From the Departments of Biochemistry and Molecular
Biology and ¶ Pathology, Georgetown University School of Medicine,
Washington, D. C. 20007 and the
United States Army Medical
Research Institute of Chemical Defense,
Aberdeen Proving Ground, Maryland 21010
Received for publication, September 17, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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DNA damaging agents up-regulate
levels of the Fas receptor or its ligand, resulting in recruitment of
Fas-associated death domain (FADD) and autocatalytic activation of
caspase-8, consequently activating the executioner caspases-3, -6, and
-7. We found that human epidermal keratinocytes exposed to a vesicating
dose (300 µM) of sulfur mustard (SM) exhibit a
dose-dependent increase in the levels of Fas receptor and
Fas ligand. Immunoblot analysis revealed that the upstream caspases-8
and -9 are both activated in a time-dependent fashion, and
caspase-8 is cleaved prior to caspase-9. These results are
consistent with the activation of both death receptor (caspase-8) and
mitochondrial (caspase-9) pathways by SM. Pretreatment of keratinocytes
with a peptide inhibitor of caspase-3 (Ac-DEVD-CHO) suppressed
SM-induced downstream markers of apoptosis. To further analyze
the importance of the death receptor pathway in SM toxicity, we
utilized Fas- or tumor necrosis factor receptor-neutralizing antibodies
or constructs expressing a dominant-negative FADD (FADD-DN) to inhibit
the recruitment of FADD to the death receptor complex and block the
Fas/tumor necrosis factor receptor pathway following SM exposure.
Keratinocytes pretreated with Fas-blocking antibody or stably
expressing FADD-DN and exhibiting reduced levels of FADD signaling
demonstrated markedly decreased caspase-3 activity when treated with
SM. In addition, the processing of procaspases-3, -7, and -8 into their
active forms was observed in SM-treated control keratinocytes, but not
in FADD-DN cells. Blocking the death receptor complex by expression of
FADD-DN additionally inhibited SM-induced internucleosomal DNA cleavage
and caspase-6-mediated nuclear lamin cleavage. Significantly, we
further found that altering the death receptor pathway by expressing
FADD-DN in human skin grafted onto nude mice reduces vesication and
tissue injury in response to SM. These results indicate that the death
receptor pathway plays a pivotal role in SM-induced apoptosis and is
therefore a target for therapeutic intervention to reduce SM injury.
Sulfur mustard (bis-(2-chloroethyl) sulfide;
SM),1 the vesicant agent used
as recently as 1988/1989 in the Iraq/Iran conflict and implied to have
been used in the Gulf War, induces vesication in human skin by its
ability to cause cytotoxic, genotoxic, or a combination of both effects
in the skin. SM is a highly reactive compound that induces the death
and detachment of the basal cells of the epidermis from the basal
lamina (1-6). SM causes blisters in the skin via poorly understood
mechanisms. In an effort to help develop medical countermeasures for
potential exposure of military personnel and civilians, we have been
attempting to define the molecular series of events leading to SM
toxicity in cell culture, in transgenic animal models, and in grafted
human epidermis.
Whereas human dermal fibroblasts may contribute to the vesication
response by releasing degradative cytosolic components extracellularly after a poly(ADP-ribose) polymerase
(PARP)-dependent SM-induced necrosis (7),
keratinocytes display markers of an apoptotic death, as well as those
of terminal differentiation (8). SM-induced apoptosis in keratinocytes
appears to be controlled by both death receptor and mitochondrial
pathways (9). The targets of these apoptotic pathways are a family of
aspartate-specific cysteine proteases or caspases (10). Caspase-3
appears to be a converging point for different apoptotic pathways
(11). In most apoptotic systems, caspase-3 is proteolytically
activated, and in turn cleaves key proteins involved in the structure
and integrity of the cell, including PARP (11-14).
In the present study, we demonstrate that SM induces both Fas and its
ligand (FasL) in primary human epidermal keratinocytes. We also
observed the activation of markers of apoptosis that are consistent
with a Fas-FasL-receptor interaction, including cleavage of caspase-8,
caspase-3, and PARP. Utilizing a combination of techniques including
the stable expression of a dominant-negative inhibitor of
Fas-associated death domain protein (FADD), we demonstrate a role for
the Fas/TNF receptor family in mediating the response of human
keratinocytes to SM. Stable expression of FADD-DN blocks SM-induced
markers of keratinocyte apoptosis, such as caspase-3 activity and
proteolytic processing of procaspases-3, -7, and -8, internucleosomal
DNA cleavage, and caspase-6-mediated nuclear lamin cleavage.
We have shown earlier that NHEK as well as an immortalized line, Nco,
could be used to establish a histologically and immunocytochemically normal epidermis when grafted onto nude mice (8, 9, 15). The present
study demonstrates that markers of apoptosis are induced in basal cells
of SM-exposed grafts, particularly in regions where microvesicles are
formed. We have now also utilized the graft system to genetically
engineer human keratinocytes prior to grafting to ectopically express a
dominant-negative FADD and generate a human epidermis containing
FADD-DN keratinocytes. These human grafts were exposed to SM, and
showed a reduced vesication response compared with control
keratinocyte. Topical SM exposure of Fas-deficient mice in the current
study also indicates the viability of this strategy to suppress
vesication by using inhibitors of the death receptor pathway.
An understanding of the mechanisms for SM vesication will hopefully
lead to therapeutic strategies for prevention or treatment of SM
toxicity. Importantly, our experiments indicate that the Fas/FADD
pathway is required for caspase-3 processing, because inhibitors of
this pathway block SM-induced apoptosis. Because the FADD pathway
can be manipulated at the level of a cell surface (Fas), receptor,
Fas/FADD as well as the caspases represent attractive targets for the
modulation of the effects of SM. Inhibition of the Fas/FADD pathway
by specific pharmacological inhibitors such as neutralizing antibodies
to Fas or peptide inhibitors of caspases may therefore be of
therapeutic value in the treatment of or prophylaxis against SM injury
in humans.
Cells, Plasmids, and Transfection--
Primary human
keratinocytes were derived from neonatal foreskins and grown in
keratinocyte serum-free medium (SFM) supplemented with human
recombinant epidermal growth factor and bovine pituitary extract
(Invitrogen). Primary keratinocytes were immortalized by
transduction with the HPV16 E6/E7 genes (16) to generate the Nco cell
line as described previously (17). The FADD-DN plasmid construct in
pcDNA 3.1 (Invitrogen), a generous gift from Dr. V. Dixit,
expresses a truncated FADD protein, which lacks the N-terminal domain
that is responsible for recruiting and activating caspase-8 at the
death receptor complex (Fig. 5A). Nco cells were transfected
with empty vector or with FADD-DN using LipofectAMINE (Invitrogen), and
stable clones were selected in G418 and maintained in SFM. Cells were
grown to 60-80% confluency, and then exposed to SM diluted in SFM to
final concentrations of 100, 200, or 300 µM, with or
without pretreatment with Fas- (clone ZB-4; Upstate Biotech, Waltham,
MA) or TNFR1- (clone H398; Bender MedSystems, Vienna, Austria (18))
neutralizing antibodies. Media was not changed for the duration of the
experiments. At different time points after SM exposure, cells were
harvested for further analyses.
Chemicals--
SM (bis-(2-chloroethyl) sulfide; >98% purity)
was obtained from the United States Army Edgewood Research, Development
and Engineering Center.
Fluorometric Assay of Caspase-3 Activity--
Cells were
resuspended in lysis buffer containing 50 mM Tris-HCl (pH
7.5), 150 mM NaCl, 1 mM EGTA, 0.25% sodium
deoxycholate, 0.5% Nonidet P-40, 10 µg/ml aprotinin, 20 µg/ml
leupeptin, 10 µg/ml pepstatin A, and 1 mM
phenylmethylsulfonyl fluoride, incubated for 10 min on ice, and
freeze-thawed 3 times. The cell lysate was centrifuged at 14,000 × g for 5 min, and the protein concentration of the
cytosolic extract was determined with the Bio-Rad DC protein assay kit.
For the fluorometric caspase-3 activity assay, 25 µg of cytosolic
extract was initially diluted to a volume of 50 µl with Nonidet P-40
lysis buffer, to which 50 µl of caspase assay buffer (10 mM HEPES (pH 7.4), 2 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol) was added. The aliquots were then mixed
with equal amounts (100 µl) of 40 µM fluorescent
tetrapeptide substrate specific for caspase-3 (Ac-DEVD-AMC; BACHEM) in
caspase assay buffer and transferred to 96-well plates. Free
aminomethylcoumarin (AMC), generated as a result of cleavage of the
aspartate-AMC bond, was monitored continuously over 10 min with a
Cytofluor 4000 fluorometer (PerSeptive Biosystems, Framingham, MA) at
excitation and emission wavelengths of 360 and 460 nm, respectively.
The emission from each well was plotted against time, and linear
regression analysis of the initial velocity (slope) for each curve
yielded the activity.
Immunoblot Analysis--
SDS-PAGE and transfer of separated
proteins to nitrocellulose membranes were performed according to
standard procedures. Proteins were measured (DCA protein assay;
Bio-Rad) and normalized prior to gel loading, and all filters were
stained with Ponceau S, to reduce the possibility of loading artifacts.
They were then incubated with antibodies to the p17 subunit of
caspase-3 (1:200; Santa Cruz Biotechnology), caspase-7 (1:1000; BD
Pharmingen), caspase-8 (1:1000; BD Pharmingen), caspase-9 (1:1000;
Trevigen), or caspase-10 (1:1000; Trevigen), lamin A (1:100; Santa Cruz
Biotechnology), DNA fragmentation factor (DFF) 45 (1:500; BD
Pharmingen), or PARP (1:1000; BD Pharmingen). Immune complexes were
detected by subsequent incubation with appropriate horseradish
peroxidase-conjugated antibodies to mouse or rabbit IgG (1:3000) and
enhanced chemiluminescence (Pierce). Immunoblots were sequentially
stripped of antibodies by incubation for 30 min at 50 °C with a
solution containing 100 mM 2-mercaptoethanol, 2% SDS, and
62.5 mM Tris-HCl (pH 6.7), blocked again, and reprobed with
additional antibodies to accurately compare different proteins from the
same filter. Typically, a filter could be reprobed three times before
there was detectable loss of protein from the membrane, which was
monitored by Ponceau S staining after stripping.
Analysis of DNA Fragmentation--
Cells were harvested and
lysed in 0.5 ml of 7 M guanidine hydrochloride, and total
genomic DNA was extracted and purified using a Wizard Miniprep DNA
Purification Resin (Promega). After RNase A treatment (20 µg/ml) of
the DNA samples for 30 min, apoptotic internucleosomal DNA
fragmentation was detected by gel electrophoresis on a 1.5% agarose
gel at 4 V/cm. DNA ladders were visualized by staining with ethidium
bromide (0.5 µg/ml) and images were captured with the Kodak EDAS 120 (Kodak) gel documentation system.
Annexin V and Propidium Iodide Staining, and FACS
Analysis--
Cells were plated in culture plates and exposed to
various concentrations of SM. 16 h after induction of apoptosis,
the cells were trypsinized, washed with ice-cold phosphate-buffered
saline (PBS), and subsequently resuspended in and incubated in the dark with 100 µl of annexin V incubation reagent that includes fluorescein isothiocyanate-conjugated annexin V (Trevigen, Gaithersburg, MD) and
propidium iodide for 15 min at room temperature. Flow cytometric analyses were conducted on a BD Biosciences FACStar Plus
cytometer using a 100-milliwatt air-cooled argon laser at 488 nm.
Grafting Protocols and Exposure of Human Skin Grafts to
SM--
A 1-cm diameter piece of skin was removed from the dorsal
surface of athymic mice, and a pellet of cells containing 8 × 106 fibroblasts + 5 × 106 keratinocytes
(NHEK or Nco) was pipetted on top of the muscular layer within a
silicon dome to protect the cells during epithelization (Fig.
10A). The dome was removed after a week and the graft was allowed to develop for 6-8 weeks. SM exposure was performed by placing
a small amount of SM liquid into an absorbent filter at the bottom of a
vapor cup, which was then inverted onto the dorsal surface of the
animal, to expose the graft site to the SM vapor. Frozen and fixed
sections were derived from punch biopsies taken from the graft site,
and analyzed for the expression of FADD-DN using the AU1 antibody,
which recognizes the specific AU1 epitope tag on the FADD-DN protein.
Histological analysis of the SM-exposed human skin grafts transplanted
onto nude mice was also performed utilizing an end point of micro- or
macroblisters or SM-induced microvesication.
Assays for in Vivo Markers of Apoptosis on Human Skin
Grafts--
Paraffin-embedded sections derived from SM-exposed human
skin grafts were subjected to analysis for markers of in
vivo apoptosis, including indirect immunofluorescence microscopy
with antibodies to the active form of caspase-3 (Cell Signaling
Technology, Beverly, MA). Sections were deparaffinized, incubated
overnight in a humid chamber at room temperature with antibodies to
active caspase-3 (1:250 dilution) in PBS containing 12% bovine serum
albumin. After a PBS wash, slides were incubated for 1 h with
biotinylated anti-mouse IgG (1:400 dilution in PBS/bovine serum
albumin), washed, and incubated for 30 min with streptavidin-conjugated
Texas Red (1:800 dilution in PBS/bovine serum albumin). Cells were
finally mounted with PBS containing 80% glycerol and observed with a
Zeiss fluorescence microscope.
DNA breaks characteristic of the late stage of apoptosis were detected
in situ using a Klenow fragment-based assay system (DermaTACS; Trevigen). For fixation, slides were equilibrated to
room temperature and redried for 2 h on a slide warmer at
45 °C, rehydrated in 100, 95, then 70% ethanol, washed in PBS,
fixed in 3.7% buffered formaldehyde for 10 min at room temperature, and washed in PBS. Slides were then incubated with 50 µl of Cytonin for 30 min at room temperature, washed twice in deionized water, and
immersed in quenching solution containing 90% methanol and 3%
H2O2 for 5 min at room temperature. After a PBS
wash, slides were incubated in terminal deoxynucleotidyltransferase
labeling buffer for 5 min at room temperature, and visualized under a
bright field microscope.
Characterization of the Sequence of Events during SM-induced
Apoptosis--
We determined the sequence of events involved in
SM-induced apoptosis by performing dose-response and time course
experiments. Fas, a cell-surface receptor found in most cell types
including keratinocytes, mediates some forms of apoptosis. Upon
activation by its specific ligand (FasL), or by agonist antibody, Fas
forms a homotrimeric complex, which in turn recruits the FADD to the membrane-bound complex. In turn, one or more of the upstream caspases (caspase-8 or -10) localize to the Fas-FADD complex, and become autocatalytically activated. We first determined whether SM induces expression of the Fas receptor or its ligand because enhanced expression of Fas or FasL has been shown to occur in cells exposed to
DNA damaging agents, leading to activation of upstream caspase-8 and
downstream apoptotic events such as caspase-3-mediated PARP cleavage
(19, 20). Immunoblot analysis of extracts derived from keratinocytes
exposed to different doses of SM revealed a dose-dependent
increase in the levels of both Fas receptor and FasL in response to SM
(Fig. 1A).
By immunoblot analysis using antibodies that recognize both the
full-length (116 kDa) and 89-kDa cleavage products of PARP, we also
demonstrated that SM-induced apoptosis is accompanied by complete
cleavage of PARP into 89- and 24- kDa fragments that contain the active
site and the DNA-binding domain of the enzyme, similar to the
caspase-3-mediated cleavage of PARP induced by exposure to anti-Fas
(Fig. 1C).
The central signaling proteins for many of the pathways that coordinate
apoptosis are the caspases, cysteine proteases named for their
preference for aspartate at their substrate cleavage site (10), which
cleave key proteins involved in the structure and integrity of the
cell. We previously focused on caspase-3 activation in the SM apoptotic
response (8, 9), because caspase-3 has been shown to be a converging
point for different apoptotic pathways (11). In a number of apoptotic
systems, caspase-3 cleaves key proteins involved in the structure and
integrity of the cell. To further understand the apoptotic
response of keratinocytes following SM exposure, we assayed for the
activation of other key caspases, in particular the upstream
caspases-8, -9, and -10, and the executioner caspases-3, -6, and -7. When the blot in Fig. 1A was stripped of antibodies and
reprobed with anti-caspase-8, SM-induced proteolytic processing of
caspase-8 was noted in cells exposed to vesicating doses of SM (200 and
300 µM; Fig. 1B).
The sequence of caspase activation provides insight into the mechanism
of apoptosis because caspase-8 is first activated following engagement
of death receptors, whereas caspase-9 is activated via a mitochondrial
pathway. We therefore investigated the molecular ordering of caspase
activation in response to SM. NHEK were exposed to 300 µM
SM for various times, and cell extracts were derived and subjected to
immunoblot analysis utilizing antibodies specific to caspases-3, -7, -8, -9, or -10. Upstream caspases-8 and -9 were both activated in a
time-dependent fashion, with caspase-8 cleaved prior to
caspase-9 (1 versus 4 h) (Fig.
2). Because activation of caspase-8
correlates with a Fas-mediated pathway of apoptosis and activation of
caspase-9 is consistent with a mitochondrial pathway, these results are
in agreement with the activation of both death receptor and
mitochondrial pathways by SM. In contrast, no cleavage of caspase-10
was observed (Fig. 6C).
The executioner caspases-3 and -7 were both proteolytically
activated after SM exposure, with caspase-3 activation detectable 3 h after SM exposure, and caspase-7 cleavage noted 4 h after exposure. To detect caspase-6 activity, we utilized antisera specific for lamin A, which is cleaved in vivo by active caspase-6 at
the peptide sequence VEID. Caspase-6 activity is essential for lamin A
cleavage, which is necessary for chromatin condensation during apoptotic execution (21). Fig. 3 shows
the time course of caspase-6-mediated lamin A cleavage in NHEK
following SM exposure. Surprisingly, this substrate was one of the
first to be cleaved (within 1 h), relative to cleavage of PARP (6 h), or the apoptotic DFF/inhibitor of caspase-activated DNase (16 h;
Fig. 3). PARP has been shown to be a substrate of caspase-3 and -7, whereas DFF 45 is primarily cleaved by caspase-3. Taken together, these
data suggest that caspase-6 may be the first of the executioner
caspases to be activated following exposure of NHEK to SM, followed by
caspase-3 and -7.
Caspase-6-mediated Cleavage of Epidermal Keratin K1 following SM
Exposure--
We previously found that the suprabasal-specific
keratins, K1 and K10, are induced upon exposure of NHEK to 100 µM SM, using monoclonal antibodies (8). In the current
study, we utilized a polyclonal antibody directed against the C
terminus of K1, and found that exposure of cells to higher
concentrations of SM resulted in proteolytic cleavage of keratin K1
(Fig. 4A). The size of the K1
cleavage product maps near a perfect consensus sequence for a site of
cleavage by caspase-6 (Fig. 4B). Moreover, point mutations near this region of K1 give rise to a genetic blistering disorder, epidermolytic hyperkeratosis, very similar to SM-induced vesication (22). K1 may therefore be a substrate for caspase-6 and a target during
SM-induced keratinocyte apoptosis.
Expression of FADD Dominant-negative in Human Keratinocytes
Inhibits SM-induced Activation and Processing of Caspases-3 and
-8--
Up-regulation of the Fas ligand or receptor (23) causes
recruitment of FADD (24), FLASH (25), and caspase-8 (26), to the
death-inducing signaling complex (27), and induces the activation of
caspase-8 (26), followed by the activation of the executioner
caspases-3, -6, and -7. SM induces a dose-dependent increase in the levels of both Fas receptor as well as FasL (Fig. 1),
and caspase-8 is activated within 2 h after exposure of NHEK to SM
(Fig. 2). To further analyze the importance of the death receptor
pathway for SM toxicity, we utilized a dominant negative inhibitor of
FADD (FADD-DN), which expresses a truncated FADD protein containing an
AU1 epitope tag and lacking the N-terminal domain necessary for
recruitment and activation of caspase-8 at the death receptor complex
(Fig. 5A). Thus, the
recruitment of FADD to the death receptor complex is inhibited in cells
expressing FADD-DN. Nco cells were transfected with empty vector or
with FADD-DN; stable clones were selected in G418 and maintained in SFM. Immunoblot analysis of extracts derived from different FADD-DN clones with antibodies to FADD confirmed the presence of both FADD and
FADD-DN in positive clones, whereas parental Nco cells expressed only
full-length FADD protein (Fig. 5B, left panel). Expression of the AU1 tag in one clone (DN3), which was chosen for high
levels of FADD-DN and used in subsequent experiments, was further
confirmed by immunoblot analysis with anti-AU1 (Fig. 5B,
right panel).
We first tested whether expression of the FADD-DN construct could in
fact suppress the death receptor pathway of apoptosis. Control Nco
(transfected with vector alone) or Nco stably expressing FADD-DN were
incubated with a Fas agonist antibody (clone CH11) to induce apoptosis.
We measured caspase-3 activity as a marker of apoptosis, by
quantitative fluorometric analysis with DEVD-AMC as a substrate.
Cytosolic extracts were derived 16 h after SM exposure and
analyzed for caspase-3 activity. Fig.
6A (right panel) shows that, following incubation with agonist antibodies to Fas, caspase-3 activity is suppressed in cells expressing the FADD-DN protein. Control Nco and Nco-FADD-DN keratinocytes were then treated with increasing doses of SM for 16 h, and extracts were analyzed for caspase-3 activity. Similar to Fas-mediated apoptosis,
SM-induced caspase-3 activity was markedly inhibited by expression of
FADD-DN (Fig. 6A, left panel). At all SM doses,
Nco keratinocytes displayed substantially more caspase-3 activity
than cells expressing FADD-DN.
We next analyzed whether expression of FADD-DN in keratinocytes could
suppress the proteolytic processing of procaspase-3 into its
catalytically active form. An immunoblot analysis was performed, using
an antibody specific for the larger subunits (p17/p20) of active
caspase-3. Fig. 6B shows that treatment of control Nco
keratinocytes with 100, 200, or 300 µM SM resulted in the
dose-dependent increase in processing of procaspase-3 into the active p17/p20 forms. On the other hand, this processing was almost
completely suppressed in cells stably expressing FADD-DN.
To further analyze the effects of FADD-DN on caspase processing, cells
were exposed to SM and extracts were harvested after the indicated
times and analyzed for the proteolytic cleavage of procaspase-8.
Immunoblot analysis with antibodies to the intact form of caspase-8
revealed that proteolytic activation of caspase-8 is suppressed in
FADD-DN keratinocytes (Fig. 6C). Caspase-8 processing can
clearly be observed as early as 2 h after SM exposure in control Nco cells but not in the FADD-DN keratinocytes. As expected, caspase-10 is not activated during SM-induced apoptosis.
FADD Dominant-negative Expression in Keratinocytes Inhibits
SM-induced Internucleosomal DNA Fragmentation and Caspase-6-mediated
Lamin Cleavage--
A hallmark of apoptosis is the generation of
multimers of nucleosome-sized DNA fragments as the result of the
activation of apoptotic endonucleases, which cleave the chromatin in
the internucleosomal linker region. We treated Nco or Nco-FADD-DN
keratinocytes with increasing concentrations of SM, after which DNA was
isolated and resolved on 1.5% agarose gels. Fig.
7A shows that SM-induced internucleosomal DNA fragmentation is clearly visible in control Nco
keratinocytes even at lower concentrations of SM, but not in those
expressing FADD-DN. At higher SM concentrations, a characteristic apoptotic pattern of internucleosomal cleavage was observed in SM-exposed control Nco cells, whereas DNA extracted from FADD-DN cells
appeared as a smear, characteristic of necrotic death.
Another well established marker of apoptosis is the fragmentation of
nuclei, which occurs partly because of the caspase-6-mediated cleavage
of nuclear lamin A at a specific sequence (21). We therefore analyzed
the cleavage of lamin A following exposure to SM. Whereas control Nco
keratinocytes displayed a dose-dependent increase in the
caspase-6-mediated cleavage of lamin A in response to SM (Fig.
7B), this cleavage was almost completely inhibited in
keratinocytes that stably expressed FADD-DN. Thus, blocking the death
receptor complex by expression of FADD-DN inhibits SM-induced internucleosomal DNA cleavage, as well as caspase-6-mediated nuclear lamin cleavage.
Expression of FADD-DN in Keratinocytes Suppresses SM-induced
Cleavage of PARP and Caspase-7, an Effect That Is Dependent on
Caspase-3--
To verify whether cleavage of downstream targets of
caspase-3 is also blocked by expression of FADD-DN, immunoblot analysis was performed on extracts from control and SM-exposed cells with antibodies to PARP and caspase-7. Whereas both caspase-3-mediated cleavage of PARP and caspase-7 were observed following exposure of
control Nco keratinocytes to 300 µM SM, these apoptotic
markers were completely abolished by expression of FADD-DN (Fig.
8).
To examine whether caspase-3 was in fact responsible for SM
cytotoxicity in human keratinocytes, we next determined whether pretreatment of keratinocytes with the peptide inhibitor of caspase-3 (Ac-DEVD-CHO; Biomol) could block SM-induced cleavage of PARP and
caspase 7. A 30-min pretreatment of cells with 50 µM
Ac-DEVD-CHO prior to SM exposure suppressed activation of caspase-7 and
PARP cleavage, which are both cleaved by caspase-3 (Fig. 8).
Inhibition of the Fas, but Not TNFR1, Pathway with
Blocking Antibodies Inhibits Markers of SM-induced
Apoptosis--
Elevation of both Fas and FasL suggested that
activation of Fas is responsible for SM toxicity. To directly test the
role of Fas and TNFR1 in SM-induced apoptosis, we utilized neutralizing antibodies specific for each receptor. Because phosphatidylserine is
exposed on the surface of apoptotic cells, and the presence of these
residues can be detected by their ability to bind to annexin V, we
analyzed the cells for annexin V binding by FACS analysis 16 h
after SM exposure. Fig. 9A
shows that untreated NHEK are more sensitive to SM-induced apoptosis at
the doses tested than those pretreated with Fas-blocking antibody
(ZB4). A plot of the survival rates (propidium iodide negative, annexin
V-negative) also confirms that control cells are more sensitive to
SM-mediated killing (Fig. 9B). Fig. 9, C and
D, further show that pretreatment of NHEK to ZB4 attenuates
caspase-3 activity and proteolytic processing. In contrast,
TNFR1-blocking antibody had no effect on SM-induced apoptotic markers
and cell survival. Thus, SM exerts its effects primarily through a
Fas-mediated pathway.
FADD-DN Expression in Human Keratinocytes Partially Blocks the
Vesication Response in Grafted Human Keratinocytes--
Human skin
grafts transplanted onto nude mice have been used successfully to
examine SM-induced biochemical alterations, utilizing an end point of
micro- or macroblisters (1-6, 28). We also previously determined that
NHEK as well as Nco cells could be used to establish a histologically
and immunocytochemically normal epidermis when grafted onto nude mice
that exhibits SM-induced vesication (8, 9, 15). Utilizing human
keratin-specific antibodies, we additionally demonstrated the correct
expression of human keratins K1, K10, and K14 within the grafted
epidermis previously (15). In an attempt to test the effects of
inhibitors of the death receptor pathway on apoptosis and
vesication in intact human epidermis, we utilized this system to
genetically engineer human keratinocytes prior to grafting to
ectopically express FADD-DN. Nco and Nco-FADD-DN human grafts were
subsequently exposed to SM by the vapor cup method 6-8 weeks after
grafting. Frozen and fixed sections derived from graft sites of these
animals were first analyzed for the expression of FADD-DN using the AU1
antibody, which recognizes the specific AU1 epitope on the FADD-DN
protein. Immunofluorescence analysis of these sections with antibodies to FADD or AU1 verified that Nco keratinocytes stably expressing FADD-DN attached with an AU1 epitope tag could be grafted, and that the
AU1 epitope could be detected within the grafted human skin (Fig.
10).
Significantly, histological analysis of SM-exposed animals grafted with
Nco (control), and those grafted with the FADD-DN clone of Nco revealed
that SM microvesication is reduced by FADD-DN. Table
I shows that while there was no
difference in the response of the athymic nude mouse host epidermis to
SM (bottom half of Table I), there was a decrease in the amount of
microvesication in the FADD-DN grafts (Table I, sixth column,
boldface).
SM Induces Markers of Apoptosis in Basal Cells in Human Skin
Grafts, Particularly in Regions of Microvesication, an Effect That Is
Inhibited by FADD-DN Expression--
Because the epidermis comprises
less than half of the weight of the grafted skin, it is difficult to
measure epidermal-specific markers of apoptosis by immunoblot analysis.
We thus performed cytochemical and immunofluorescent analysis to
examine the expression of markers within individual cells. In addition
to increased sensitivity, cytochemical staining and
immunofluorescent labeling of individual cells allowed us to localize
and identify the cell type within the epidermis undergoing
apoptosis (i.e. basal, spinous, granular, or
cornified). This information coupled with the vesication data ultimately permits correlation between the apoptotic pathways and blistering.
DNA breaks can be detected in situ using a Klenow
fragment-based assay system (DermaTACS; Trevigen). We tested the
relationship between apoptotic DNA breaks, vesication, and the Fas/TNF
pathway by two different approaches. In the first approach, we grafted control Nco keratinocytes, or FADD-DN-expressing Nco, followed by
exposure to SM. 24 h after exposure, animals were sacrificed and
skin biopsies were obtained, fixed, and sectioned. DNA breaks were
then detected by the DermaTACS method as described under "Materials
and Methods." Fig. 11A
shows that SM induces apoptosis in basal cells of grafts derived from
Nco. In addition, apoptotic cells were concentrated in the areas of
microvesication. In contrast, Nco-FADD-DN skin grafts did not display
the same degree of apoptosis or microvesication.
The second approach involved exposing control and
Fas-knockout (lpr) newborn pups to SM by the
vapor cup method. SM strongly induced apoptosis primarily in the basal
cells of control animals in the areas of microvesication, but DNA
breaks were markedly diminished in skin derived from genetically
matched mice with a disrupted Fas gene (Fig 11B).
Taken together, the data suggest that SM activates a Fas/TNF
apoptotic pathway resulting in the activation of caspase-3 and
apoptosis of basal cells, contributing to the vesication response.
To observe caspase-3 activation in skin sections, we performed
immunofluorescent staining utilizing antibodies that recognize the
cleavage products of caspase-3 but not the full-length protein to
localize active caspase-3 in individual cells following exposure of
human skin grafts to SM. Immunostaining of mouse epidermis exposed to
SM by the vapor cup method using anti-active caspase-3 reveals that
caspase-3 is activated in basal epidermal cells of control mouse skin
treated with SM (Fig. 12). On the other
hand, caspase-3 activation in basal cells was markedly diminished in skin derived from genetically matched mice with a disrupted
Fas gene
(knockout).2 These results
suggest that the Fas/TNF pathway of apoptosis is activated in
individual basal cells by SM, particularly in regions of
microvesication. We also obtained similar results in which basal
cells of SM-treated human skin grafts derived from Nco keratinocytes displayed immunostaining for active caspase-3 in areas of
microvesication in the skin grafts. In contrast, preliminary results
indicate that grafts derived from FADD-DN keratinocytes exhibit less
active caspase-3 in the basal cells, consistent with the results of
immunoblot analysis.2
SM vesication involves both cytotoxicity and detachment of the
epidermal basal cell layer in vivo. Using a cell culture
model in the present study, we have described a potential mechanism for
SM-induced keratinocyte basal cell death and detachment: apoptosis in
keratinocytes via a Fas/TNF death receptor pathway. Keratinocyte basal
cell death is primarily because of apoptosis at the doses tested
(100-300 µM SM), contributing to SM vesication (8). We
have further observed the activation of markers of apoptosis that are
consistent with a Fas ligand-receptor interaction, including caspase-8, caspase-3, and PARP cleavage (7-9). Several investigators have also examined the mode of cell death induced by SM in other cell
types. SM induces an apoptotic response in HeLa cells (10-100 µM) (29), peripheral blood lymphocytes (6-300
µM) (30), keratinocytes (50-300 µM) (8,
17), and endothelial cells (<250 µM) (31). However, a
time-dependent shift to necrosis was observed in SM-treated lymphocytes (30), whereas markers of necrosis were observed at higher
levels of SM in endothelial cells (>500 µM) (31) and HeLa (1 mM) (29).
SM is a strong bifunctional alkylating agent with a high affinity for
DNA, and has been shown to induce DNA strand breaks in keratinocytes
(8, 32), which is confirmed by our results showing the presence of DNA
breaks in SM-exposed human skin grafts. It is therefore likely that DNA
strand breaks play a role in the SM-induced apoptosis in human
keratinocytes. In an attempt to define the molecular series of events
leading to SM vesication, we elucidated important pathways by which SM
induces cell death in cultured keratinocytes, as well as in intact
mouse and grafted human skin. Members of the Fas/TNFR family and their
ligands may be induced at the level of transcription following
stimulation by apoptosis-inducing agents, such as doxorubicin (19, 20), and p53 has been shown to play a role in the up-regulation of Fas (33).
Consistently, we have shown that p53 is also rapidly up-regulated in
keratinocytes following SM treatment, and that p53 may play a role in
SM-induced apoptosis (9, 17). Similarly, ectopic overexpression of
either Fas or FasL directly leads to apoptosis. In the present paper,
we observed activation of a death receptor pathway for apoptosis, in
which Fas receptor and FasL play a role. Following SM exposure,
keratinocytes significantly up-regulate levels of both Fas receptor and
FasL, followed by the rapid activation of the upstream caspase-8,
mediated by recruitment of the adaptor protein FADD, and the consequent
activation of the executioner caspases-3, -6, and -7.
To better understand the contribution of FADD-regulated pathways in the
cutaneous response to SM, we blocked the death receptor pathway
utilizing keratinocytes stably expressing a truncated FADD adaptor
protein (FADD-DN); this protein lacks the N-terminal domain responsible
for recruitment and activation of caspase-8 at the death receptor
complex. Keratinocytes expressing FADD-DN exhibited reduced levels of
FADD signaling and were found to be more resistant to SM-induced PARP
cleavage and processing of caspases-3, -6, -7, and -8 into their active
forms. In most apoptotic systems, caspase-3, the primary executioner
caspase, is proteolytically activated, and in turn cleaves key proteins
involved in the structure and integrity of the cell, including PARP,
DFF 45, fodrin, gelsolin, receptor-interacting protein, X-linked
inhibitor of apoptosis protein, topoisomerase I, vimentin, Rb, and
lamin B (11-14, 34). Caspase-3 is also essential for
apoptosis-associated chromatin margination, DNA fragmentation, and
nuclear collapse (34).
Utilizing the stable expression of a dominant-negative inhibitor of
FADD, we also demonstrated a role for the Fas/TNF receptor family in
mediating the response of grafted human keratinocytes to SM.
Significantly, we noted that blocking the Fas/FADD death receptor
pathway in human skin grafted onto nude mice reduces vesication and
tissue injury in response to SM, thus indicating that this pathway is
an excellent target for therapeutic intervention to reduce SM injury.
Fas-blocking antibody experiments in cultured keratinocytes also show
that SM partially exerts its apoptotic effect via a Fas-FasL
interaction (Fig. 9). In addition, our recent studies with
Fas-deficient mice indicate the viability of this strategy to prevent
vesication by using inhibitors of the death receptor pathway.
Both SM and UV, another agent that induces apoptosis in keratinocytes,
have been shown to up-regulate the levels of another member of the
Fas/TNF family, TNF An understanding of the mechanisms for SM-induced cell death in
keratinocytes will hopefully lead to strategies for prevention or
treatment of SM vesication. The present study suggests that inhibition
of FADD (upstream) or caspase-3 (downstream) may alter the response of
the epidermis to SM. With an understanding of the biochemical pathways
for SM vesication and having attenuated SM-induced toxicity in
vivo using a genetic approach, we are currently further testing
the effects of specific pharmaceutical inhibitors of Fas/caspase death
receptor pathway of apoptosis to block this pathway, and alter the
cytotoxic response of keratinocytes to SM in cell culture, as well as
the vesication response in vivo. To assay whether the
SM-induced apoptotic response is altered upon treatment with inhibitors
of the Fas/caspase pathway, we are examining the biochemical,
morphological, and structural changes that we have previously
established as characteristic markers of apoptosis (7, 8, 17). Our
present study shows that we can detect activation of caspase-3 in
single cells, thus, whether other caspases of the Fas/TNF receptor
pathway are coactivated by SM in vivo, and whether this
activation can be prevented by using inhibitors of this pathway, also
remain to be clarified.
Toxic epidermal necrolysis, a blistering lesion similar to that
resulting from SM exposure, has been successfully treated with
intravenous immunoglobulins, containing naturally occurring neutralizing antibodies specific for human-Fas (46). FasL blocking antibodies, 5 mg/kg, injected into the tail vein, have also been shown
to be effective in blocking ethanol-induced liver apoptosis in mice
(47). Using antibodies that have been clinically used for other
lesions, such as toxic epidermal necrolysis, systemic lupus
erythematosus, rheumatoid arthritis, and psoriasis (40-46), we are
currently testing the effects of inhibiting Fas/TNF binding to their
ligands with neutralizing antibodies to Fas/TNFR in grafted human epidermis.
The effects of suppressing the function of the upstream caspases-8 and
-9 as well as the downstream central execution caspase-3 with
cell-permeable peptide inhibitors are also currently being investigated. An inhibitor that blocks the activity of all caspases, N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)-fluoromethyl
ketone (zVAD-fmk) has been used in a number of cell culture studies and in mouse in vivo studies. For example, three intraperitoneal
injections of 0.25 mg/mouse on days 0, 5, and 10 were recently found to
be sufficient to prevent silicosis (48). For in vivo
inhibition of Fas/TNFR, systemically administered neutralizing
antibodies against Fas/TNF, as well as systemic and topical peptide
inhibitors of caspases are presently being evaluated. The use of
pharmacological Fas/TNF/caspase inhibitors to study SM pathology, in
the context of the whole animal grafted with human skin offers a better
understanding of the mechanism of this damage for human personnel.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Exposure of human keratinocytes to SM results
in a dose-dependent up-regulation of Fas and FasL
expression, caspase-8 activation, and caspase-3-mediated PARP
cleavage. Human keratinocytes (NHEK) were incubated for 16 h
with the indicated concentrations of SM in SFM
(A-C) or agonistic Fas antibody (C),
after which cell extracts were prepared and assayed for the presence of
Fas and FasL (A), and proteolytic cleavage of caspase-8
(B) or PARP (C) by immunoblot analysis. The
positions of molecular size standards (in kilodaltons) and of the
various proteins are indicated.
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Fig. 2.
Exposure of human keratinocytes to SM induces
proteolytic processing of procaspases-8, -9, -3, and -7 in a
time-dependent fashion. Human keratinocytes (NHEK)
were incubated with 300 µM SM in SFM and, after the
indicated times, cell extracts were prepared and assayed for the
proteolytic cleavage and activation of upstream caspases-8 and -9, as
well as effector caspases-3 and -7 by immunoblot analysis. The
positions of the various procaspases and their cleavage products (for
caspases-3 and -9) are indicated.
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Fig. 3.
Exposure of human keratinocytes to SM induces
proteolytic cleavage of downstream targets of caspase-3
(PARP and DFF 45) as well as of
caspase-6 (lamin A). NHEK were incubated with 300 µM SM in SFM and, after the indicated times, cell
extracts were prepared and assayed for the proteolytic cleavage of
downstream targets of caspase-3: PARP and DFF 45, and
caspase-6-mediated lamin A cleavage by immunoblot analysis with
antibodies specific for these proteins. The positions of the various
proteins and their cleavage products are indicated.
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Fig. 4.
Exposure of human keratinocytes to SM results
in a dose-dependent cleavage of epidermal keratin K1.
A, NHEK were incubated with 300 µM SM in SFM
and, after the indicated time, cell extracts were subjected to
immunoblot analysis with antibodies to keratin K1. The positions of K1
and its cleavage product are indicated. B, schematic diagram
of the K1 consensus sequence containing a putative site of cleavage by
caspase-6 (VEID).
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Fig. 5.
Positive clones of human keratinocytes
(Nco) stably transfected with FADD-DN express truncated
FADD (FADD-DN) and epitope tag AU1. A,
schematic representation of FADD, and a dominant-negative inhibitor of
FADD (FADD-DN), which expresses a truncated FADD lacking the death
effector domain (DED) responsible for recruitment and
activation of caspase-8 at the death receptor complex, thereby blocking
the recruitment and activity of endogenous FADD. B, Nco
cells, derived from NHEK were transfected with empty vector or with
FADD-DN, and stable clones were selected in G418. Extracts of different
FADD-DN clones were subjected to immunoblot analysis with antibodies to
FADD, confirming the presence of both FADD and FADD-DN in positive
clones, whereas parental Nco cells expressed only full-length FADD
(left panel). Expression of the AU1 tag in one clone
(DN3), which was chosen for high levels of FADD-DN and used
in subsequent experiments, was confirmed by immunoblot analysis with
anti-AU1 (right panel). The positions of FADD and FADD-DN
are indicated.
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Fig. 6.
Stable expression of FADD-DN in Nco
keratinocytes inhibits SM-induced activation and proteolytic processing
of procaspases-3 and -8 to their active forms. A, Nco
and FADD-DN keratinocytes were incubated for 16 h with the
indicated concentrations of SM in SFM (left panel) or to
agonist antibodies to Fas (right panel), after which whole
cell extracts were prepared and assayed for caspase-3 activity with the
specific substrate DEVD-AMC. Cell extracts from the experiment in
A were subjected to immunoblot analysis with antibodies
specific for caspases-3 (B), -8, and -10 (C). The
positions of the caspases and their proteolytic cleavage products are
indicated.
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Fig. 7.
Stable expression of FADD-DN in Nco
keratinocytes inhibits SM-induced internucleosomal DNA fragmentation
and caspase-6-mediated lamin cleavage. A, control
keratinocytes, or Nco stably expressing FADD-DN were exposed to the
indicated concentrations of SM in SFM for 16 h, after which total
genomic DNA was extracted, purified, and apoptotic internucleosomal DNA
fragmentation was detected by gel electrophoresis on a 1.5% agarose
gel and ethidium bromide staining. B, cell extracts from the
experiment in A were subjected to immunoblot analysis with
antibodies to lamin A. The positions of lamin A and its cleavage
product are indicated.
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Fig. 8.
Expression of FADD-DN in Nco keratinocytes
inhibits SM-induced PARP cleavage and proteolytic activation of
caspase-7. Control or Nco keratinocytes stably expressing FADD-DN
were exposed to 300 µM SM in SFM for 16 h, with or
without a 30-min pretreatment of cells with a peptide inhibitor of
caspase-3 (Ac-DEVD-CHO). Cell extracts were derived and subjected to
immunoblot analysis with antibodies to FADD, PARP, caspase-7, and
caspase-10. The positions of FADD and FADD-DN, as well as PARP,
caspases-7 and -10, and their cleavage products are indicated.
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Fig. 9.
Inhibition of the Fas, but not TNFR1, pathway
with blocking antibodies inhibits caspase-3 activity and
processing. Human keratinocytes (NHEK) were incubated for 16 h with the indicated concentrations of SM in SFM in the presence or
absence of Fas- or TNFR1-neutralizing antibodies, after which cells
were prepared and assayed for annexin V binding plus propidium iodide
staining by FACS analysis (A and B). Percentage of cells
exhibiting annexin V binding (A) or that were negative for
annexin V binding PI staining (B) as determined by FACS
analysis are shown. All the data in A and B are
presented as mean ± S.D. of three replicates of a representative
experiment; essentially the same results were obtained in three
independent experiments. Whole cell extracts were also prepared and
assayed for caspase-3 activity with the specific substrate DEVD-AMC
(C), or subjected to immunoblot analysis with antibodies
specific for caspases-3 (D). The positions of the caspases
and their proteolytic cleavage products are indicated.
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Fig. 10.
Detection of AU1-tagged FADD-DN and human
K14 in human keratinocytes grafted to nude mice.
A, schematic diagram of grafting protocol wherein a 1-cm
diameter piece of skin is removed from the dorsal surface of athymic
mice, and a pellet of cells containing 8 × 106
fibroblasts + 5 × 106 keratinocytes (NHEK or Nco) are
pipetted on top of the muscular layer within a silicon dome to protect
the cells during development. The dome is removed after 1 week.
B, Nco human keratinocytes were stably transfected with a
FADD-DN construct, containing a FADD-DN insert in pCDNA 3.1 linked
to a sequence encoding the AU1 epitope. Six weeks after grafting, skin
was harvested, fixed in formalin, and embedded in paraffin. 5 µM sections were deparaffinized, and stained with
antisera specific for AU1 (middle), or human keratin 14 (right). No staining was observed in host mouse skin.
Level of epidermal damage and microvesication in human skin grafts
derived from Nco or Nco-FADD-DN keratinocytes
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Fig. 11.
SM induces markers of apoptosis in basal
cells in human skin grafts, particularly in regions of microvesication,
an effect that is inhibited by Fas-knockout or FADD-DN expression.
A, control human keratinocytes (Nco), or FADD-DN-expressing
Nco were grafted onto nude mice, which were then exposed to SM by vapor
cup. The SM-exposed human skin grafts were obtained, fixed, sectioned,
and subjected to DNA break detection by DermaTACS. Slides were then
observed by bright field microscopy. The positions of the basal cells,
the dermis, and areas of vesication are indicated. B,
control and Fas knockout newborn pups were exposed to SM by the vapor
cup method. 24 h after exposure, animals were sacrificed, and skin
biopsies were obtained, fixed, and sectioned. DNA breaks were then
detected by the DermaTACS method as described under "Materials and
Methods."
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Fig. 12.
Caspase-3 is activated in basal epidermal
cells of mouse skin treated with SM by vapor cup, particularly in
regions of microvesication. Newborn mice were exposed to SM by the
vapor cup method, and paraffin-embedded sections were derived from the
sites of SM-exposed mouse skin. Sections were deparaffinized, incubated
with antibodies to active caspase-3 with biotinylated anti-mouse IgG,
and with streptavidin-conjugated Texas Red, and then observed with a
Zeiss fluorescence microscope as described under "Materials and
Methods." Immunostaining of mouse epidermis treated with SM by vapor
cup exposure using anti-active caspase-3 (left) or
phase-contrast (right) are shown. The positions of the basal
cells, cells with active caspases-3, as well as areas of
microvesication are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and partial protection of keratinocytes from UV
can be obtained by incubating keratinocytes with antibody that
neutralizes TNF (35, 36). Targeted gene disruption (knockout) studies
have shown that the majority of pathophysiological responses to TNF
are mediated by the p55 TNF receptor (TNFR1) (37, 38). TNF
was also
shown to be elevated in SM-treated epidermal cells (39), and
TNF
-blocking treatments have demonstrated a clinical usefulness for
a wide variety of lesions, including systemic lupus erythematosus (40),
rheumatoid arthritis (41), psoriasis (42-45), and cutaneous necrosis.
However, in the current study, TNFR1-neutralizing antibody was unable
to block SM-induced apoptosis.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Wen Fang Liu and Ruibai Luo for technical assistance.
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FOOTNOTES |
---|
* This work was supported by United States Army Medical Research and Materiel Command contract DAMD17-00-C-0026 (to D. S. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Georgetown University School of Medicine, 3900 Reservoir Rd. NW, Washington, D. C. 20007. Tel.: 202-687-1056; Fax: 202-687-7186; E-mail: rosenthd@georgetown.edu.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M209549200
2 D. S. Rosenthal, A. Velena, F-P. Chou, R. Schlegel, R. Ray, B. Benton, D. Anderson, W. J. Smith, and C. M. Simbulan-Rosenthal, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: SM, sulfur mustard; NHEK, normal human epidermal keratinocytes; FADD, Fas-associated death domain; DN, dominant-negative; PARP, poly(ADP-ribose) polymerase; FasL, Fas ligand; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; SFM, serum-free medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AMC, aminomethylcoumarin; DFF, DNA fragmentation factor; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline.
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