1 Gastrointestinal Research Laboratory, Division of Gastroenterology Hepatology and Nutrition, Department of Medicine, Winthrop-University Hospital, Mineola 11501; and 2 Department of Medicine, State University of New York-Stony Brook School of Medicine, Stony Brook, New York 11794-8430
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ABSTRACT |
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The regulatory
mechanisms of nontransformed intestinal epithelial cell
apoptosis have not been thoroughly investigated. We determined
the susceptibility and mechanism of Fas-mediated apoptosis in
nontransformed human intestinal epithelial cells (HIPEC) in the
presence and absence of inflammatory cytokines. Despite ample expression of Fas, HIPEC were relatively insensitive to Fas-mediated apoptosis in that agonist anti-Fas antibody (CH11) induced a
<25% increase in HIPEC apoptosis. Pretreatment of HIPEC with
interferon (IFN)-, but not tumor necrosis factor-
or
granulocyte-macrophage colony-stimulating factor, significantly
increased CH11-induced apoptosis of these cells without
increasing Fas expression. Increased apoptosis correlated with
increased caspase 3 activation but not expression of procaspase 3. Also, there was a significant delay in the onset of Fas-mediated
apoptosis in HIPEC, which correlated with the generation of an
activated caspase 3 p22/20 subunit. HIPEC required both initiator
caspases 8 and 9 activity but expressed significantly less of the
zymogen form of these caspases than did control cells. IFN-
-mediated
sensitization of HIPEC occurred upstream of caspase 9 activation and
correlated with a small increase in procaspase 8 expression (<1-fold
increase) and a significant increase in expression of an intermediate
form (p35) of caspase 4 (3.3-fold increase).
intestinal epithelium; interferon- regulation of
apoptosis; caspases
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INTRODUCTION |
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THE EPITHELIAL CELLS lining the intestine (IEC) are the first line of defense against contamination by bacteria, viruses, and food antigens present in the lumen. Consequently, a regulated process of epithelial cell turnover is critical in maintaining mucosal integrity and avoiding inflammation of the intestinal epithelium. Normally, proliferating progenitor crypt cells differentiate, migrate, and replace the outgoing surface epithelium every 3-5 days. This balanced process of cell renewal in the intestine is believed to be maintained through apoptosis (programmed cell death). In fact, apoptotic IEC have been detected on the tips of the villi of the small intestine, the luminal surface of the colon, and within the crypts at the level of the progenitor cells (17, 40, 48). Altered regulation of either IEC proliferation or apoptosis can lead to crypt hyperplasia, villous atrophy, and disruption of IEC barrier function. In some immune-mediated intestinal disorders, such as ulcerative colitis and graft-versus-host disease, the number of apoptotic IEC may be greatly increased (16, 22, 25, 26), suggesting that apoptosis may contribute to the disruption of intestinal function and the pathophysiology of these diseases. However, the effects of inflammatory cytokines or intestinally derived growth factors on IEC apoptosis, and regulatory mechanisms involved in IEC apoptosis, are not clearly understood.
It has recently been proposed that the death receptor Fas (CD95, APO-I) may play a role in IEC apoptosis. Fas is constitutively expressed on the basolateral membranes of IEC in normal intestinal mucosa (27), and we have previously reported that freshly isolated IEC express Fas on their surface (36). IEC in organ cultures or isolated intestinal crypt epithelial cells have been shown to be sensitive to Fas-mediated apoptosis (42, 49). The potential involvement of Fas in programmed epithelial cell death in the intestine is further suggested by the finding that apoptotic, Fas-expressing IEC colocalize with Fas ligand (Fas-L)-expressing mononuclear cells in the intestinal tissue of patients with ulcerative colitis (49, 55).
The pathways for Fas-mediated apoptosis have been extensively studied. Stimulation through Fas initiates the sequential activation of a series of cysteine proteases, called caspases, the effectors of apoptotic cell death (24, 31, 52). Cross-linking of Fas by Fas-L or agonist antibody leads to the formation of a death-inducing signaling complex (DISC) that contains aggregated Fas, the Fas adaptor protein Fas-associated death domain (FADD), and the proenzyme form of caspase 8 (5, 19, 57). Recruitment of procaspase 8 to the DISC results in its autoproteolysis and activation of this initial caspase in the apoptotic cascade. Events downstream of caspase 8 activation include activation of effector caspases 3, 6, and 7 as well as mitochondrial release of cytochrome c (20, 29, 47). The release of cytochrome c from mitochondria can induce the activation of an alternative branch of the caspase cascade through the activation of caspase 9. Cytochrome c binds and activates the adaptor protein apoptotic protease activating factor 1 (Apaf-1) such that it can recruit and activate procaspase 9 (21, 61). Caspase 9 then cleaves and activates downstream effector caspases, amplifying the cascade (46). Although both pathways of Fas-mediated caspase activation are functional in most cell types, in some, the mitochondrial events are not required for efficient Fas-mediated apoptosis, but in others, apoptosis is dependent on the release of cytochrome c (44).
Although enormous progress has been made in identifying effectors and
regulators of Fas-mediated apoptosis in various cell systems,
our understanding of how these processes are regulated in maintaining
epithelial integrity (under normal physiological conditions) or in
causing epithelial injury (in diseased states) is still incomplete.
Also, the caspases required for Fas-mediated apoptosis of IEC
have so far not been completely defined. Studies using colon
carcinoma and adenocarcinoma cell lines have allowed some insight into
the role of Fas in IEC renewal (1, 2, 34). However, these
studies do not always represent the physiological scenario since
immortalized cell lines frequently display abnormalities in
apoptosis. Also, the findings for one malignant epithelial cell
line are frequently not reproducible for another malignant/transformed cell line (35). These abnormalities in apoptosis
may contribute to the immortality of transformed cell lines in vitro as
well as contribute to their resistance to tumor-specific immune
responses, including apoptosis through the Fas death receptor,
in vivo. In fact, it has been shown that most colon carcinomas are
resistant to Fas-mediated apoptosis, and the mechanisms by
which these lines have acquired resistance are numerous (reviewed in
Ref. 32). It has also been shown that this resistance can
be overcome by exposing cultures to inflammatory cytokines such as
interferon (IFN)- (1, 2, 33, 34). Multiple mechanisms
of cytokine-mediated Fas sensitization have been identified using
transformed cell lines (32). These mechanisms of
cytokine-mediated sensitization include regulation of expression of Fas
(1, 2, 10, 54, 58), Fas-L (6, 28),
procaspases (7, 34), and Bcl-2 family proteins (33,
34). Cytokines might also regulate the expression of proteins
involved in DISC formation, as well as inhibitors of caspases such as
the inhibitor of FADD-like interleukin-1
-converting enzyme
(I-FLICE) (33, 34). Recently, Ruemmele et al.
(42) have demonstrated that some of these mechanisms of
cytokine-mediated sensitization may take place in nontransformed IEC
cultures as well.
Alternative in vitro models for the study of Fas-mediated apoptosis of IEC include primary cultures of freshly isolated IEC, intact colonic crypts, or organ culture. Isolation of IEC requires the disruption of tissue integrity, which can result in stress-induced forms of apoptosis (13, 14), making it difficult to investigate the mechanism of apoptosis induced by alternative stimuli such as through death receptor ligation. Although it has been shown that IEC in organ cultures and intestinal crypts are sensitive to Fas-mediated apoptosis (42, 49), the IEC-specific regulatory effects of cytokines are difficult to determine using multicellular specimens. Also, isolated IEC do not survive in culture long enough to determine the effects of cytokine on these cells.
We have recently developed a protocol for the isolation and maintenance of nontransformed human intestinal primary epithelial cells (HIPEC) in long-term culture and have established a number of HIPEC lines from small and large intestine (37). Using this cellular model, we determined the susceptibility of HIPEC to anti-Fas agonist antibody (CH11), identified the caspases required for Fas-mediated apoptosis of HIPEC, and systematically determined whether the mechanisms of cytokine-mediated sensitization for Fas-mediated apoptosis previously reported for transformed cell lines were involved in regulation of HIPEC susceptibility.
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MATERIALS AND METHODS |
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Tissue culture. Human IEC were isolated from surgical specimens, and HIPEC were derived and maintained as previously described (37). Briefly, isolated crypt cells were cultured in mucosal tissue-derived growth factor containing F-12 medium supplemented with epidermal growth factor, insulin, transferrin, retinoic acid, and hydrocortisone (HIPEC medium). Cells were grown in serum-free HIPEC medium for at least four passages and then in HIPEC medium supplemented with 2% dialyzed fetal calf serum (Summit Biotech, Atlanta, GA). Jurkat cells, LS180, and HT-29 were maintained in RPMI (GIBCO BRL, Grand Island, NY) with 10% fetal calf serum (Sigma, St. Louis, MO), supplemented with glutamine (2 mM), penicillin (50 U/ml), and streptomycin (50 µg/ml). Adherent cells were harvested using 0.25% trypsin containing 1 mM EDTA.
Antibodies and reagents.
Monoclonal anti-Fas (DX2, IgG1), isotype control murine IgG1,
polyclonal anticaspase 3 (procaspase and large subunits), monoclonal anticaspase 8 (procaspase), polyclonal (procaspase and large subunit) anti-caspase 9, monoclonal anticaspase 1 (proenzyme and intermediate fragment), monoclonal anti-FADD, and polyclonal anti-I-FLICE were purchased from BD Pharmingen (San Diego, CA). Polyclonal anti-caspase 8 (procaspase and large subunit), antibodies to the inhibitor of
apoptosis proteins c-IAP1 and c-IAP2, Bcl-2,
Bclx, Bak, Bax, caspase 4 (proenzyme and p20 fragment), and
horseradish peroxidase-labeled anti-goat antibodies were from Santa
Cruz Biotechnology (Santa Cruz, CA) and anti-XIAP (human IAP-like
protein) was purchased from BD Transduction Laboratories (San
Diego, CA). Agonist anti-Fas antibody CH11 was purchased from Upstate
Biotechnology (Lake Placid, NY). Horseradish peroxidase-labeled
anti-mouse and anti-rabbit antibodies were from Amersham (Arlington
Heights, IL). FITC-labeled anti-mouse IgG was purchased from Jackson
Immunoresearch Laboratories (West Grove, PA). Anti-human -actin
antibody and murine ascites IgM were purchased from Sigma. Additional
reagents, unless otherwise stated, were from Sigma.
Immunocytochemistry. Cultures were harvested, washed in PBS containing 0.1% bovine serum albumin and 0.1% sodium azide (PBSA), and then incubated with either DX2 anti-Fas monoclonal antibody or isotype control murine IgG1 for 30 min on ice. Cells were washed twice in PBSA and incubated with FITC-labeled secondary antibody for another 30 min on ice. Cells were washed three times in PBSA and then resuspended in PBS or PBS with 2% paraformaldehyde and analyzed on a FacScan (Becton Dickinson, San Jose, CA).
Induction and detection of apoptosis.
Jurkat cells (0.5 × 106/ml), and HIPEC, LS180, and
HT-29 monolayers were grown in 12-well tissue culture plates and
incubated with medium alone or medium containing either recombinant
human IFN- (200 U/ml), human tumor necrosis factor
(TNF)-
(10 ng/ml), or human granulocyte-macrophage
colony-stimulating factor (GM-CSF; 20 ng/ml) (R&D Systems,
Minneapolis, MN) for 18-20 h before the addition of CH11 agonist
anti-Fas antibody. CH11 was used at a concentration of 100 ng/ml,
approximately three times the concentration required for maximum
apoptosis of Jurkat after 20 h incubation. Cytokine-primed and
unprimed HIPEC were incubated with CH11 for 18-24 h before both
adherent and nonadherent cells were harvested for assays to measure
apoptosis or for preparation of lysates. Apoptosis was
measured by staining with FITC-labeled annexin V (Pharmingen) following
the manufacturer's directions. Alternatively, cells were stained with
FITC-labeled dUTP in terminal deoxynucleotidyl transferase dUTP
nick-end labeling (TUNEL) assays, following the manufacturer's
instructions for the Death Detection kit from Boehringer Mannheim
(Mannheim, Germany). Labeled cells were quantitated by flow cytometry.
Western blot analysis.
Cell pellets were lysed in 10 mM NaH2PO4, 150 mM NaCl buffer containing 1% SDS, 1% Triton X-100, 1% sodium
deoxycholate, 2 mM EDTA, and protease inhibitors (2 mM
phenylmethylsulfonyl fluoride, 10 mM benzamidine, and 10 mg/ml trypsin
inhibitor, leupeptin, antipain, and aprotinin). Total protein was
determined by the BCL method (Pierce Chemical, Rockford, IL). Protein
(20-50 µg) was run on 12% SDS-PAGE gels (Bio-Rad, Hercules, CA)
and then transferred to Trans-Blot nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% milk in Tris-buffered saline (TBS) before incubation with antibodies overnight at 4°C. After being washed (TBS with 0.5% Tween), blots were incubated with horseradish peroxidase-labeled secondary antibody, washed, and developed by the
enhanced chemiluminescence method (Amersham). Autoradiographs were
analyzed by densitometry. Blots were then stripped and reprobed with
murine anti-human -actin, against which all immunoblot data were normalized.
Peptide inhibition of caspase activity.
Caspase activity was irreversibly inhibited with fluoromethylketone
(fmk)-derived tetrapeptides containing benzyloxycarbonyl (z) groups to
enhance cell permeability (R&D Systems). Untreated and IFN--treated
HIPEC or Jurkat cells (control) were incubated with peptide inhibitors
for 15 min before the addition of CH11 antibody. After 20-h
incubation, cells were harvested and stained with FITC-labeled annexin
V. Inhibitory peptides were dissolved in DMSO (final dilution of DMSO
was >1:400). Therefore, cultures treated with vehicle alone were run
as controls in each experiment. Background apoptosis was
defined as the percentage of annexin V-positive cells in cultures that
did not contain CH11 antibody or inhibitors. Percent inhibition of
apoptosis was calculated using the formula
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Caspase activity assay.
Colon HIPEC were grown in 100-mm tissue culture dishes, and confluent
monolayers were stimulated with IFN-, CH11, or IFN-
followed by
CH11 as described in Induction and detection of
apoptosis. Cultures were lysed in 50 mM HEPES,
100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% and glycerol containing 1.0%
NP-40. An aliquot was reserved for protein quantitation (BCL assay),
and then dithiothreitol was added to a final concentration of 10 mM.
Caspase activity was assayed using caspase 3 (Ac-DEVD-pNA) colorimetric
substrate (BIOMOL Research Laboratories, Plymouth Meeting, PA)
following the manufacturer's recommendations. Recombinant active
caspase 3 was used as a control. Optical density
readings were performed at 0, 1, 2, and 3 h. The data are reported
as the change in optical density for lysates from treated cells
compared with untreated cells (defined as 1.00) normalized to total protein.
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RESULTS |
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HIPEC lines are relatively insensitive to Fas-mediated
apoptosis.
We examined whether cross-linking of Fas antigen could induce HIPEC
apoptosis. As seen in Fig. 1,
treatment of HIPEC lines with agonist anti-Fas antibody CH11 for
18-20 h induced a <25% increase in the number of annexin
V-positive cells above background. This increase in cell death was
significant in jejunum HIPEC (P < 0.001) but not in
colon HIPEC (P > 0.05). Incubation with control IgM
antibody had no effect on HIPEC lines (data not shown). Treatment of
HIPEC lines with up to 300 ng/ml CH11 did not induce any further increase in the number of apoptotic cells, indicating that the difference in HIPEC and Jurkat sensitivity to CH11 was not due to
differences in antibody dose requirements (data not shown). These
results suggest that additional stimuli may be required for
sensitization of HIPEC for Fas-mediated apoptosis. Therefore, we also assessed the effects of proinflammatory cytokines on HIPEC sensitivity to Fas-mediated apoptosis. HIPEC lines were
incubated with IFN- or TNF-
for 18-20 h before the addition
of CH11 (Fig. 1). Cytokines alone did not induce apoptosis of
either colon or jejunum HIPEC. Addition of CH11 to IFN-
-treated
colon and jejunum HIPEC induced significant numbers of annexin
V-positive cells compared with either medium or IFN-
alone. Also,
the level of apoptosis in jejunum HIPEC treated with IFN-
and CH11 was significantly increased compared with jejunum HIPEC
treated with CH11 alone. Similar results were obtained using another
colon HIPEC line (data not shown). In contrast to IFN-
, treatment
with TNF-
did not alter HIPEC susceptiblity to CH11-induced cell
damage (Fig. 1). HIPEC lines were not primed for Fas-mediated
apoptosis if treated with IFN-
for only 6 h before
addition of CH11 or if treated with IFN-
and CH11 concurrently for
18-20 h (data not shown). In addition, we determined the effects
of GM-CSF and combinations of TNF-
+ IFN-
on HIPEC
susceptibility (data not shown). Preincubation with GM-CSF did not
enhance CH11-induced apoptosis of HIPEC lines. Incubation of
HIPEC with a combination of IFN-
+ TNF-
induced a
significant level of cell death by 48 h. However, preincubation with IFN-
+ TNF-
(20 h) followed by CH11 induced a level of cell death that was less than the added values for IFN-
+ TNF-
and IFN-
followed by CH11. This indicated that IFN-
+ TNF-
was no better at priming HIPEC for Fas-mediated
apoptosis than was IFN-
alone and that IFN-
-treatment
primed HIPEC for TNF-
-mediated cell damage as well as Fas-mediated
cell apoptosis.
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HIPEC lines express surface Fas, which is not altered by treatment
with proinflammatory cytokines.
To determine if HIPEC resistance to Fas-mediated apoptosis was
related to a decrease in Fas expression in culture, colon and jejunum
HIPEC lines were stained with anti-Fas DX2 monoclonal antibody or an
isotype control and analyzed by flow cytometry. As seen in Fig.
2, colon and jejunum HIPEC lines
consistently expressed high levels of Fas antigen, comparable with that
of freshly isolated colon IEC. Fas expression on HIPEC was also
comparable with that of Jurkat cells, the prototypical Fas-sensitive
cell line. Previous studies have demonstrated that proinflammatory cytokines may induce or enhance the expression of Fas by IEC lines (1, 2, 34, 42). Therefore, we evaluated whether a similar regulatory mechanism of Fas expression existed in our HIPEC system. In
contrast to these previous reports, treatment of HIPEC with IFN-,
TNF-
, or GM-CSF had no effect on Fas antigen expression (Fig. 2).
Addition of lipopolysaccharide or the combination of IFN-
+ lipopolysaccharide to HIPEC cultures did not alter Fas expression
either (data not shown). Lack of response to TNF-
and GM-CSF was not
due to a lack of receptor expression because, in a series of
independent experiments, TNF-
and GM-CSF were both found to bind
HIPEC in a specific manner (data not shown). Also, IFN-
treatment
induced upregulation of HIPEC major histocompatability complex
class II expression, providing functional evidence for expression of
IFN-
receptor on HIPEC (data not shown).
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Fas-mediated death of HIPEC is apoptotic.
To confirm that the HIPEC death caused by IFN- + CH11
stimulation was truly apoptotic and not necrotic, we assayed for
the presence of caspase 3 activation fragments or subunits. We chose to
look at caspase 3 for two reasons. First, caspase 3 is an effector caspase commonly activated by many apoptotic stimuli. Secondly, we
wanted to determine whether IFN-
-mediated increase in
apoptosis correlated with an increase in effector caspase
activation. Jurkat was chosen as a control line because it is known to
utilize caspase 3 activation for Fas-mediated apoptosis
(5, 57). As seen in Fig.
3A, Jurkat cells (control)
treated with CH11 for 4 h contained caspase 3 proenzyme (36 kDa) and three large subunits, p25, p22, and p20. These
fragments correspond to the p24 intermediate form and activated p20 and
p17 subunits, previously reported (9, 23). HIPEC contained
ample amounts of caspase 3 proenzyme, comparable to that found in the
Fas-sensitive Jurkat cell line (Fig. 3A) as well as HT-29
(data not shown). Untreated or IFN-
-treated HIPEC contained no large
subunits of caspase 3, and IFN-
treatment did not induce increased
expression of procaspase 3. HIPEC treated with CH11 alone contained
proenzyme and the intermediate p25 fragment, indicating that caspase 3 cleavage had taken place and implying caspase 3 activation. However,
only low levels of p22, and no p20 fragment, were detected in lysates
from CH11-treated HIPEC. In contrast, HIPEC primed with IFN-
followed by CH11 contained more completely activated caspase 3, as
indicated by the detection of increased levels of the p25 intermediate
fragment as well as the detection of p22 and p20 activation fragments.
These results confirm that Fas-mediated death of HIPEC was
apoptotic and indicate that the level of caspase activation
correlated with the level of apoptosis in HIPEC lines.
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Apoptosis of HIPEC was delayed and correlated with the
generation of the p22/20 activation fragment of caspase 3.
In initial experiments, we found that when untreated or IFN--treated
HIPEC were incubated with CH11 for 4-6 h there was no increase in
the number of annexin V-positive cells above background. By comparison,
CH11-induced apoptosis of Jurkat was morphologically evident by
2 h and IFN-
-primed HT-29 showed evidence of apoptosis 4-6 h after addition of CH11, as previously reported (2,
34). To further investigate the kinetics of Fas-mediated
apoptosis in HIPEC, we performed a time-course study using
IFN-
-treated HIPEC as well as Jurkat cells for comparison. Annexin
V-positive Jurkat cells were detected 2 h after the addition of
CH11 (Fig. 4A). By comparison,
apoptosis was not detected in IFN-
-primed HIPEC lines until
after 8-h incubation with CH11. Immunoblots for the detection of
caspase 3 subunits (Fig. 4B) show that the p25 subunit was
generated by 4 h, but p22/20 subunits were not detected until the
onset of apoptosis at or after 8 h of treatment. These
data demonstrate that the onset of Fas-mediated apoptosis in
HIPEC was significantly delayed and correlated with the generation of
the active subunits of caspase 3.
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Initiator caspases 8 and 9 as well as effector caspase 3 are
required for Fas-mediated apoptosis of HIPEC.
Fas-mediated apoptosis may require activity of both initiator
caspases 8 and 9 (44). To identify the specific caspases
required for CH11-induced apoptosis in HIPEC, we used synthetic
peptide inhibitors, z-IETD-fmk, z-LEHD-fmk, and z-DEVD-fmk, to
irreversibly inhibit caspase 8, 9, and 3 activity, respectively (Fig.
5). Jurkat cells were run as controls, and our findings were consistent
with those of previous reports (5, 19, 57). All three
peptides inhibited CH11-induced apoptosis in untreated as well
as IFN--treated HIPEC lines, suggesting that HIPEC require both
initiator caspases 8 and 9 as well as effector caspase 3 for
Fas-mediated apoptosis. All of the peptides tested inhibited
apoptosis in a dose-dependent fashion from 1 µM to 50 µM
(1, 15, 30, and 50 µM) in primed and unprimed HIPEC (data not shown).
It should also be noted that z-IETD-fmk (caspase 8) and z-LEHD-fmk
(caspase 9) were more inhibitory in untreated and IFN-
-treated HIPEC
than in Jurkat cells. Although not all of these differences were
statistically significant, the findings suggest that HIPEC lines might
contain less active caspase 8 and 9 than Jurkat. In addition,
z-DEVD-fmk inhibited more apoptosis in untreated HIPEC than in
IFN-
-treated HIPEC or Jurkat. Again, although the differences were
not statistically significant, the data suggest that untreated HIPEC
contained less activated caspase 3 than did Jurkat cells, and that
IFN-
treatment of HIPEC resulted in increased production of active
caspase 3. Indeed, as shown in Fig. 3A, lysates from Jurkat
cells contained more active caspase 3 (p22/20 fragment) than lysates
from untreated HIPEC. Furthermore, lysates from IFN-
-treated HIPEC
contained increased amounts of p22 and p20 subunits compared with
untreated HIPEC (Fig. 3A).
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HIPEC express comparatively low levels of procaspase 8, and IFN-
treatment induces a less than onefold increase in procaspase 8 expression but does not alter expression of FADD or I-FLICE.
The peptide inhibition data indicated that HIPEC required caspase 8 activity for Fas-mediated apoptosis and suggested that HIPEC
lines might contain less activated caspase 8 than Jurkat cells. To
determine if IFN-
-mediated sensitization of HIPEC for apoptosis was dependent on upregulation of procaspase 8 expression, we performed immunoblot analysis of lysates from untreated
and IFN-
-treated HIPEC as well as HT-29 and Jurkat cells (controls). As seen in Fig. 6A, HIPEC
expressed two isoforms of procaspase 8 (45).
HIPEC expressed substantially lower levels of procaspase 8 than
either Jurkat or HT-29. Also, IFN-
treatment of HIPEC resulted in a
less than onefold increase in expression of procaspase 8 compared with
untreated HIPEC (Fig. 6B). IFN-
treatment also induced an
increase in procaspase 8 in HT-29 (Fig. 5A), as previously reported (34).
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IFN--mediated sensitization of HIPEC is independent of
procaspase 9, Bcl-2 family proteins, or IAP expression.
Because the peptide inhibition data suggested that Fas-mediated
apoptosis of HIPEC was dependent on caspase 9 activity and, by
extension, release of mitochondrial cytochrome c, we
determined whether IFN-
treatment modulated expression of procaspase
9 or Bcl-2 family proteins. These data (Fig.
5) also suggested that HIPEC expressed
decreased levels of caspase 9. As seen in Fig. 7A, HIPEC expressed less
procaspase 9 than either Jurkat or HT-29 and IFN-
-priming did not
upregulate expression of this initiator caspase. Also, HIPEC expressed
BCLx, BAX, and BAK, but not BCL-2 (Fig.
7B). However, IFN-
treatment did not induce an
increase in expression of any of these apoptosis regulators.
IAP functions, in part, by binding to procaspase 9 (8, 9).
However, as seen in Fig. 7C, IFN-
-treatment did not alter
expression of XIAP or c-IAP2 (HIAP-1). In addition, we found
that HIPEC did not express c-IAP1 (HIAP-2), and IFN-
treatment did
not induce its expression (data not shown).
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IFN- treatment induced an increase in expression of caspase 4 intermediate subunit but not caspase 1.
It has been suggested that caspase 1 or caspase 4 may play a role in
Fas-mediated apoptosis (18, 32-34). To assess
whether these caspases were involved in IFN-
-mediated sensitization
of HIPEC to Fas-mediated apoptosis, untreated and
IFN-
-treated colon HIPEC were incubated with z-YVAD-fmk peptide
inhibitor before addition of anti-Fas antibody and assayed for
apoptosis by annexin V-FITC staining. As seen in Fig.
9A, z-YVAD-fmk inhibited
apoptosis in a dose-dependent fashion in both untreated and
IFN-
-treated colon HIPEC, suggesting that an
interleukin-1
-converting enzyme (ICE) family caspase
(caspase 1-like) was involved in Fas-mediated apoptosis of
HIPEC. Western blotting for caspase 1 showed that, although caspase 1 was presumably activated during Fas-mediated apoptosis, as
indicated by the detection of a p33 intermediate fragment, IFN-
treatment did not increase the level of procaspase 1 expression nor the
level of its activation. In contrast, the proenzyme form (p45) and
intermediate-sized fragments (p40 and p35) of caspase 4 were
consistently detected in both untreated and CH11-treated HIPEC. IFN-
treatment did not induce a significant increase in procaspase 4 and a
less than onefold increase in the p40 band. However, IFN-
treatment
did induce a significant increase in expression of the p35 intermediate
fragment (up to a 3.3-fold increase above untreated HIPEC) (Fig.
9C). Despite the apparent activation of caspase 4, as
suggested by the detection of an intermediate fragment, the active
fragment of caspase 4 (p20) was not detected in lysates from any HIPEC
culture.
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DISCUSSION |
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We have used an in vitro model of nontransformed HIPEC to study Fas-mediated epithelial apoptosis in the intestine. As previously reported for IEC (27, 36), HIPEC constitutively expressed Fas antigen on their surface. However, Fas expression did not correlate with susceptibility to Fas-mediated apoptosis in that agonist antibody induced only marginal amounts of HIPEC death, as detected by two independent types of assays for apoptosis: detection of phosphatidylserine inversion (annexin V staining; Fig. 1) and fragmentation of DNA (TUNEL assay; Fig. 3B). These results are supported by the finding that caspase 3 was poorly activated after addition of anti-Fas, as indicated by the low level of p22/20 fragment detected by Western blot as well as by caspase 3 activity assays. Previous work using organ culture of isolated intestinal crypts suggested that IEC are sensitive to Fas-mediated apoptosis (42, 49), and although it is known that many transformed IEC lines are often resistant to Fas-mediated apoptosis, it is somewhat surprising to find that nontransformed primary cells are relatively resistant as well. In addition, the onset of apoptosis in HIPEC was significantly delayed compared with Jurkat as well as HT-29, further demonstrating that HIPEC lines are insensitive to Fas-mediated apoptosis.
HIPEC are relatively resistant not only to Fas-mediated
apoptosis but also to apoptosis induced by TNF-,
camptothecin, and etoposide, and although the level of Fas
apoptosis is significantly increased in IFN-
-sensitized
cells, the onset of apoptosis is still delayed. The
comparatively low level of caspase 3 activation fragments detected in
lysates from CH11-treated HIPEC lines, and the observation that caspase
3 activity in IFN-
+ CH11-treated HIPEC is about half that of
CH11-treated Jurkat, suggests that caspase activation was inefficient
in HIPEC. Although the level of expression of the proenzyme form of a
caspase is not indicative of its activation status, the availability of
that procaspase form may be a limiting factor in the efficiency of
activation of the caspase cascade. HIPEC lines express a level of
procaspase 3 comparable to that of Jurkat and HT-29 cell lines,
indicating that this is not a limiting factor in the effector caspase
activation. In contrast, our results demonstrate that the levels of
procaspases 8 and 9 are decreased in HIPEC compared with both Jurkat
and HT-29. In addition, caspase 8 and 9 activation fragments could not
be detected in lysates from unprimed CH11-treated HIPEC and these fragments were just barely detectable in lysates from IFN-
-primed, CH11-treated cells, even when 75-100 µg of total protein were loaded onto gels for Western blotting (data not shown). These observations lend support to the hypothesis that caspase activation is
inefficient in HIPEC lines and suggest that decreased initiator procaspase expression may contribute to HIPEC resistance to
apoptosis. Also to be considered is the observation that HIPEC
express multiple inhibitors of apoptosis, including the caspase
8 inhibitor I-FLICE (15) as well as the caspase 9 and 3 inhibitors XIAP and c-IAP2 (8, 9). Whether HIPEC
resistance to apoptosis and poor activation of the caspase
cascade is due to low initiator caspase expression or the action of
apoptosis inhibitors has yet to be determined.
IEC express receptors for multiple cytokines (38, 41) that
regulate IEC proliferation, differentiation, and function (39, 43). In transformed cell lines, inflammatory cytokines have been
reported to modulate apoptotic pathways by regulating the expression of Fas (1, 2, 10, 34, 42, 54, 58) and Fas-L
(6, 28). This IFN--induced increase in Fas expression frequently correlates with increased sensitivity to apoptosis induced by agonist anti-Fas antibody (1, 2, 34). In a recent report, Ruemmele et al. (42) report that both
IFN-
and TNF-
upregulate Fas expression and sensitize
nontransformed, primary cultures of human IEC for Fas-mediated
apoptosis. Consistent with these reports (1, 2, 34,
42), we found that IFN-
treatment primed HIPEC for
anti-Fas-stimulated apoptosis. However, in direct contrast to
these reports, IFN-
-mediated sensitization of HIPEC did not
correlate with regulation of Fas expression (1, 2, 34,
42). Our finding that proinflammatory cytokines do not induce an
increase in HIPEC Fas expression is in accordance with the observation
that Fas expression is not upregulated on epithelial cells of the
intestinal mucosa in patients with inflammatory bowel disease compared
with normals (49, 55).
In addition to regulation of surface death receptor expression,
inflammatory cytokines have been shown to sensitize IEC lines for
apoptosis by modulating intracellular regulators, or effectors, of apoptosis (32-34). We found that IFN-
priming of HIPEC resulted in enhanced generation of caspase 3 activation fragment p22/20 and a 2.9-fold increase in caspase 3 activity above that for unprimed HIPEC. Also, the onset of Fas-mediated
apoptosis correlated with appearance of the caspase 3 p22
activation subunit, and inhibition of caspase 3 activity blocks
Fas-mediated apoptosis of HIPEC. These data suggest that
caspase 3 is the principle effector caspase in Fas-mediated
apoptosis in HIPEC. However, we have not excluded the
possibility that IFN-
-enhanced apoptosis was due to
upregulation of expression or activation of additional caspases, such
as caspase 6 or 7. In cell-free systems, caspase 8 and 9 have been
shown to directly or indirectly cleave and activate caspases 2, 3, 6, 7, and 10 (31, 52). Also, z-DEVD-fmk is not an exclusive
inhibitor of caspase 3 (11, 53). Nonetheless, our data
strongly suggest that caspase 3 is a primary effector caspase in
Fas-mediated apoptosis of HIPEC.
HIPEC expressed high levels of procaspase 3, comparable to that of
Jurkat (Fig. 3) and HT-29 (data not shown), and IFN- priming did not
induce any further increase in procaspase 3 expression. This indicates
that IFN-
-mediated sensitization occurs proximal to caspase 3 cleavage. The data indicate that Fas-mediated apoptosis of
HIPEC is dependent on two initiator caspases, caspase 8 and caspase 9. Therefore, IFN-
treatment could have upregulated apoptosis by affecting either caspase 8 or caspase 9 activation.
Caspase 8 activation requires autoproteolysis. This activation through
self-cleavage is dependent on procaspase 8 recruitment to the DISC,
which in turn is dependent on the adaptor protein FADD (5, 19,
57, 60). Caspase 8 activity is also regulated by I-FLICE, a
mammalian homologue of a family of viral proteins called FLIPs
(FLICE-inhibitory proteins) that inhibits autoproteolysis by blocking
caspase 8 recruitment by FADD (15). We determined that the
expression of FADD and I-FLICE is unaltered by incubation with IFN-.
However, IFN-
treatment did induce a small increase in expression of
procaspase 8 (<1-fold). It is possible that this small increase in
caspase 8 concentration was sufficient to promote more efficient
autoactivation with a subsequent increase in activation of downstream
caspases (caspase 9 and 3).
IFN- treatment of HIPEC did not induce an increase in procaspase 9 expression. However, this does not indicate whether caspase 9 activity
was increased or not. Regulators of caspase 9 activation or activity
include Bcl-2 family proteins (3) and IAP family proteins
(8, 9). IFN-
priming of HIPEC did not alter expression of proapoptotic BAX or BAK or antiapoptotic BCLx,
suggesting that IFN-
treatment did not affect the release
of cytochrome c and subsequently the activation of caspase
9. IAP proteins, such as XIAP, have been shown to bind the proenzyme
form of caspase 9 as well as the activated form of caspase 3 and
inhibit proteolytic activity (8, 9). However, we found
that although HIPEC express XIAP and c-IAP2, IFN-
treatment did not
modulate their expression. These data suggesting that IFN-
did not
prime HIPEC through the direct regulation of caspase 9 activation are
supported by the finding that IFN-
did not sensitize HIPEC for
apoptosis induced by either camptothecin or etoposide
(50). It also indicates that the target(s) of
IFN-
-mediated priming was upstream of caspase 9 activation. These
findings differ from those of several studies using IEC tumor lines in
which IFN-
treatment sensitized for both Fas- and drug-mediated
apoptosis (32-34).
Caspases 1 and 4, two closely related proteases with similar amino acid
sequences and substrate specificity (30, 52), have
previously been implicated in Fas-mediated apoptosis (18, 32-34). IFN--mediated sensitization of IEC tumor lines
has been shown to involve upregulation of caspase 1 or 4 expression
(33, 34). Although caspase 1 is activated in CH11-treated
HIPEC, we found no evidence of IFN-
-mediated regulation of caspase 1 expression or activation. In contrast, IFN-
-mediated sensitization of HIPEC to Fas-mediated apoptosis correlated with the
detection of significantly increased levels of an intermediate-sized
fragment (p35) of caspase 4. Interestingly, two intermediate-sized
fragments, p40 and p35, were detected in untreated as well as
CH11-treated colon HIPEC cultures. It has been suggested that, like for
caspase 1 (59), these intermediate-sized fragments are
indicative of caspase 4 activation (18) or perhaps partial
activation. It is probable that, as for caspase 1 (59) and
caspase 3 (23), the intermediate fragment of caspase 4 is
proteolytically active, but to a lesser extent than the fully activated
p20/p10 subunit aggregate. Also, this intermediate fragment may not
have the same substrate specificity as the fully activated form but is
primarily involved in autoproteolysis (59). However, a
caspase 4 p20 activation fragment was not detected in lysates from
either treated or untreated HIPEC. The concentration of p20 fragment
may have been too low for detection by Western blotting but high enough
for proteolytic activity, in which case it could cleave and activate
effector caspase 3 (18). However, the fact that IFN-
treatment alone does not induce apoptosis of HIPEC or sensitize
cultures for drug-induced apoptosis suggests that the priming
effect of elevated caspase 4 p35 expression requires caspase 8 activity. An alternative possibility is that an increase in expression
of caspase 4, independent of its activation status, may contribute to
the activation of caspase 8. This has been demonstrated for caspase 1 (51). HeLa cells transfected with only the prodomain of
caspase 1 are more sensitive to Fas-mediated apoptosis, and
this sensitization was specific for apoptosis triggered through
Fas but not by etoposide (51). Tatsuta et al.
(51) demonstrated that this caspase 1 prodomain-mediated Fas sensitization correlated with an increase in activity of caspase 8 as well as downstream increases in caspase 3 activity. Therefore, in
the case of IFN-
-sensitized HIPEC, a caspase 4-mediated increase in
caspase 3 activity may occur through the enhanced activation of caspase
8 and not require caspase 4 activation (generation of a p20 subunit).
How an increase in caspase 4 p35 expression can regulate caspase 8 activation remains to be determined.
Our finding that nontransformed IEC are relatively resistant to
Fas-mediated apoptosis has physiological relevance in that it
suggests that Fas may not play a significant role in normal IEC
turnover. However, in conditions in which inflammatory cytokines are
increased in the intestinal mucosa, such as inflammatory bowel disease
or graft-versus-host disease, Fas-mediated apoptosis may contribute significantly to the pathophysiology of these diseases. Also, our finding that IFN--mediated sensitization of nontransformed IEC correlates with expression of caspase 4 intermediate subunit contributes to our understanding of the diversity of regulatory mechanisms that may be involved in controlling apoptosis in the intestinal mucosa.
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ACKNOWLEDGEMENTS |
---|
This work was supported by a First Award Grant (to A. Panja) from the Crohn's and Colitis Foundation of America and Winthrop-University Hospital Intramural Funding (to C. A. Martin and A. Panja).
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FOOTNOTES |
---|
Address for reprint requests and other correspondence: A. Panja, Gastrointestinal Research Laboratory, Winthrop-University Hospital, 222 Station Plaza North, Suite 511, Mineola, NY 11501 (E-mail: apanja{at}winthrop.org).
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.
Received 21 December 2000; accepted in final form 11 September 2001.
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