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* INSERM E9910, Institut Claudius Régaud, 31052 Toulouse, France;
Institut de Pharmacologie et de Biologie Structurale, UPR 9062 CNRS, Toulouse, France;
Laboratoire de Biochimie Médicale, INSERM U466, CHU Rangueil, 31403 Toulouse, France; and
§ Service d’Hématologie, CHU Purpan, 31059 Toulouse, France
2Correspondence: INSERM E9910, Institut Claudius Régaud, 20 rue du Pont St. Pierre, 31052 Toulouse, France. E-mail: jaffrezou{at}icr.fnclcc.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: myeloid cells • nuclear signaling • ionizing radiation • resistance
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
.
Radiobiologists have generally admitted that the induction of apoptosis
is mediated by a cascade of events initiated by the direct effect of IR
on the target cell nucleus (3
, 4)
. The prevailing paradigm
of the lethal effects of IR identifies double-stranded DNA breaks as
the critical lesions (5)
. Differences in the intrinsic
radiosensitivity of human cells to IR is believed to be related mostly
to the rate and fidelity of double-strand break rejoining in which
transcriptional activation of early response genes plays a key role
(reviewed in ref 6
). This has led to the present concept
that IR therapy is determined by the effective activation of an
apoptotic process in tumor cells in response to the induced DNA damage
and that genotypic alterations in tumor cells that interfere with DNA
damage-induced apoptosis confer resistance to IR (7
, 8)
.
Several groups provide ample evidence that the signaling cascade
consisting in sphingomyelin (SM) hydrolysis through the activation of a
sphingomyelinase (SMase) with concomitant generation of ceramide (CER)
can mediate the IR-triggered apoptotic response (9
10
11
12
13)
.
The specific steps along the CER signal transduction pathway have,
however, yet to be delineated. Identification of the SMase(s) involved
in CER generation has also proved to be equivocal (reviewed in ref
14
).
To date, both acid SMase and neutral SMase have been implicated in
apoptosis. Recent reports presented conflicting conclusions,
demonstrating either a defective or a normal response in
A-SMase-deficient human cells (Niemann-Pick disease) or in cells
derived from A-SMase knockout mice, even though the same stimulus and
the same cell lines were sometimes used (reviewed in ref
14
). Moreover, in a study using A-SMase knockout mice, a
defective apoptotic response to IR was observed in the lung but not in
the thymus (10)
. This conflicted with a previous report by
the same group that suggested a role for neutral SMase in irradiated
bovine endothelial cells (9)
. More recently, two studies
designated neutral but not acid SMase as responsible for SM hydrolysis,
CER generation, and apoptosis in irradiated myeloid TF-1 cells and
lymphoid WEHI 231 JM cells (11
, 12)
. Regardless of the
identity of the SMase, the apparent cytosolic or plasma membrane
location of these enzymes argues for an extranuclear origin in IR
mediated apoptosis signaling. Indeed, the description that IR can
trigger CER generation independently of IR–DNA interaction
(9)
and that the cytosol is solely required for
IR-mediated apoptosis (11)
has astounded the field of
radiobiology and has profound implications in our understanding of
radiosensitivity and radioresistance.
To address the question of the role of the nucleus in IR-triggered CER
generation and apoptosis, and in light of the fundamental importance of
IR toxicology in hematopoietic cells, we used in the present study two
previously described IR-sensitive and IR-resistant myelocytic cell
lines TF-1–33 and TF-1–34, respectively (12)
. We show
that CER generation through the activation of a neutral SMase in
IR-sensitive TF-1–33 cells was only observed in highly purified nuclei
preparations, and not in other subcellular preparations. Moreover, the
lack of an apoptotic response in TF-1–34 cells was correlated with the
failure in the induction of nuclear CER signaling. These results point
to the existence of a novel SM-CER signaling pathway implicated in
IR-triggered DNA laddering and protease activation of myeloid cells
that may be independent of the classical ‘central cytosolic
executioner of apoptosis’ (e.g., cytoplasmic caspase activation,
mitochondrial disruption, cytochrome c release)
(15)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
The human erythro-myeloblastic cell line TF-1 (16)
was generously provided by Dr. W. Vainchenker, INSERM U362, Villejuif,
France. TF-1 subclones were obtained by granulocyte-macrophage
colony-stimulating factor withdrawal and limited dilution. Two clones
were chosen based on autonomous growth and phenotypic characteristics:
TF-1–34 (CD34+, CD33-, CD38+, CD41+, glycophorin A-); TF-1–33 (CD34-,
CD33+, CD38+, CD41+, glycophorin A-) (12)
. Cells were
cultured in RPMI 1640 medium supplemented with 10% heat-inactivated
fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100
µg/ml streptomycin (all from Eurobio, Les Ulis, France). Cells were
maintained at 37°C in a humidified atmosphere containing 5%
CO2. Cell stocks were screened routinely for
Mycoplasma (Stratagene Mycoplasma PCR kit, La Jolla,
Calif.).
Isolation of nuclei
Isolation and purification of intact TF-1–33 and TF-1–34
nuclei were performed essentially as described previously
(17)
. Briefly, 5 x 106 cells
were pelleted and resuspended in extraction buffer [20 mM
N-(2-hydroxyethyl) piperazine-N' (2-ethane) sulfonic acid (HEPES), pH
7.4, 10 mM MgCl2, 2 mM ethylene
diaminetetra-acetic acid (EDTA), 5 mM DTT, 100 mM sodium orthovanadate,
100 mM sodium molybdate, 10 mM ß-glycerol-phosphate, 750 µM ATP, 1
mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 1 µg/ml
pepstatin A (0.02% Nonidet-40, which significantly increased yield,
was also added)]. Cells were incubated on ice for 20 min and disrupted
with 20 strokes of a Dounce homogenizer. The homogenate was layered
onto 1 ml of 1 M sucrose and centrifuged at 2000 g for 15
min to pellet the nuclei. The nuclei pellet was then resuspended in
complete RPMI 1640 medium and gently washed twice in complete medium.
Only fresh nuclei preparations were used in experiments.
Preparations of nuclei-free lysates
Preparation of nuclei-free cell membranes was performed as
described previously with slight modifications (18)
.
5 x 106 cells were pelleted, washed in
phosphate-buffered saline (PBS), and resuspended in hypotonic buffer
(25 mM Tris-HCl, pH 7.4, 2 mM EDTA, 0.05% bovine serum albumin, 0.2 mM
phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 1 µg/ml
pepstatin A). Cells were then disrupted using a Balch ball-bearing
device (18)
, which essentially peels open the plasma cell
membrane while conserving organelle integrity. Lysates were centrifuged
at 500 g for 30 min at 4°C and the postnuclear
supernatants were recuperated and used fresh.
Cytoplast preparation
Cytoplasts were prepared essentially as described previously
(19)
. 1 x 107 cells were
incubated at 37°C for 30 min in complete RPMI 1640 medium containing
20 µM cytochalasin B. Cells were then layered on top of a
discontinuous Ficoll density gradient containing 20 µM cytochalasin B
(12.5%/16%/25%). These were centrifuged for 1 h at 46,000
g in a TH641 swing-out rotor (Dupont, Wilmington, Del.).
After centrifugation, cytoplasts were harvested from the interface of
the 12.5%/16% Ficoll gradient and resuspended in complete RPMI 1640
media. Absence of intact cells was confirmed by 4', 6'-diamidino
2-phenylindole (DAPI) staining.
Quality control of nuclei, cytoplast, and lysate preparations
Purity of nuclei, cytoplasts, and nuclei-free lysates was
monitored by enzymatic assays: lysosomal ß-glucosidase
(20)
, mitochondrial succinate dehydrogenase
(21)
, Golgi galactosyltransferase (22)
,
endoplasmic reticulum glucose-6-phosphatase (23)
, and
plasma membrane [14C]-concanavalin binding
(24)
; as well as immunoblot analysis using the ECL
detection system (Amersham, France) with anti-PDGF receptor (plasma
membrane marker), anti-Golgi, anti-human mitochondria, anti-histone
monoclonal antibodies, and a polyclonal anti-acid sphingomyelinase
(lysosome marker). Analysis of the structural integrity of nuclei and
cytoplast preparations was assessed by means of electron microscopy.
Electron microscopy
Cells, nuclei, or cytoplast preparations were fixed with 2.5%
glutaraldehyde (w/v) in PBS. After 24 h at 4°C samples were
treated with 1% osmium tetraoxide in PBS, dehydrated in ethanol,
followed by epoxy-1,2-propane, and embedded in Epon for 48 h at
60°C. Ultrathin sections obtained with a diamond knife (Diatome Co.,
Bienne, Switzerland) were counterstained with 5% uranyl acetate in a
30% ethanol solution, followed by lead citrate. These sections were
observed with a Philips EM 301 electron microscope (Eindhoven,
Holland).
Irradiation
Irradiations were performed using a 60Co
source (1.25 MeV; Alcyon, General Electric) at a dose rate of 
1
Gy/min.
Metabolic cell labeling and sphingolipid quantitation
SM quantitation was performed by labeling cells to isotopic
equilibrium with 0.4 µCi/ml of
[methyl-3H]choline (specific activity 81.0
Ci/mmol, DuPont NEN, Les Ulis, France) for 48 h in complete medium
as described previously (25)
. Cells were then washed; cell
preparations were performed and resuspended in serum-free medium for
kinetic experiments. Aliquots were taken for protein determination
(26)
. Radioactive SM was extracted with
chloroform:methanol (2:1,v/v), vortex mixed and centrifuged at 1000
g for 15 min (27)
. The lower (organic) phase
was removed, washed with chloroform:methanol:water (3:48:47), and
evaporated. SM was quantitated by scintillation counting. Similar
results for SM quantitation were obtained after thin-layer
chromatography, where SM spots were identified based on Rf and
comigration with authentic standards (data not shown) and with SM
quantitation determined through phosphorus measurements after
mineralizing phosphorus and spectrophotometric analysis
(29)
.
Total cellular CER quantitation was performed by labeling cells to
isotopic equilibrium with 1 µCi/ml of [9,
10-3H]palmitic acid (53.0 Ci/mmol, Amersham,
France) for 48 h in complete medium as described previously
(25)
. Cells were then washed and resuspended in serum-free
medium for kinetic experiments. Lipids were extracted and resolved by
thin-layer chromatography (29)
, CER was scraped and
quantitated by liquid scintillation spectrometry. Alternatively,
intracellular CER was quantitated using Escherichia coli
diacylglycerol kinase (Amersham, kit no. RPN200) and
[33P]
-ATP (3300 Ci/mmol, Isotopchim,
Ganagobie, France) according to previously published procedures
(30)
. Statistical analyses were performed by the
Student’s t test.
Quantitation of acid and neutral sphingomyelinase activities
SMase assays were performed as described previously
(25)
. To measure acid SMase activity, cell pellets were
resuspended in 0.1% Triton X-100 and incubated for 15 min at 4°C before homogenization. About 100 µg of cellular lysate protein
was incubated for 1 h at 37°C in a buffer containing 250 mM
sodium acetate (pH 5.0) and 1 mM EDTA and
[choline-methyl-14C]SM (54.5 mCi/mmol, NEN DuPont; 120,000 dpm/assay). To measure neutral SMase activity, cells
were resuspended in 0.1% Triton X-100, 20 mM HEPES (pH 7.4), 10 mM
MgCl2, 2 mM EDTA, 5 mM dithiothreitol, 0.1 mM
Na3VO4, 0.1 mM
Na2MoO4, 10 mM
ß-glycerophosphate, 750 µM ATP, I mM phenylmethylsulfonyl
fluoride, 10 µM leupeptin, and 10 µM pepstatin. After incubation
for 5 min at 4°C, cells were homogenized and 100 µg of
cellular lysate protein was incubated for 2 h at 37°C in 20 mM
HEPES (pH 7.4), 1 mM MgCl2, and
[choline-methyl-14C]SM (120,000 dpm/assay). The
radioactive phosphocholine produced was extracted with
chloroform/methanol (2:1) and quantitated by scintillation counting
(27)
. Under these conditions, enzymatic activities were
linear during incubation times.
Measurement of phosphatidylserine externalization by annexin V
binding
5 x 106 cells or cytoplasts were
pelleted and resuspended in HEPES buffer (10 mM HEPES-NaOH, pH 7.4, 150
mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM
CaCl2), then incubated for 5 min with 1 µg/ml
annexin V-FITC (Bender Medsystem, Vienna, Austria) and 10 µg/ml
propidium iodide, followed by flow cytometry on a FACScan (Becton
Dickinson, Rutherford, N.J.) (31)
.
Morphological analysis
Changes in nuclear chromatin were evaluated by fluorescence
microscopy by DAPI staining (32)
.
DNA analysis
DNA was resolved on a 1.8% agarose gel and visualized with
ethidium bromide as previously reported (33)
. Quantitative
DNA fragmentation was determined by the spectrofluorometric DAPI
procedure as described previously (34)
.
PARP cleavage assay
Analysis of poly(ADP-ribose)polymerase (PARP) proteolysis was
assessed by resuspending cells in sample buffer (62.5 mM Tris, pH 6.8,
4 M urea, 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 0.04%
bromphenol blue). Samples were boiled for 5 min, loaded onto a 10%
SDS-polyacrylamide gel, electrophoresed, and transferred to a
nitrocellulose membrane. PARP and its cleaved fragment were detected by
using a rabbit polyclonal antiserum (Boehringer-Mannheim, Meylan,
France) and a donkey anti-rabbit secondary antibody (Immunotech,
Marseille, France). The signal was visualized by enhanced
chemiluminescence (Amersham, Buckinghamshire, U.K.).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, markers for Golgi, mitochondria, plasma membrane, and lysosomes were
essentially absent in nuclei fractions. Only a small
glucose-6-phosphatase activity was observed, which, as
expected, reflected that minor endoplasmic reticulum material was still
associated to the nuclear membranes. Furthermore, as seen in Fig. 1A
, both TF-1–33 cells and nuclei-free lysates, but not the
purified nuclei preparations, were positive for anti-PDGF receptor
(plasma membrane marker), anti-Golgi, anti-human mitochondria, and
anti-acid sphingomyelinase (lysosomal marker). Moreover, both TF-1–33
cells and purified nuclei, but not the nuclei-free lysate preparations,
presented a positive signal for anti-histones (nucleus marker). The sum
of these data indicated that the nuclear preparations were devoid of
contaminating plasma membrane, mitochondrial, Golgi, and lysosomal
material. Similarly, the nuclei-free lysates contained all cellular
organelles except for the nucleus. Finally, cell disruption triggered
neither CER generation, SMase activation (basal sphingolipid levels and
SMase activities are listed in Table 2
), nor cytochrome c release in either cell line (data not
shown).
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The purity of the cytoplasts assessed by DAPI staining
demonstrated that these were essentially free (> 95%) of
contaminating intact cells or isolated nuclei (data not shown).
Analysis by electron microscopy revealed a well-preserved cellular
architecture with little to no damage of organelles (Fig. 1B
). Analysis of the structural integrity of nuclei
preparations revealed the presence of essentially intact nuclei.
Identical results were obtained with TF-1–34 preparations (data not
shown).
Ceramide generation in irradiated nuclei-free cell lysates,
cytoplasts, and purified nuclei preparations
To identify whether CER generation was triggered by IR, TF-1–33
and TF-1–34 cells were prelabeled with
[9,10-3H]palmitic acid to equilibrium for
48 h. Cells were washed and resuspended in serum-free medium;
lysates, cytoplasts, and nuclei were prepared, after which cells and
cell preparations were irradiated at 12 Gy. In the IR-sensitive cell
line TF-1–33, basal CER level was measured at 1.9 nmol/mg protein (95
pmol/µg DNA) (Table 2)
and, as expected (12)
, a 42%
increase in CER was observed 8 min post-IR in TF-1–33 but not in
the IR-resistant cell line TF-1–34 (Fig. 2A
). Remarkably, we failed to demonstrate CER generation in
the nuclei-free lysates as described previously (9
, 11)
or
in cytoplasts. However, we did observe a distinct 194% CER peak in the
purified nuclei preparations of TF-1–33 cells, where basal CER levels
were measured at 54.3 pmol/mg nuclear protein (4.0 pmol/µg DNA). No
increase in CER was found in TF-1–34 nuclei preparations (Fig. 2A
).
|
To ascertain whether IR-triggered CER generation resulted from SM
hydrolysis, cells were labeled with
[methyl-3H]choline, and cells and cell
preparations were irradiated at 12 Gy as described above. Basal SM
level in TF-1–33 cells was 23 nmol/mg protein (1.1 nmol/µg DNA)
(Table 2)
. A cycle of SM hydrolysis (> 22%) was observed in the
sensitive cell line TF-1–33, but not in the resistant cell line
TF-1–34. We did not observe SM hydrolysis in the nuclei-free lysates
or in cytoplasts. However, we did find significant SM hydrolysis
(30%) in the purified nuclei of TF-1–33 cells but not TF-1–34
(Fig. 2B
).
Since the product of IR-induced SM hydrolysis is CER, the enzyme
involved in this reaction is a SMase. To determine the identity of the
SMase implicated, we measured both acid and neutral SMase activities
(Table 1)
. No significant acid SMase activity was measured in purified
nuclei of either cell line. Acid SMase activities were unaffected by IR
in both TF-1–33 and TF-1–34 cells and cell preparations (Fig. 3A
). However, we did observe an increase in neutral SMase
activity triggered by IR in both TF-1–33 cells and purified nuclei,
but not in nuclei-free lysates or cytoplasts (Fig. 3B
).
Neutral SMase activity in TF-1–34 cells remained unaffected.
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IR induced apoptosis in cell preparations
Since phosphatidylserine externalization generally precedes the
nuclear changes that define apoptosis (31)
, we measured
annexin V binding in TF-1–33 cytoplasts irradiated at 12 Gy. The
objective was to determine whether, in the absence of cytoplasmic CER
generation, TF-1–33 cytoplasts could still externalize
phosphatidylserine. Intact TF-1–33 cells gated on the propidium iodine
negative population presented a significant increase in annexin V
binding 24 h post-12 Gy IR, as we previously described
(12)
, whereas TF-1–34 cells did not. However no
significant effect was observed in irradiated TF-1–33 cytoplasts
(Fig. 4
). The functional demonstration that these cytoplasts were sensitive to
stimulus-triggered phosphatidylserine externalization was provided by
anti-Fas treatment, where we observed increased annexin V binding as
previously reported (32)
.
|
Our results strongly suggested that the CER-mediated apoptotic pathway
triggered by IR in these myeloid cells was activated at least in part
in the nuclei. We confirmed this hypothesis by demonstrating that IR
produced biological changes characteristic of apoptosis (chromatin
fragmentation, DNA laddering, PARP cleavage) in TF-1–33 nuclei
preparations 24 h after exposure to 12 Gy IR, whereas TF-1–34
nuclei remained essentially unchanged (Figs. 5
, 6
, 7
).
Furthermore, both TF-1–33 and TF-1–34 isolated nuclei were sensitive
to the apoptotic effect of a cell-permeant short chain CER, C6-CER.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. IR doses
between 6 and 12 Gy led to interphase apoptotic cell death in TF-1–33
cells, neutral SMase-induced SM hydrolysis, and subsequent CER
production (12)
. In TF-1–34 cells, IR was unable to
induce SMase stimulation, CER production, or apoptosis. Furthermore, we
demonstrated that the response to IR (sensitivity or resistance) did
not correlate with DNA damage repair capacity (12)
.
Overall, these observations supported the hypothesis whereby CER acts
as a second messenger contributing to the signaling pathway of
IR-induced apoptosis in both normal and tumor cells
(9
10
11
12
13)
.
The absence of an IR-triggered SM-CER pathway in TF-1–34 cells
probably accounts for the lack of apoptosis (12)
. Similar
observations have been described for IR-resistant murine lymphoid cells
(11)
, Burkitt’s lymphoma cells, and glioma cells
(13)
. All three studies concluded that neutral SMase
activation or lack thereof was responsible at least in part,
respectively, for sensitivity or resistance to IR-triggered apoptosis.
Most intriguingly, evidence has been presented that apoptosis signaling
(i.e., neutral SMase activation) induced by IR originates within the
cytoplasm independent of the nucleus (9
, 11)
. This
observation was somewhat unexpected. Indeed, present dogma dictates
that the induction of apoptosis is mediated by a cascade of events
initiated directly by effector/target interaction. In the case of IR,
double-stranded DNA breaks are the critical lesions that lead to cell
death and apoptosis (3
4
5)
. Hence, differences in
radiosensitivity to IR are believed to be mostly related to DNA repair
capacity (6
7
8
9)
. The description that IR can trigger CER
generation independent of IR/DNA interaction has profound implications
in our understanding of radiosensitivity and radioresistance.
Therefore, we attempted to determine in our model the origin of the
SM-CER signaling pathway.
To reconcile these conflicting reports, it is possible that IR
triggers apoptosis through two distinct pathways (cytosolic and/or
nuclear). Indeed, IR may stimulate a cytosolic SMase activity, plasma
membrane-associated SM hydrolysis and CER generation. This CER would in
turn activate a cascade of cytosolic events, including protease
activation and mitochondria alterations leading to enzymatic digestion
of DNA and nuclear proteins. Within this pathway, the cellular control
of CER-induced apoptosis would take place in the cytosol through
several mechanisms involving protein kinase C (PKC) (9)
,
Bcl-2 (35
36
37)
, and the oxidative balance (38
, 39)
. IR-mediated DNA damage may also be the trigger for an
apoptotic pathway implicating nuclear proteins such as p53 and other
cell cycle control proteins. Within this pathway, the nucleus would
play a central role even if cytosolic events were also involved in the
activation and translocation of proteins such as p53 (40)
,
tyrosine kinases (41)
, cyclin-associated kinases
(42)
, and transcription factors (43
, 44)
.
Alternatively, the nucleus could also play a role in activating
the SM-CER pathway. Indeed, an attractive hypothesis could be that IR
stimulates a nuclear chromatin-associated SMase leading to nuclear SM
hydrolysis and the generation of nuclear CER responsible for
IR-triggered apoptosis. Although the presence of both nuclear SMase and
SM have been well documented (45
46
47
48
49
50
51)
, this hypothesis may
appear rather provocative since it is generally believed that the
molecular targets of CER reside within the cytosol (52)
.
However, there are two exceptions in the literature that argue for a
role of a nuclear SMase (50
, 51)
. Furthermore, this would
imply that the cytosolic control mechanisms of CER-mediated apoptosis
are not operative, at least in some forms of IR-triggered apoptosis.
Our study demonstrates that in purified TF-1–33 nuclei, IR
triggered CER production. We did not observe CER generation in
nuclei-free cell lysates or in enucleated cells (cytoplasts).
Furthermore, we show that treatment of these nuclei with CER could
induce nuclear fragmentation. The direct quantitative comparison of CER
generation between whole TF-1–33 cells and purified nuclei, normalized
on a per microgram DNA basis, reveals approximately a 10-fold lower
amount: 0.04 vs. 0.004 nmol/µg DNA, respectively. The reasons
for this are unclear; perhaps this is due to a rapid loss of nuclear
CER in this cell-free system, or one could speculate that in intact
cells the initial nuclear CER signal induces an immediate cytosolic
response leading to the amplification of the apoptotic signal
(necessary for a cellular apoptotic process). Indeed, this study does
not negate the role of cytosolic or plasma membrane CER in IR-triggered
apoptosis in whole cells. Nevertheless, in isolated nuclei (devoid of
potential cytoplasmic antiapoptotic mediators), the CER generated is
sufficient to activate a nuclear fragmentation process. These results
demonstrate that IR can trigger a proapoptotic-like response in a
distinctive manner compared to that, for example, described for Fas
agonists, which induce apoptotic features in enucleated cells
(53)
. Our data also show that IR can stimulate the
hydrolysis of nuclear SM through the activation of a nuclear SMase, and
therefore leads to the generation of a nuclear CER.
The present study identifies nuclear SMase as a potentially
contributing mediator for IR-triggered apoptosis. As for SM, it has
previously been shown that tumor cells are rich in nuclear SM and that
this SM is associated with the nuclear matrix (45
46
47
48
49
50
51)
.
The originality of our report is double: first, it presents a new role
for nuclear SMase and SM and, second, suggests that CER can originate
from a non-plasma membrane-bound SM. IR appears to be unique compared
to tumor necrosis factor 
and interleukin 1, for example, which
stimulate the hydrolysis of SM situated within the inner plasma
membrane (24
, 54
, 55)
.
In TF-1–34 cells, IR failed to stimulate CER production and
nuclear fragmentation in both whole cells and isolated nuclei. This
suggests that the resistance to IR-triggered apoptosis of TF-1–34
cells might be due, at least in part, to the inability of IR to
activate a nuclear SMase. It does not appear that this is linked to a
deficiency in nuclear SMase since basal levels were detected in both
TF-1–33 and TF-1–34. This observations suggest that negative
regulatory elements are blocking IR-mediated SMase activation. For
example, our group showed that PKC activation could block SMase
stimulation triggered by cytotoxic agents such as IR (14)
.
Although both TF-1–33 and TF-1–34 present similar basal PKC
activities (unpublished observations), it is possible that the status
in nuclear PKC (or isoforms thereof) differs between the two cell
lines. The potential antiapoptotic implication of a constitutively high
nuclear PKC activity in TF-1–34 cells is presently under investigation
in our laboratory.
We demonstrate that IR not only triggers CER production in purified
nuclei, but also induces DNA fragmentation and PARP cleavage,
suggesting that CER may play a role in endonuclease and protease
activation in purified nuclei. These observations are intriguing since
several studies have led to the general hypothesis that apoptosis is
under mitochondrial control (15)
. Furthermore, the
described targets for CER have all been assumed to be solely cytosolic,
such as protein kinase ceramide-activated protein kinase, CAP
phosphatase, SAP kinase, and caspases ced-3/interleukin-1ß-converting
enzyme.
In conclusion, we present evidence for the occurrence of nuclear SMase-mediated events in IR-triggered apoptosis of myeloid cells. This observation underscores a novel CER-mediated apoptotic-like nuclear signaling pathway that is independent of a cytosolic intermediate. It remains to be determined whether this in vitro observation illustrates an in vivo event and how it relates to the hypothesis of a ‘central cytosolic executioner of apoptosis’.
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ACKNOWLEDGMENTS |
|---|
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FOOTNOTES |
|---|
Received for publication May 30, 2000.
Revision received July 6, 2000.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced ceramide generation and apoptosis in resistant human leukemia KG1a cells by the P-glycoprotein blocker PSC833. FASEB J 12,101-109
ttcher, C. J. F., van Gent, C. M., Pries, C. A. (1961) Rapid and sensitive sub-micro phosphorus determination. Anal. Chim. Acta 699,57-62
B. J. Clin. Invest. 88,691-695
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) and TF-1–33
(•) cells and cell preparations were resuspended in serum-free
medium, irradiated at 12 Gy, and incubated for kinetic experiments.
After incubation, aliquots were collected and lipids were extracted.
Ceramide (A) and sphingomyelin (B)
quantification was performed by thin-layer chromatography as described
in Materials and Methods. Results are representative of three to seven
independent experiments. Values are the mean of triplicate
determinations ± SE. Asterisks mean significant at
P<0.01.
) and
irradiated (
) cells observed at peak ceramide generation. Results
are representative of five to eight independent experiments. Values are
the mean of triplicate determinations ± SE. Asterisks
mean significant at P<0.01.
