| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Technical Advance |
From the Departments of Medicine,*
Pathology,¶
Physiology and
Biophysics,§
and Epidemiology and Biostatistics
and The Ireland Cancer Center,
Case Western
Reserve University School of Medicine, and the Cleveland Clinic
Foundation,
Cleveland, Ohio
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, granzyme B, chemotherapeutic agents, and growth factor
deprivation.5-7
Poly(ADP-ribose) polymerase (PARP), an
enzyme involved in genome surveillance and DNA repair, is a substrate
of caspase 3 and other members of its subfamily.8
PARP
cleavage is a commonly assessed molecular event to demonstrate caspase
activation and represents an early molecular hallmark of programmed
cell death.6 Intestinal epithelial cells (IECs) derive from stem cells at the base of the crypt and migrate along the crypt-villus axis as they differentiate. On reaching the luminal surface after 3 to 5 days, they detach and are shed.9 As IECs show evidence of DNA fragmentation and morphological features of apoptosis in the region of shedding, programmed cell death is thought to play an integral role in completing the life cycle of these cells.10 A form of apoptosis, referred to as detachment-induced cell death, or anoikis,11 is likely to contribute to this process, as the configuration of the basement membrane, the expression of transforming growth factor-ß, and the composition of integrins at the luminal surface create conditions that promote loss of anchorage and anoikis.12-14
Research on apoptosis of human IECs has predominantly been carried out
using adenocarcinoma cell lines; however, these transformed cells
cannot be considered ideal models for studies of physiological cell
death. Apoptosis in IEC lines can be induced by a variety of
triggers, including interferon-
, tumor necrosis
factor-/Fas,15
transforming growth
factor-ß,14
short-chain fatty acids,16
p53,17
cyclooxygenase 2,18
and bak19
as well as detachment from
extracellular matrix (ECM)14
and loss of cell-cell
contact.20,21
Studies on apoptosis in nontransformed human IECs have been largely performed using immunohistochemical techniques. In the colon, bcl-2, a known inhibitor of apoptosis, is highly expressed in the region of the stem cells and is undetectable at the luminal surface.22,23 No expression of bcl-2 is detected in the small intestine of humans24 or mice.25 The expression of bak, a promoter of apoptosis, displays an inverse pattern to that of bcl-2, with strongest expression at the luminal surface of the colon.19 Recently, the susceptibility to Fas-mediated apoptosis has also been demonstrated in freshly isolated human IECs.26 Progress of studies on human IECs, however, has been limited by the fact that freshly isolated IECs die of apoptosis within hours of isolation, caused by loss of ECM anchorage or cell-cell contact.27,28 Thus, human IECs undergoing anoikis in vitro may represent a useful model to study the molecular mechanisms of cell death in IECs at the luminal surface.
In this report, a novel protocol, specifically designed to elucidate early molecular events of apoptosis in nontransformed human IECs, is described. Using this method, highly purified intact intestinal crypts can be isolated before activation of caspase 3 family members and cleavage of intracellular caspase substrates. Elucidating the molecular mechanisms of anoikis in IECs may enhance our understanding of homeostasis in the intestinal epithelium as well as facilitate long-term cultures of human nontransformed IECs.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Surgical specimens were obtained from patients undergoing large bowel resection for colon cancer. Macroscopically normal tissue, at least 10 cm from the tumor margins, was studied. All specimens were processed within 1 hour of the resection.
Isolation of IECs
IECs were isolated by three existing protocols (methods A, B, and C) and one newly designed protocol (method D), characterized in this report.
Method A
This method was carried out according to the protocol of Youngman et al.29 After washing the specimen in Ca2+-, Mg2+-free, Hanks' balanced salt solution (CMF-HBSS; BioWhittaker, Walkersville, MD), the mucosa was dissected from the submucosa and cut into strips. These were washed at room temperature in 10 mmol/L dithiothreitol (DTT; Fisher Biotech, Fair Lawn, NJ) for 30 minutes, followed by two 90-minute washes in 1 mmol/L EDTA (Sigma Chemical Co., St. Louis, MO). Cells liberated from both washes were harvested by centrifugation at 500 x g for 5 minutes at room temperature, followed by a 30-minute incubation in 3 mg/ml dispase (Boehringer Mannheim, Indianapolis, IN) and 1 mg/ml DNase (Worthington, Freehold, NJ) at 37°C. Cells were harvested by centrifugation and washed twice with CMF-HBSS, followed by further purification with a 50% Percoll gradient (Pharmacia Biotech, Uppsala, Sweden). After centrifugation for 20 minutes at 300 x g, purified epithelial cells were harvested at the interphase and washed three times in CMF-HBSS.
Method B
This method was carried out according to the protocol of Gibson et al.30 After washing the specimen in CMF-HBSS, the epithelial cell layer was scraped from the submucosa with a glass slide and finely minced using a crossed scalpel technique, followed by a 90-minute incubation at 37°C in 1.2 U/ml dispase (Boehringer Mannheim) and 50 U/ml collagenase type IV (Worthington). After passing through a 21-gauge needle, the digest was washed in CMF-HBSS and filtered through a 300- 400-µm mesh to remove undigested tissue fragments. The cells were then washed twice in CMF-HBSS, separating crypts from single cells by repeated centrifugations at 75 x g for 2 to 3 minutes.
Method C
This method was carried out according to the protocol of Whitehead et al.31 Tissue was washed several times in 0.1 mol/L phosphate-buffered saline (PBS; BioWhittaker), and the mucosa was dissected from the submucosa, followed by a 30-minute wash in 0.04% sodium hypochloride. The mucosal strips were then cut into 1-cm2 pieces and incubated for 1.5 hours at 22°C in 1 mmol/L EDTA, 1 mmol/L EGTA (Sigma) and 0.5 mmol/L dithiothreitol in PBS. Crypts were then liberated by vigorous hand shaking of the beaker. This procedure was repeated six times with 10-ml aliquots of PBS. Liberated crypts were harvested by centrifugation and washed once in PBS.
Method D
Specimens were thoroughly rinsed in CMF-HBBS. Mucus and debris were removed by gently tapping the tissue with paper towels between rinses. The mucosa was dissected from the submucosa, cut into strips that were washed in 10 mmol/L dithiothreitol for 30 minutes at room temperature, and then incubated for 60 minutes in 1 mmol/L EDTA in CMF-HBBS at 4°C. Epithelial cells were detached as intact crypts by 10 vigorous shakes of the vessel. The cells suspended in EDTA were immediately passed through filtering cylinders prepared by taping a piece of 80-µm nylon mesh (Nytex, Tetko, Elmsford, NY) to a plastic ring (5 cm diameter, 2.5 cm height). This allowed single cells to pass through while retaining the crypts. The mesh was rinsed with 20 ml of Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY) and inverted, and the purified crypts were backwashed from the cylinder with 15 ml of keratinocyte serum-free medium (K-SFM; Gibco BRL), supplemented with 2.5% penicillin-streptomycin-fungizone (PSF; BioWhittaker) and 1% gentamicin (BioWhittaker). To confirm the importance of anchorage for epithelial cell survival, in some experiments, surgical specimens were divided in half. From one half ("early"), IEC crypts were isolated immediately and purified IECs were maintained in a polypropylene container at 37°C. The other half of the tissue ("late") was incubated intact for 5 hours under the same conditions as the isolated crypts. At the end of this period, IECs were isolated from the second half of the tissue.
Ethidium Bromide/Acridine Orange Stain
Ethidium bromide/acridine orange staining was performed as described.32 Briefly, 2 µl of a combined dye of 100 µg/ml acridine orange (Sigma) and 100 µg/ml ethidium bromide (Sigma) were added to 20 µl of the cell suspension, and 5 µl of the stained cell suspension was rapidly transferred to a glass slide for immediate analysis using an ultraviolet fluorescence microscope (Olympus-BH2, Olympus, Lake Success, NY). Cells were scored into four categories: (C1, cells with large, green, noncondensed nuclei as non-apoptotic, viable cells; C2, cells with green nuclei that showed signs of nuclear condensation or nuclear bead formation as apoptotic, viable cells; C3, cells with red/orange nuclei that showed signs of nuclear bead formation as apoptotic, nonviable cells; and C4, cells with large red nuclei that did not show signs of nuclear condensation or bead formation as necrotic cells. At least 200 cells/sample were counted and scored. The apoptotic index (percent) was calculated by dividing the sum of apoptotic cells (C2 + C3) x 100 by the total number of cells scored.
DNA Extraction and Electrophoresis
DNA extraction was performed as described.33 Freshly isolated IECs were pelleted by centrifugation and lysed with 0.4 ml of DNA extraction buffer (10 mmol/L Tris/HCl, pH 8.0 (Boehringer Mannheim), 0.1 mol/L EDTA, pH 8.0, and 0.5% sodium dodecyl sulfate (SDS; Boehringer Mannheim)), followed by gentle pipetting. Proteinase K (200 µg/ml; Gibco BRL) was added, and samples were incubated overnight at 42°C. An equal volume of ultra-pure phenol (Gibco BRL) was added and gently agitated for 30 minutes, followed by centrifugation for 5 minutes at 14,000 rpm. The upper aqueous phase was carefully harvested, and an equal volume of chloroform/isoamyl alcohol (Sigma) was added. After 30 minutes of gentle agitation and centrifugation for 5 minutes at 14,000 rpm, the upper, aqueous phase was harvested and incubated for 1 hour at 37°C after addition of 50 µg/ml RNAse (Sigma). An equal volume of a 1:1 mixture of phenol and chloroform/isoamyl alcohol was added. The samples were gently agitated for 30 minutes and centrifuged as above. The upper phase was again harvested, and DNA was precipitated by adding a 2.5-fold volume of ice-cold 100% ethanol. DNA was harvested by centrifugation for 10 minutes at 14,000 rpm, and the supernatant was decanted. DNA was washed twice with 1 ml of 70% ethanol, followed by centrifugation. The DNA was resuspended in 20 µl of TE buffer (10 mmol/L Tris/HCl, 1 mmol/L EDTA, pH 8.0) and quantified spectrophotometrically. Two micrograms of DNA was fractionated on a 1.6% agarose gel (Gibco BRL) by electrophoresis, stained with 0.5 µg/ml ethidium bromide (Sigma), and visualized under ultraviolet light.
Fluorogenic Substrate Assays
Synthetic tetrapeptide substrates, specifically cleaved by members of the caspase 3 and caspase 1 subfamilies, respectively, were added to IEC cytosol. N-Acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC; Biomol Research Laboratories, Plymouth Meeting, PA) was used as a substrate specific for caspase 3 family members, and N-acetyl-Tyr-Val-Ala-Asp-4-methyl-coumaryl-7-amide (YVAD-AMC; Biomol) served as a substrate for caspase 1 family members.34 Freshly isolated IECs were lysed in 0.25 ml of lysis buffer (100 mmol/L HEPES, 10% sucrose, 0.1% 3-[(3-cholanidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS), 1 mmol/L phenylmethanesulfonyl fluoride (PMSF), 10 mmol/L dithiothreitol (DEVD-AMC fluorogenic assay only), 100 µmol/L pepstatin, 100 µmol/L leupeptin, 1 mmol/L EDTA at pH 6.8), followed by a 30-minute incubation on ice. Lysis was completed by two 10-second sonication pulses (Sonicator XL, Heat Systems, Farmingdale, NY). Cellular debris was removed by centrifugation, and the cytosolic extract was stored at -20°C. Protein content of the lysates was determined by the BioRad protein assay (BioRad, Hercules, CA) according to the manufacturer's instructions. Twenty micrograms of cytosolic protein and 50 µmol/L DEVD-AMC or YVAD-AMC were incubated in a total volume of 500 µl in 100 mmol/L HEPES, 10% sucrose, 0.1% CHAPS, pH 6.8, for 30 minutes on an orbital shaker at 37°C. Samples were diluted with lysis buffer to a final volume of 1 ml immediately before measurement of fluorescence by fluorospectrophotometry (Perkin Elmer LS-3; excitation, 380 nm; emission, 480 nm; slit width, 3.0). For inhibition of caspase 3 family activity, 20 µg of protein from lysates was incubated with 150 µmol/L DEVD-CHO (Biomol) for 20 minutes at 37°C before incubation with the fluorescent substrate. Standards containing 0 to 5000 pmol of AMC were used to determine the amount of fluorochrome released. Measurements were recorded over the linear range of the assay. Bovine serum albumin (50 µg) served as negative control. As a positive control, a monocytic cell line (U937) was treated with lipopolysaccharide (100 µg/ml) to induce caspase 1 activation. Eight hours after stimulation, cytosol from U937 cells was extracted by the same protocol used for IECs and tested for caspase 1 activity.
Western Blotting
Fifty micrograms of cytosolic protein was fractionated on an 8 to 14% SDS-polyacrylamide gel and electrotransferred to Immobilon p15 membranes (Millipore Corp., Bedford, MA). Membranes were blocked overnight at 4°C with 5% milk in 0.1% Tween-20/Tris-buffered saline (TBS; Fisher), followed by incubation with the mouse monoclonal primary antibodies (anti caspase-3 at 1:2000; Transduction Laboratories, Lexington, KY; and 1 µg/ml anti-PARP35) for 60 minutes at ambient temperature. Membranes were washed six times with 0.1% Tween-20/TBS and then incubated for 1 hour with horseradish-peroxidase-conjugated goat anti-mouse IgG secondary antibody (1:5000; Transduction Laboratories), washed again, and incubated with chemiluminescent substrate (Super Signal, Pierce, Rockford, IL) for 5 minutes. The membranes were exposed to film (Amersham, Arlington Heights, IL).
Immunohistochemistry
Freshly isolated IECs were suspended in PBS, and 50 µl of the cell suspension was transferred to glass slides (Superfrost Plus, Fisher). The slides were air dried for 10 minutes, fixed for 10 minutes in ice-cold acetone, and incubated for 10 minutes in 3% hydrogen peroxide to block endogenous peroxidase activity. All staining was performed using the Dako Autoimmunostainer (Dako, Carpinteria, CA). Slides were rinsed between each step twice for 5 minutes with TBS (Dako). The following primary antibodies were used: anti-CD3 at 1:500 for 20 minutes (Ao452, Dako), a pan-cytokeratin cocktail (anti-AE1/AE3) at 1:25 for 10 minutes (MU071-UC, BioGenex, San Ramon, CA), and anti-CD68, a macrophage marker at 1:500 for 10 minutes (M814, Dako). After two rinses with TBS, slides were incubated with a universal secondary antibody solution of biotinylated anti-rabbit and anti-mouse immunoglobulin (LSAB-2, Dako) for 10 minutes, rinsed with TBS, and labeled with horseradish-peroxidase-conjugated streptavidin (LSAB-2, Dako) for 10 minutes. Slides were rinsed with TBS and developed with diaminobenzidine chromogen substrate (Dako) for 5 minutes in hydrogen peroxide and counterstained with hematoxylin and eosin (H&E). Human tonsillar tissue served as a positive control. Percentage of positive staining was determined by scoring 200 intact single cells from three different specimens.
Electron Microscopy
Freshly isolated IECs were fixed with glutaraldehyde and suspended in agar. IECs were post-fixed with OsO4, dehydrated with a series of ethanol washes, rinsed with propylene oxide, and embedded in LX-112 (Ladd Research Industries, Burlington, VT), which polymerized at 65°C. Samples were thin sectioned (80 nm), counterstained with uranyl acetate and lead citrate, and examined with a JEOL 100CX transmission electron microscope.
Terminal Deoxynucleotidyl Transferase (TdT) Nick End Labeling (TUNEL)
Terminal deoxynucleotidyl transferase (TdT) nick end labeling was performed using the Apoptag-plus kit (Oncor, Gaithersburg, MD) according to the manufacturer's instructions. IECs were fixed on glass slides (Superfrost Plus) in 4% paraformaldehyde. Bovine aortic endothelial cells, treated with 25 µmol/L ketocholesterol for 24 hours to induce apoptosis,36 were processed simultaneously as positive controls.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Colonic IECs were isolated in strict accordance to three existing
protocols (methods A to C) and the newly developed protocol (method D),
characterized in these studies. Although the viability of the cells
isolated by all these protocols, as assessed by trypan blue staining,
is greater than 95%, the degree of apoptosis varied considerably.
Isolation of IECs using method A requires 6 hours and yields greater
than 80% apoptotic cells when stained with acridine orange/ethidium
bromide (Figure 1A)
. This high level of
apoptosis was confirmed by analyzing the DNA from isolated IECs by gel
electrophoresis (Figure 1B)
. IECs isolated using method A proved to be
unsuitable for the study of early events in apoptosis. Therefore, the
degree of apoptosis was determined in IECs isolated by two other
protocols (methods B and C).
|
, the cells obtained using protocol B were
less apoptotic (apoptotic index = 53% versus 83% as
determined for protocol A) with less DNA fragmentation. These data
suggested that reducing the processing time is critical to decrease the
degree of apoptosis in IEC. Using method C, IECs could be isolated
within 2 hours at room temperature. The apoptotic index of these cells
was 15% with little detectable DNA fragmentation (Figure 1, A and B)
.
The lower apoptotic index in IECs isolated using method C suggested
that an additional reduction in both the time and temperature of
isolation further decreases the rate of apoptosis. Development of a New IEC Isolation Protocol
To develop a new procedure to isolate non-apoptotic colonic IECs,
the time required for isolation, the temperature at which the protocol
was performed, and the use of enzymes were systematically minimized.
The final protocol (method D, as described in Materials and Methods)
evolved by first reducing the time of EDTA incubation outlined in
method A, then lowering the temperature of the tissue and eliminating
the protease digestions as recommended in method C, and finally adding
a filtration step to rapidly harvest and purify colonic crypts. A
comparative study of each protocol is detailed in Table 1
.
|
.
Figure 1
compares the four methods for the degree of apoptosis as
determined by acridine orange/ethidium bromide staining (Figure 1A)
and
gel electrophoresis of epithelial cell DNA (Figure 1B)
. An important
observation derived from these data is that DNA fragmentation is a late
event in IEC apoptosis. Loss of Anchorage Induces Apoptosis of IECs
To confirm the critical role of anchorage for epithelial cell
survival, surgical specimens were divided in half. From one half
("early"), IEC crypts were isolated immediately and maintained in a
polypropylene container at 37°C. The other half of the tissue was
incubated intact for 5 hours under the same conditions. At the end of
this period, IECs were isolated from the second half of the tissue
("late"). The degree of IEC apoptosis was assessed by acridine
orange/ethidium bromide stain and DNA electrophoresis. As shown in
Figure 2A
, epithelial cells, purified
immediately ("early"), displayed an apoptotic index of 2%. Five
hours later, 89% of these IECs were apoptotic. In contrast, when IECs
remained anchored during the incubation period, 95% of these cells
remained viable and non-apoptotic ("late"). These observations were
confirmed by DNA electrophoresis (Figure 2B)
.
|
IECs isolated using method D were further analyzed for
evidence of apoptosis by TUNEL assay. No staining was detected,
demonstrating lack of fragmented DNA (data not shown). Electron
microscopy assessing the morphology and ultrastructure of IECs
confirmed the lack of apoptosis. In particular, the large, oval nuclei
located at the basolateral surface revealed no signs of chromatin
condensation at the nuclear membrane or formation of apoptotic bodies
(Figure 3A)
. IECs were preserved as a
single layer of columnar-shaped cells. Major organelles (mitochondria,
endoplasmic reticulum, and lysosomes), microvilli, tight junctions, and
desmosomes were perfectly preserved (Figure 3B)
.
|
Light microscopy examination of the isolated epithelium revealed
tubular structures, typical of colonic crypts, formed by a single layer
of columnar cells lining the crypt lumen (Figure 4A)
. As the distribution of apoptotic
IECs varies along the crypt axis, the isolation of selected areas of a
crypt might give an unrepresentative estimate of apoptosis. After
isolation, mucosal strips were fixed and stained with H&E. Microscopic
examination showed that crypts were removed from the lamina propria in
their entire length (Figure 4B)
. To assess the purity of cells isolated
using the new protocol, the percentage of epithelial cells, T cells,
and macrophages was determined by immunohistochemistry. IECs were
positive for cytokeratin and represented greater than 97% of the cells
isolated (data not shown). Contamination by CD3+ T cells
was less than 2%, and none of the cells stained positive for the
macrophage marker CD68 (data not shown).
|
DNA fragmentation represents a relatively late event during the
execution phase of apoptosis.37
In contrast, activation of
caspases initiates the apoptotic cascade and amplifies the death
signal, thereby representing an early event. DEVD-AMC is a synthetic,
fluorescent substrate specific for the caspase 3 subfamily. On
cleavage, fluorescence is released, reflecting proteolytic
activity.38
To study the activation of caspases in IECs
isolated using the four methods described above, cytosolic extracts
were prepared and the enzymatic activity of caspase 3 family members
was determined. IECs purified using methods A to C contained high
levels of caspase-3-like enzymatic activity, whereas the caspase-3-like
enzymatic activity in IECs isolated using the new method showed only
background levels (Figure 5)
. To confirm
the specificity of DEVD-AMC cleavage, an inhibitor of caspase-3-like
proteases (DEVD-CHO) was added to lysates before incubation with the
substrate. Absence of fluorescence indicated that caspase-3-like
specific cleavage of DEVD-AMC was totally inhibited (Figure 5)
. These
findings confirm that method D yields non-apoptotic IECs before caspase
activation. Furthermore, they demonstrate that caspase-3-like cysteine
proteases are activated in IECs undergoing anoikis. To determine
whether IECs isolated by method D contain activated caspase 1 family
members, the fluorogenic assay was performed using YVAD-AMC, a
synthetic substrate of the caspase 1 family. YVAD-AMC was not cleaved
when incubated with cytosol from IECs isolated by any method,
suggesting that members of the caspase 1 family are not involved in IEC
anoikis (Figure 6)
. As a positive
control, cytosolic protein from LPS-stimulated U937 was used. As active
caspase 1 is present in stimulated monocytic cells, the substrate was
readily cleaved, and this activity was inhibited by YVAD-CHO, a
selective caspase 1 inhibitor.
|
|
Although the fluorescence assay described above indicated the
activation of various members of the caspase 3 subfamily, we performed
Western blot analysis to assess in particular the activation of caspase
3, which is abundantly expressed in the intestinal epithelium. The
inactive 32-kd proenzyme of caspase 3 is specifically cleaved during
apoptosis to 17- and 12-kd fragments that reassemble to form the active
enzyme. As shown in Figure 7
, the 17-kd
cleavage product was readily detectable in lysates from methods A, B,
and C, whereas in lysates of IECs isolated by method D only the intact,
non-activated 32-kd form of caspase 3 was detectable, demonstrating
that method D yielded IECs before activation of caspase 3. These
findings also indicate the involvement of caspase 3 in IEC anoikis.
Furthermore, they show that caspase 3 activation is an early event as
IECs from method C display marked cleavage of caspase 3 within 2 hours
after detachment.
|
PARP is a 116-kd protein, which is cleaved during apoptosis by
members of the caspase 3 family of cysteine proteases to 85- and 31-kd
fragments. To study the biological activity of caspase 3 family members
in freshly isolated IECs, lysates of IECs, isolated according to the
four methods, were assessed by Western blot for evidence of PARP
cleavage. In agreement with the data presented above, PARP cleavage was
readily detected in lysates from IECs isolated using methods A and B.
Cleavage of PARP was markedly less evident in IECs purified using
method C and undetectable in lysates of IECs isolated by method D
(Figure 8)
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our study demonstrates that anoikis in IECs involves the rapid activation of caspase 3 family members and, in particular, caspase 3. We were unable to detect caspase 1 family activity in the four populations of IECs isolated, suggesting that anoikis involves the select activation of distinct caspases. The lack of caspase 1 family activation is seemingly contradictory to the results of two previous reports, which suggest that members of the caspase 1 family of cysteine proteases may be involved in mammary epithelial cell apoptosis39 and programmed cell death of the intestinal epithelial tumor cell line HT-29.40 The conclusions drawn from those studies, however, are based on indirect evidence, using cytokine response modifier A (CrmA) and Boc-aspartyl(benzyl) chloromethylketone (BACMK) to inhibit apoptosis. At the time these investigations were reported, CrmA and BACMK were thought to be specific inhibitors of caspase 1. However, subsequent studies demonstrate that CrmA also inhibits caspase 3, 7, and 8.41-43 Although caspase 1 has been shown to induce apoptosis when overexpressed in fibroblasts44 and mediates Fas-induced apoptosis in W4 cells,45 other investigators have not been able to confirm these findings.46 In addition, caspase-1-deficient mice develop normally.47 Therefore, the existence of multiple caspase activation pathways seems likely.48 Notably, IECs isolated by the new method displayed no evidence of PARP cleavage even though cleavage of PARP is considered to be a particularly early molecular event of apoptosis.6,49 Ongoing studies employ this new method to characterize the kinetics of activation for various caspases and cleavage of caspase substrates in IECs undergoing anoikis.50
Epithelial cells isolated by the new protocol are fully preserved as whole crypts by light and electron microscopy, displaying intact junctional complexes and organelles without evidence of degeneration, ischemia, or apoptosis. Contamination with T cells, macrophages, and red blood cells is effectively avoided by using an 80-µm mesh, which retains only intact crypts. The high purity, as judged by morphological examination, is confirmed by immunohistochemistry. Importantly, the isolation procedure does not involve any centrifugation, a technique that we observed to be disruptive to the delicate structure of the intestinal crypts. We could not perform flow cytometry to assess purity or evidence of apoptosis because our method isolates intact crypts, which are not suitable for single-cell analysis.
The degree of apoptosis in IECs correlated strongly with the length of time required to purify the cells. This observation is not surprising, as the initial step of IEC isolation inherently involves the detachment of the epithelial cells, which initiates the apoptotic cascade. In this study, we provide additional evidence that detachment is the pivotal trigger of IEC apoptosis during isolation as prolonged anchorage to the mucosa can effectively prevent apoptosis for several hours despite lack of blood supply and incubation of the tissue at 37°C. Furthermore, our data indicate that not only the duration of the protocol but also the temperature during cell isolation is of crucial significance to determine the degree of apoptosis. IECs isolated using method C are less apoptotic than those purified by method B (16% versus 53%), even though method C is only 30 minutes shorter. The decreased degree of apoptosis using method C probably reflects the fact that these cells were never incubated at 37°C. Both observations led to the design of a new protocol to purify IECs within the shortest possible time and at a low temperature (method D).
As changes in Ca2+ metabolism have been shown to induce apoptosis,51 we specifically addressed this issue by studying a protocol that does not employ chelating agents (method B). IECs isolated by mechanical disruption underwent extensive apoptosis within 5 hours of detachment (>90%). Ongoing experiments using dispase in the absence of EDTA to detach IECs also confirm this observation, demonstrating that loss of anchorage, not the technique of isolation, is the critical trigger of IEC apoptosis in vitro.52
The data in this report underscore that assays based on DNA fragmentation, such as oligonucleosome electrophoresis and TUNEL assays, are useful only to detect late stages of apoptosis. Notably, caspase 3 and PARP were already cleaved in IECs isolated using method C, even though these cells showed no DNA fragmentation. Therefore, caution is warranted when classifying cells as non-apoptotic on the basis of assays assessing DNA fragmentation alone. It should also be noted that the viability-based trypan blue stain cannot be used to identify apoptotic cells, as IECs isolated by method A (extensive DNA fragmentation and 83% apoptotic cells by acridine orange/ethidium bromide stain) were >95% viable according to trypan blue stain.
In summary, the new method described in this report provides an in vitro model to explore the mechanisms underlying homeostasis of the intestinal epithelium in vivo, as apoptosis due to loss of anchorage is likely to play a pivotal role in the physiological life cycle of IECs, preventing their ectopic growth after shedding.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by grants DK30399 and DK50984 from NIH (C. Fiocchi) and grants 1523/1–1 and 1523/1–2 from the Deutsche Forschungsgemeinschaft, Bonn, Germany (J. Grossmann).
Accepted for publication April 24, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ligation of Fas antigen. J Immunol 1995, 155:4147-4154[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  M. Toruner, M. Fernandez-Zapico, J. J. Sha, L. Pham, R. Urrutia, and L. J. Egan Antianoikis Effect of Nuclear Factor-{kappa}B through Up-regulated Expression of Osteoprotegerin, BCL-2, and IAP-1 J. Biol. Chem., March 31, 2006; 281(13): 8686 - 8696. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. B. Seidelin, T. Horn, and O. H. Nielsen Simple and efficient method for isolation and cultivation of endoscopically obtained human colonocytes Am J Physiol Gastrointest Liver Physiol, December 1, 2003; 285(6): G1122 - G1128. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yang, Y. Fan, and D. H. Teitelbaum Intraepithelial lymphocyte-derived interferon-gamma evokes enterocyte apoptosis with parenteral nutrition in mice Am J Physiol Gastrointest Liver Physiol, April 1, 2003; 284(4): G629 - G637. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Uesaka, H. Lu, O. Katoh, and H. Watanabe Heparin-binding EGF-like growth factor gene transcription regulated by Cdx2 in the intestinal epithelium Am J Physiol Gastrointest Liver Physiol, October 1, 2002; 283(4): G840 - G847. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Jara, P. Esponda, and R. Carballada Abdominal Temperature Induces Region-Specific p53-Independent Apoptosis in the Cauda Epididymidis of the Mouse Biol Reprod, October 1, 2002; 67(4): 1189 - 1196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Dieterich, M. Schandar, M. Noll, F.-J. Johannes, H. Brunner, T. Graeve, and S. Rupp In vitro reconstructed human epithelia reveal contributions of Candida albicans EFG1 and CPH1 to adhesion and invasion Microbiology, February 1, 2002; 148(2): 497 - 506. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y Hyoh, S Ishizaka, T Horii, A Fujiwara, T Tegoshi, M Yamada, and N Arizono Activation of caspases in intestinal villus epithelial cells of normal and nematode infected rats Gut, January 1, 2002; 50(1): 71 - 77. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Martin and A. Panja Cytokine regulation of human intestinal primary epithelial cell susceptibility to Fas-mediated apoptosis Am J Physiol Gastrointest Liver Physiol, January 1, 2002; 282(1): G92 - G104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Rosignoli, R. Fabiani, A. De Bartolomeo, F. Spinozzi, E. Agea, M.A. Pelli, and G. Morozzi Protective activity of butyrate on hydrogen peroxide-induced DNA damage in isolated human colonocytes and HT29 tumour cells Carcinogenesis, October 1, 2001; 22(10): 1675 - 1680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Grossmann, K. Walther, M. Artinger, S. Kiessling, and J. Schölmerich Apoptotic Signaling during Initiation of Detachment-induced Apoptosis ("Anoikis") of Primary Human Intestinal Epithelial Cells Cell Growth Differ., March 1, 2001; 12(3): 147 - 155. [Abstract] [Full Text]
|
|
|
|
 |
|
 L. A. Scheving, J. R. Thomas, and L. Zhang Regulation of intestinal tyrosine phosphorylation and programmed cell death by peroxovanadate Am J Physiol Cell Physiol, September 1, 1999; 277(3): C572 - C579. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||
