The theory of
differing thresholds is supported by the data presented here on
HUVECs and on the normal and transformed mesangial cells.
Normal cells were not as susceptible to apoptosis induced by
sphingolipids as transformed cells or cancer cell lines, yet after
longer treatments some apoptosis was apparent. Furthermore,
we have evidence that Sph and DMS inhibit MAP kinase
activity, which is known to be involved in many signaling
pathways, and that the endogenous level of this enzymatic
activity in malignant cells might correlate with susceptibility to
apoptosis (data not shown).
ICE-like proteases, including apopain/CPP32, have been
implicated in the apoptosis pathway(s) in many cell systems
(Kumar, 1995; Tewari et al., 1995). Specific inhibitors of these
proteases are effectively able to block apoptosis induced by
ligands binding to the receptors TNFRl or Fas. Apoptosis
induced by sphingolipids can be inhibited by the serine
protease inhibitors TPCK and TLCK and, to some degree, by
the peptide inhibitor of cysteine proteases Leu, but not by the
366
SWEENEY ETAL.
less specific ALLnM or PMSF. This supports our theory that
Sph and DMS elicit a physiological signal on the cells and that
one or both may be signal messengers in apoptosis. It further
suggests that this messenger acts up-stream in the pathway
from one or more cellular proteases. It is also significant that
DMS-induced apoptosis is less inhibited than that of Sph by
both TLCK and TPCK. DMS may act more quickly or bind
more tightly with its target than Sph. It is unclear at present,
however, whether DMS and Sph are acting as different
messengers or if DMS is merely mimicking Sph.
Finally, addition of Sph has been shown to elicit similar
cellular responses as Cer, including regulation of protein
kinases (Pushkareva et al., 1992). Spiegel and co-workers have
also shown that Sph and S-1-P induce large increases in Ca2+
concentrations in cells (Zhang et al., 1990). Our studies using
HL60 cells and neutrophils show that TNF induces apoptosis
and raises cellular Cer levels and, interestingly, cellular Sph
levels after the rise in Cer (Ohta et al., 1994). Okazaki et al.
(1995) have reported differences in c-jun message induction in
response to Sph and Cer. That evidence, together with the data
reported here, supports the theory that Sph and/or DMS may
act as a messenger in the apoptosis pathway distinct from Cer
and that more than one pathway may be involved.
ACKNOWLEDGEMENTS
This work was supported in part by funds from The
Biomembrane Institute, in part under a research contract with
Otsuka Pharmaceutical Co. and Seikagaku Co. and by National Institute of Health Outstanding Investigator Grant
CA-42505 (to S. Hakomori).
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