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Regular Articles |
From the Departments of Pathology*
and
Anatomy,
Seoul National University College of
Medicine, and Institute of Allergy and Clinical
Immunology,
Seoul, and the Department of
Otolaryngology,§
Inje University College of
Medicine, Kimhae, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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m). These results suggest that CD99 engagement induce
CsA-inhibitable mitochondrial permeability transition pore
opening, followed by a reduction of m and caspase
activation, thereby leading to apoptosis. Based on these
results, we suggest the possible involvement of CD99 in the
apoptotic processes that occur during nervous system development and
also its application in immunotherapeutic trials for ES
cases.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, various triggering factors and processes of apoptosis have
been widely investigated in vitro and in
vivo.4,5
One of the common initial manifestations of
the apoptotic process, irrespective of cell types and inducing stimuli,
is a disruption of mitochondrial membrane function, including a
dissipation of the mitochondrial transmembrane potential (m) due
to the opening of the mitochondrial permeability transition (PT)
pores.4
In many systems, mitochondrial PT pore opening is
inhibited by cyclosporin A (CsA)6-8
via a mechanism
involving a mitochondrial cyclophilin, but not by
calcineurin.6
Despite the extensive studies on PCD during
development, little is known about the major cell surface proteins
controlling PCD and its intracellular signaling pathway at the early
stage of neural ontogeny.
Ewing's sarcoma (ES) is a rare, small-round-cell undifferentiated tumor of bone and soft tissues. ES has been described to represent the stage of either very early pluripotential cells or primitive neuroectodermal cells9,10 that can differentiate along a neuronal, glial, Schwannian, melanocytic, neuroendocrine, or even ectomesenchymal pathway. ES is considered to be closely related to primitive neuroectodermal tumor (PNET), because they share a common chromosomal abnormality and the high expression of CD99 molecules on their cell surfaces. However, they have some differences in neuronal differentiation potential. PNET has neuronal features such as dense core granules, whereas ES lacks any trace of neuronal differentiation.11,12 Recently, in vitro culture studies have described that ES cell lines possess the ability to differentiate along neuronal pathways in response to various stimuli of differentiating agents.12-14 One report has shown that the mRNA expression patterns of a neural differentiation marker, NF-L, in ES cell lines were different in PNET, but similar in undifferentiated neural tissues.12
CD99 is a ubiquitous 32-kd transmembrane protein encoded by the mic2 gene,15 in particular, highly expressed in human cortical thymocytes, Ewing's sarcoma/primitive neuroectodermal tumor (ES/PNET) cells, pancreatic islet cells, and Leydig and Sertoli cells.16,17 Recently, it has been reported that engagement of CD99 induces homotypic cell aggregation,18,19 up-regulation of T cell receptor and major histocompatibility complex molecules,20 and apoptosis in immature thymocytes.21
In the present study, we demonstrate that CD99-induced apoptosis occurs only in undifferentiated ES cells, not in differentiated ES cells, as it does in immature cortical thymocytes. We suggest that CD99 might trigger apoptosis in a certain developmental stage during neural ontogeny and also that CD99 might be used as a target for immunotherapy of ES patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The monoclonal antibody (MAb) DN16 (IgG1) used for CD99 activation was produced in our laboratory and described previously.22 We purchased rabbit anti-mouse immunoglobulin Ab and FITC-conjugated goat anti-mouse immunoglobulin Ab from Sigma Chemical Co. (St. Louis, MO) and mouse anti-NF-H MAb (NCL-NF200) from Novocastra Laboratories (New Castle upon Tyne, UK). N6-O2-Dibutyryladenosine-3,5'-cyclic monophosphate (db-cAMP) and 3,3'dihexyloxacarbocyanine iodide (DiOC6(3)) were purchased from Sigma Chemical Co. Calcium ionophore A23187 was from Boehringer Mannheim Biochemicals (Mannheim, Germany). Apoptosis inhibitors, such as actinomycin D (Act D), cycloheximide (CHX), cyclosporin A (CsA), and EGTA, were also from Sigma Chemical Co. FK-506 was kindly provided by Dr. J. K. Shin (Dana-Farber Cancer Institute). Z-VAD-fmk and Z-FA-fmk were obtained from Enzyme System Products (Livermore, CA).
Cell Culture
RD-ES (human Ewing's sarcoma), SK-N-MC (human PNET), and SK-N-SH (human neuroblastoma) cells were obtained from the American Type Culture Collection (Rockville, MD), and CADO-ES1 cells (human Ewing's sarcoma) were obtained from the German Collection of Microorganism and Cell Cultures (Braunschweig, Germany). Two human neuroblastoma cell lines, SK-N-AS and SH-SY5Y, were generous gifts of Dr. Chong-Jae Kim (Seoul National University College of Medicine, Seoul, Korea). RD-ES and CADO-ES1 cells were maintained in RPMI 1640 supplemented with 15% and 10% fetal bovine serum (FBS; Life Technology, Gaithersburg, MD), respectively. SK-N-MC, SK-N-SH, SK-N-AS, and SH-SY5Y cells were cultured in Dulbecco's minimal essential medium supplemented with 10% FBS. For differentiation induction of RD-ES and CADO-ES1 cells, the cells were cultured in Dulbecco's minimal essential medium supplemented with 10% FBS in the presence of 2.5 mmol/L and 0.25 mmol/L db-cAMP, respectively. The medium was changed every 3rd day and maintained for up to 12 days.12
Immunofluorescence Staining and Confocal Analysis
For immunofluorescence staining, db-cAMP-treated and untreated ES
cells were fixed in 95% methanol for 15 minutes at room temperature,
permeabilized with 0.1% Triton X-100 in PBS, and washed three times
with PBS containing 1% FBS. The cells were then blocked in 10% FBS
for 30 minutes and stained with mouse anti-NF-H MAb overnight at 4°C.
After washing three times in PBS containing 1% FBS for 15 minutes, the
cells were incubated with fluorescein isothiocyanate (FITC)-conjugated
goat anti-mouse immunoglobulin Ab and mounted on the glass with
mounting media (GEL/MOUNT, Biomeda Corp., Foster City, CA). Confocal
analysis was performed with a 600MRC equipped with an argon/krypton
laser (BioRad Labs, Hercules, CA). Green fluorescence was detected at
> 515 nm after excitation at 488 nm.
Trypan Blue Dye Exclusion Assay
For the MAb-induced cell death assay, cells (5 x 105 cells/well) were placed in 24-well plates and incubated with 10 µg/ml anti-CD99 MAb and the secondary Ab for cross-linking of CD99 or the secondary Ab alone for the indicated period. In the inhibition experiment of cell death induced by CD99 or calcium ionophore A23187, RD-ES cells were treated with 10 µg/ml anti-CD99 MAb or 10 µmol/L A23187, respectively, at 37°C in the presence or absence of 0.1 µg/ml Act D, 1.0 µg/ml CHX, 0.1 to 200 µmol/L CsA, 1.5 mmol/L EGTA containing 1.5 mmol/L MgCl2, and various concentrations of FK-506 for the indicated period. To examine the requirement of caspases in CD99-induced apoptosis, RD-ES cells were pretreated with 20 µmol/L Z-VAD-fmk (a general caspase inhibitor) or Z-FA-fmk (a control cystein protease inhibitor) for 2 hours. The cells were then incubated for 6 hours in the presence or absence of DN16 MAb. Cell death was quantified by trypan blue dye exclusion. All experiments were performed at least three times, and a representative result of the experiments is shown in figures.
Electron Microscopy
Cells were fixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4, for 24 hours at 4°C. Then they were post-fixed in 0.1% osmium tetroxide in the same solution for 1 hour, dehydrated, transferred to propylene oxide, and embedded in epoxy resin (Polyscience Co., Warrington, PA). Ultrathin sections were stained with saturated aqueous uranyl acetate and lead citrate and examined under an electron microscope (Hitachi, H-600) at 75 kV.
Flow Cytometric Analysis
Cells (1 x 106) were incubated with anti-CD99 MAb (1 µg/100 µl) in PBS containing 1% bovine serum albumin and 0.1% sodium azide for 30 minutes at 4°C, washed with PBS, and stained with FITC-conjugated goat anti-mouse immunoglobulin Ab. After three washes, cells were analyzed on a FACScan (Becton Dickinson, San Jose, CA).
TUNEL Labeling
Apoptosis was assessed by TdT-mediated dUTP nick end-labeling (TUNEL) assay23 using the APO-DIRECT apoptosis detection kit (Pharmingen, San Diego, CA). Control or anti-CD99 Ab-treated cells (1 x 106 to 2 x 106) were fixed with 4% paraformaldehyde, washed in PBS, resuspended in 70% cold ethanol, and stored overnight. After washing the cells with PBS, the TUNEL assay was performed according to the manufacturer's protocol.
Assessment of Mitochondrial Transmembrane Potentials
After the induction of apoptosis with anti-CD99 MAb (2 µg/ml)
for 2 hours, cells (1 x 106) were incubated for 15
minutes in 1 ml of 20 nmol/L DiOC6(3) at 37°C, followed
by analysis on a FACScan (excitation, 488 nm; emission, 525
nm).24
The control experiment was performed in the presence
of 50 µmol/L carbamoyl cyanide m-chlorophenylhydrazone
(mClCCP), an uncoupling agent that abolishes m,
for 15 minutes at 37°C.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, it has been reported that CD99 mediates apoptosis in
immature thymocytes and Jurkat cells.21
The findings that
ES/PNET is a neoplastic counterpart of primitive neuroectodermal
cells9-11
and that these cells express a large amount of
CD99 on their surfaces17,25
led us to ask whether the
ligation of CD99 with anti-CD99 MAb DN16 could induce cell death in
ES/PNET cell lines. In this experiment, cells from two ES cell lines
(RD-ES and CADO-ES1) and one PNET cell line (SK-N-MC) were incubated
with DN16 in the presence of cross-linking Ab. Within 2 hours after the
DN16 treatment, cell death was clearly observed in the ES cells.
Maximal cell death was evident within 6 hours (Figure 1A)
. However, in SK-N-MC, which has
recently turned out to be PNET,26
CD99-induced cell death
did not occur within 6 hours (Figure 1B)
or even after 24 hours (data
not shown), indicating that CD99 molecules were able to deliver a death
signal only in ES cells, but not in SK-N-MC cells. In addition,
neuroblastoma cells, such as SK-N-SH, SK-N-AS, and SH-SY5Y, were also
resistant to CD99-mediated death after a 6-hour engagement (Figure 1B)
.
Among the three types of tumors tested, ie, PNET, neuroblastoma, and ES
cells, which differ in their neurogenic potentials and the levels of
CD99 expression, ES cells are considered to be the tumor of the most
undifferentiated stage.12,27
As it was necessary to prepare
cells in various stages of differentiation, we performed the in
vitro induction of neural differentiation of RD-ES and CADO-ES1
cells by db-cAMP treatment. After 12 days of the induction of ES cells
with db-cAMP, morphological changes such as elongated cytoplasmic
process and varicosity formation were observed under phase contrast
microscopic examination (Figure 2, B and D)
. Furthermore, in the indirect immunofluorescence analysis, the
expression of NF-H, a neural differentiation marker, was clearly
visualized in differentiated CADO-ES1 (Figure 2E)
and RD-ES cells (data
not shown). As shown in Figure 2E
, NF-H was mainly localized in the
perinuclear cytoplasm and in the elongated neuritic process of the
differentiated CADO-ES1 cells. Subsequent experiments were done on both
the undifferentiated and the differentiated ES cells to investigate
whether the induction of CD99-mediated apoptosis is dependent on the
degree of differentiation. Interestingly, apoptosis occurred only in
the undifferentiated form of ES cells (Figure 3A)
. The expression level of CD99 was
dramatically decreased in the differentiated ES cells (Figure 3B)
. All
these data suggest that CD99 can deliver a death signal into
undifferentiated ES cells that express CD99 on their surfaces at a high
level.
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Apoptosis has been characterized by morphological changes such as
chromatin condensation, nuclear fragmentation, internucleosomal DNA
fragmentation, and cytoplasmic blebbing.1
The electron
microscopic analysis of RD-ES cells treated with DN16 MAb clearly
showed several ultrastructural changes comparable to those of apoptotic
cells (Figure 4, A–C)
, as compared with
the control Ab-treated cells (Figure 4D)
.
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, right), whereas only 4.5% of the
control Ab-treated cells were positive for FITC-dUTP (Figure 5
, left).
It is likely that CD99 induces extremely limited endonuclease
activities, as a DNA ladder pattern was not seen on gel electrophoresis
and the labeling intensity of FITC-dUTP was weak.
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As many apoptotic processes require de novo synthesis
of RNA and/or protein,2,3
we tested whether this is also
the case in CD99-induced apoptosis in RD-ES cells. As shown in Figure 6
, the CD99-mediated apoptosis in RD-ES
cells was completely blocked after treatment with a RNA synthesis
inhibitor, Act D (Figure 6B)
, or a protein synthesis inhibitor, CsA
(Figure 6C)
, at the concentration of 0.1 µg/ml or 1.0 µg/ml,
respectively. These results indicate that de novo synthesis
of both RNA(s) and protein(s) are necessary for the process of
apoptosis via CD99 in RD-ES cells.
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An increasing amount of evidence has shown that the alteration of
mitochondrial membrane function plays a crucial role in the induction
of apoptosis.28-30
As CsA is a potent inhibitor of
mitochondrial PT pore opening,6,8
we examined the effect of
CsA in CD99-induced apoptosis. When RD-ES cells were pretreated with
CsA at the various concentrations ranging from 0.1 to 200 µmol/L,
CD99-induced apoptosis was completely inhibited at the concentration
above 100 µmol/L CsA (Figure 7)
. It has
been well known that CsA can inhibit apoptosis not only through the
modification of mitochondrial PT pores but also through inactivation of
calcineurin by complex formation with cyclophilin A.31
Therefore, to rule out the possible involvement of calcineurin in
CD99-induced apoptosis, we examined the effect of FK-506, a more
selective calcineurin inhibitor, in RD-ES cells. The apoptosis in RD-ES
cells was not protected by the treatment of FK-506 (ranging from 10 to
100 nmol/L; Figure 8A
), indicating that
apoptosis induced by CD99 cannot be blocked by inhibition of
calcineurin. Interestingly, calcium ionophore, which induces calcium
signaling and activates calcineurin in a calcium-dependent manner, was
also able to induce the death of RD-ES cells. Figure 8B
shows that both
CsA and FK-506 protect RD-ES cells from the calcium-ionophore-induced
apoptosis. These data strongly suggest that CD99-induced apoptosis is
mediated by mitochondrial PT pore opening, regardless of
Ca2+/calmodulin-dependent calcineurin activity.
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showed that removal of extracellular
calcium with the combined treatment of 1.5 mmol/L EGTA and 1.5 mmol/L
MgCl2 did not prevent CD99-induced apoptosis in RD-ES
cells, whereas ionophore-induced cell death was inhibited by EGTA and
MgCl2 pretreatment. These results suggest that CD99-induced
cell death may occur through a signal transduction pathway independent
of a sustained increase of intracellular calcium.
Reduction in the m of RD-ES Cells Is Detected after
Engagement of Anti-CD99 MAb
To confirm whether CD99-induced cell death in RD-ES is dependent
on the reduction of the m, which is mediated by the opening of
mitochondrial PT pores, we used the fluorochrome DiOC6(3).
As DiOC6(3) incorporates into cells in strict nonlinear
correlation with m,32
the reduction of m can be
determined by the uptake rate of DiOC6(3). Anti-CD99
MAb-treated RD-ES cells exhibited a reduction in the incorporation of
DiOC6(3), as compared with control Ab-treated cells (Figure 9)
. With mClCCP, an uncoupling
agent of the oxidative phosphorylation that abolishes staining with
DiOC6(3), we demonstrated that the dye uptake was driven by
m and did not involve significant binding to other cellular
components (Figure 9
, top). Therefore, we were able to confirm the
reduction of the m in DN16 MAb-treated cells.
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Recently, many reports have shown that caspases are crucial for
the execution phase of apoptosis.33,34
To examine whether
caspases are required for CD99-induced apoptosis, RD-ES cells were
treated with a cell-permeable caspase inhibitor (Z-VAD-fmk) or a
control peptide (Z-FA-fmk) for 2 hours before CD99 engagement. As shown
in Figure 10
, cell death was almost
completely abolished in Z-VAD-pretreated RD-ES cells as compared with
that in Z-FA-pretreated or only anti-CD99-treated cells (Figure 10)
.
This result suggests that CD99-induced apoptosis is executed through
caspases that are inhibited by Z-VAD-fmk.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m due to
mitochondrial PT pore opening.
Recently, it has been reported that CD99-mediated apoptosis occurs in
immature thymocytes.21
CD99-induced cell death in both
thymocytes and ES displays many common features, such as typical
ultrastructural changes, the inhibition of cell death by caspase
inhibitors, the absence of DNA ladder formation, and Fas-independent
apoptosis (data not shown). Despite the fact that internucleosomal DNA
fragmentation has been shown to be one of the widely accepted hallmarks
of the apoptotic cell death, we were not able to observe the obvious
DNA fragmentation in this type of apoptosis. Recently, several lines of
evidence have shown that caspases, particularly caspase 3, plays an
important role in the DNA fragmentation.33,35
For example,
caspase 3 induces the DNA fragmentation by the cleavage of
caspase-activated deoxyribonuclease inhibitor (ICAD) or by the
inactivation of its CAD-inhibitory effects. ICAD-transformants were
resistant to staurosporin-induced DNA degradation, although
staurosporin still induced cell death by activating
caspases.35
The findings indicate that the activation of
CAD downstream of the caspase cascade is responsible for
internucleosomal DNA degradation during apoptosis. The apoptosis
through CD99 appeared to be devoid of prominent internucleosomal DNA
fragmentation in the two types of cells, thymocytes and ES cells,
despite the inhibition by the caspase inhibitors (Figure 10)
.
Therefore, it can be suggested that CD99-induced apoptosis in RD-ES
cells might occur through a unique signaling pathway that could induce
caspase activation, chromatin condensation, and nucleus fragmentation
without the activation of calcium-dependent internucleosomal
endonucleases.
Although the CD99-induced apoptosis in both ES cells and thymocytes
displays many features in common, these two types of cells show
apparent contrast in that ES cells require de novo synthesis
of RNA and protein (Figure 6)
, unlike immature thymocytes. It implies
that CD99-induced apoptosis in ES cells might function through a
metabolically active process that is dependent on RNA and protein
synthesis.2,3
The pretreatment of CsA protected RD-ES cells from CD99-induced
apoptosis. It is well known that the functions of CsA are mediated by
the formation of a complex with one of the CsA-binding proteins,
cyclophilins, for instance, CsA-cyclophilin D complex for the
inhibition of mitochondrial PT pore opening and CsA-cyclophilin A and B
complex for the antagonization of calcineurin.6
It has been
recently reported that one of the common manifestations of the
apoptotic process is a disruption of mitochondrial membrane functions,
including a dissipation of the m due to the opening of the
mitochondrial PT pores.24
In RD-ES cells, CD99-induced
apoptosis was abrogated by CsA pretreatment (Figures 7 and 8A)
but not
by FK-506 or EGTA (Figure 8, A and C)
pretreatment, whereas
ionophore-induced apoptosis was inhibited by the pretreatment with the
inhibitors (Figure 8, B and C)
. Furthermore, the engagement of CD99 led
to a substantial reduction in the m (Figure 9)
. Therefore, the
CD99-induced apoptosis in RD-ES cells may occur through the m
disruption due to CsA-inhibitable mitochondrial PT pore opening, which
is independent of a sustained increase of intracellular calcium and
calcineurin activation. This suggestion is in agreement with the result
that the prominent activities of endonucleases, which require
intracellular calcium signaling, were virtually absent.
It is very intriguing that engagement of CD99 can trigger apoptosis
only in undifferentiated ES cells and not in differentiated ES and PNET
cells. The origin of ES has been hypothesized to be derived from a
primitive pluripotential cell that can differentiate into cells with
neural, Schwannian, melanocytic, neuroendocrine, or even mesenchymal
features.9,10
Much evidence has been accumulated for the
potential of ES cells to undergo marked neural differentiation in
vitro.11-14
One report demonstrates that ES cells
express several typical markers of neuronal precursor cells, such as
NCAM, LNGFR, and Leu-7. Furthermore, ES cells that are naturally
negative for neurofilaments induced the expression of neurofilaments
(NF-L, -M, and -H) when cultured in the presence of
db-cAMP.12
These characteristics indicate that ES cells
maintain a primitive phenotype and have a potential to differentiate
into cells with a neural phenotype. Likewise, ES cells used in our
experiment have no evidence of differentiated neuronal features, such
as long cytoplasmic processes and the presence of NF-H, a marker of a
developing neuron. Upon the treatment of ES cells with db-cAMP, the
expression of NF-H became evident (Figure 2)
. After differentiation,
the expression level of CD99 on the cell surfaces was dramatically
reduced, and the cells appeared to be resistant to CD99-induced cell
death. It is a conceivable explanation that the lack of response to
anti-CD99 MAb treatment in the differentiated ES cells might be due to
the alteration of an apoptotic signaling pathway via CD99 engagement
and/or due to the down-regulation of CD99 expression in the
differentiated ES cells. CD99 is also dramatically down-regulated in
medullary mature thymocytes in contrast with immature thymocytes. In
addition, CD99 engagement induces apoptosis only in immature thymocytes
but not in mature thymocytes,21
suggesting that CD99
induces cell death in a stage-specific manner. We observed
similar phenomena in ES cells. This finding suggests that the
CD99-induced cell death might be specific for a very narrow range of
cells during neural development.
To confirm the possibility that CD99 might function in a stage-specific manner, it is important to investigate whether this is also the case during development in normal tissues. This possibility is now being tested experimentally in this laboratory.
In summary, CD99 induces apoptosis in ES cell lines through
CsA-inhibitable mitochondrial PT pore opening, the reduction of the
m, and caspase activation. By analogy with the role of CD99 in
thymocytes, the finding that CD99-induced cell death was confined only
in undifferentiated ES cells suggests a specific role of CD99 in the
apoptosis during a particular stage of neural development. Therefore,
we hypothesize that CD99 might play a certain role in the apoptosis of
the undifferentiated form of neural progenitor cells during the
development of the nervous system in vivo. Furthermore, the
result that CD99 induced cell death only in ES cells might open up the
possibility that CD99 can be used as an immunotherapeutic tool for ES
patients.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant (KOSEF 95-0403-10-01-3) from the Korean Science and Engineering Foundation and a research grant ('95) from the Seoul National University Hospital Research Fund.
Accepted for publication August 22, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or with the
secondary Ab alone (control,
) for the indicated periods of time. All
values are means ± SD of three separate experiments. B:
Resistance to CD99-induced cell death in a PNET and three neuroblastoma
cell lines. SK-N-MC (PNET cell
line) and SK-N-SH, SK-N-AS, and SH-SY5Y
(neuroblastoma cell
lines) as well as RD-ES and CADO-ES1
(ES cell lines) cells
were treated with the secondary Ab alone
(control,
open bars) or with DN16
(filled bars) for 6 hours as described in A. Percent
cell death was assessed by trypan blue dye exclusion and shown as a
percentage of dead cells in total cells.
) or the secondary Ab
alone (
) in the
absence of both (A) or the presence of either 100 ng/ml Act D
(B) or 1 µg/ml CHX (C).
