CMLS, Cell. Mol. Life Sci. Vol. 57, 2000
Research Article
1119
2
00,000 g for 2 h in a HITACHI 55p-72 ultracentrifuge. Results
The cytosol produced was stored in aliquots under
liquid nitrogen.
Preparation of mouse liver nuclei. Mouse liver nuclei
were prepared as described previously [16]. In brief,
minced mouse liver was homogenized in homogeniza-
tion buffer (250 mM sucrose, 10 mM Hepes, pH 7.4, 15
C2-ceramide induces apoptosis in cultured HeLa
cells. C2-ceramide (50 mM) was administered to cul-
tured HeLa cells. No obvious morphological changes
occurred within 3 h. After 6 h, crevices appeared among
cells, and some cells were becoming round. With time,
more and more cells became round and detached, and
many refractive granules, not seen in normal cells, were
mM KCl, 2 mM MgCl , 5 mM EGTA, 0.5 mM PMSF,
2
0
.5 mM i-mercaptoethanol, 2 mM cytochalasin B). observed under the microscope. Many membrane-envel-
After filtration through a layer of 200-mesh silk screen, oped bodies characteristic of apoptotic bodies were
vol of homogenization buffer containing 2.3 M su- blebbed out of cells (fig. 1D). Within 24 h, more than
crose was added, mixed thoroughly and centrifuged 90% of cells were detached, while the normal cultured
RPS 50-2 rotor, 4 °C, 30 min, 124,000 g) into a cushion cells grew into a compact monolayer.
2
(
HeLa cells treated with 50 mM hydroxy-ceramides also
underwent apoptosis, but the apoptosis-inducing effect
was less. The apoptotic changes after 12 h in hydroxy-
ceramide-treated cells (fig. 1E) corresponded to those
treated with C2-ceramide after 7 h (fig. 1B).
Cells treated with nonhydroxy-ceramides at various
concentrations (5, 30, 50, and 150 mM) did not show
any morphological change, indicating that nonhydroxy-
ceramide is not able to induce apoptosis in cultured
HeLa cells (data not shown).
Cells stained with DAPI were examined with a fluores-
cent microscope. Chromatin condensation, margina-
tion, and release of apoptotic body-like granules that
were finally extruded from nuclei were observed (fig. 2).
However, there were some differences between ce-
ramide-induced HeLa apoptosis and cytochrome c-in-
duced nuclear apoptosis, in that the chromatin
consisting of homogenization buffer plus 2.3 M sucrose.
The pellet was resuspended in nuclei stock solution (10
mM Pipes, 80 mM KCl, 20 mM NaCl, 250 mM su-
crose, 5 mM EGTA, 0.5 mM spermidine, 0.2 mM
spermine, 50% glycerol) at a concentration of 5×10
nuclei/ml and stored under liquid nitrogen.
Isolation and purification of mitochondria [17]. Livers
from mouse were rinsed with ice-cold PBS, minced and
homogenized in three volumes of H-buffer [0.07 M
4
sucrose, 0.21 M
D-mannitol, 2 mM Hepes-KOH, pH
7
.4, 0.05% BSA-V (w/v) (defatted)]. The homogenates
were centrifuged at 1100 g for 3 min and the superna-
tants were centrifuged at 6780 g for 15 min. Pellets were
resuspended in 1/4 volume of H-buffer and centrifuged
at 20,200 g for 15 min. The sediments were crude
mitochondria. These were resuspended in 1/8 volume of
H-buffer, centrifuged at 3000 g for 3 min, and the condensation in cytochrome c-treated nuclei was on a
supernatant was retained. The sediments were resus- much larger scale: the chromatin marginated and subse-
pended and centrifuged once more. The two superna- quently underwent dramatic hypercondensation into
tants were pooled and centrifuged at 20,200 g for 20 sharply defined spherical domains (fig. 6B).
min. The upper fluffy pellet was removed. Then, 1/8 To confirm that the morphological changes in HeLa
cells treated with C2-ceramide reflected apoptosis, cells
were fixed with glutaraldehyde and examined under a
transmission electron microscope. Membrane blebbing
in the cytoplasm, nuclear shrinkage, and chromatin
condensation were all observed in C2-ceramide-treated
HeLa cells (fig. 3).
Analysis of chromatin DNA from C2-ceramide-treated
cells demonstrated that nuclear chromatin was frag-
mented. The cleavage of DNA increased as the concen-
tration of C2-ceramide increased (fig. 4A). Figure 4B
shows that DNA fragmentation increases with time.
Caspase inhibitors block C2-ceramide-induced HeLa
apoptosis. When caspase inhibitors AC-DEVD-CHO
and AC-YVAD-CHO were added to the culture
medium before the administration of C2-ceramide,
apoptosis was inhibited, to different degrees, with AC-
DEVD-CHO having a stronger effect than AC-YVAD-
volume of H-buffer was added to the tube, and mito-
chondria were collected by centrifugation at 20,200 g
for 20 min. They were re-suspended in H-buffer,
counted and store in liquid nitrogen.
Assay of cell-free apoptosis. The reaction mixture con-
taining 50 ml of egg extract buffer or egg extract s-200
5
and ꢀ1×10 mouse liver nuclei was supplemented
with isolated mitochondria and incubated at 22 °C for
the times indicated. In vitro apoptosis was monitored as
described previously [15]. After incubation, 10 volumes
of buffer D (100 mM Tris-HCl, pH 8.0, 5 mM EDTA,
0
.2 M NaCl, 0.4% SDS, 0.2 mg/ml proteinase K) was
added to each reaction and incubated at 37 °C
overnight. The DNA was deproteinized with phenol
and phenol-chloroform (1:1), precipitated with 2 vol-
umes of ethanol, and then loaded onto a 1.5% agarose
gel, electrophoresed, and visualized by staining with CHO. However, their effects decreased with time.
ethidium bromide and illumination with short-wave UV Within 24 h, most cells had died and detached, regard-
light.
less of the addition of caspase inhibitors (fig. 5).