Assessment of Apoptosis by Immunohistochemical Markers Compared to Cellular Morphology in Ex Vivostressed Colonic Mucosa
Department of MicrobiologyImmunology (HH,CMP,HB,KD,CB,CNW), and Department of Surgery (JAW), College of Medicine, University of Arizona, Tucson, Arizona; and Section of Hematology/Oncology, Tucson Veteran Affairs Medical Center, Tucson, Arizona (HG)
Correspondence to: Carol Bernstein, Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson, AZ 85724. E-mail: bernstein3{at}earthlink.net
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Summary |
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(J Histochem Cytochem 53:229235, 2005)
Key Words: apoptosis bile acid deoxycholate colonic mucosa caspase-3 cytokeratin-18 lamin A histone H2AX
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Introduction |
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A number of pathways appear to be important in stress-induced apoptosis, some of which depend on cell type as well as on type of apoptosis-inducing agent (Inagaki-Ohara et al. 2002). A schematic of events accompanying some known stress-induced apoptosis pathways is shown in Figure 1
. One set of early steps in apoptosis induction involves alteration of the mitochondrial membrane with release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Antonsson 2004
). Release of cytochrome c activates a caspase-dependent pathway involving cytochrome c assembly with procaspase-9 and Apaf-1 in close association with cytokeratin 18 (CK18) (Dinsdale et al. 2004
). This leads to formation of the apoptosome on CK18. Next, procaspase-9 is cleaved and activated, forming c-cas-9, which then cleaves and activates both procaspase-3, producing c-cas-3, and procaspase-7, producing c-cas-7 (Slee et al. 1999
). This is followed by c-cas-3-catalyzed cleavage of procaspase-6 (and additional cleavage of procaspase-9) (Slee et al. 1999
; Ruchaud et al. 2002
). Then, c-cas-3, c-cas-6, and c-cas-7 (c-cas-7 action not shown) cleave CK18, forming c-CK18 (Caulin et al. 1997
). C-cas-3 and c-cas-7 both cleave poly(ADP-ribose) polymerase (PARP), forming c-PARP (Soldani and Scovassi 2002
). C-cas-6, subsequent to its cleavage by c-cas-3 (Slee et al. 1999
), cleaves lamin A in the nucleus, forming c-lam-A (Ruchaud et al. 2002
).
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The modified proteins selected as potential markers for apoptosis in our colonic mucosa samples were chosen from the schematic of known steps of apoptosis outlined in Figure 1 and include c-CK18, c-cas-3, c-lam-A, H2AX, c-PARP, and AIF. This set of apoptosis-related proteins represents events taking place in the nucleus (
H2AX, c-PARP, c-lam-A, AIF, and c-cas-3) and cytoplasm (c-cas-3, c-CK18) and represents distinct events in the apoptotic process. It should be noted, however, that cytokeratin 18 is present in the simple (glandular) epithelium of the colonic mucosa but present only at low levels or not present at all in many other cell types (Bosch et al. 1988
; Chu and Weiss 2002
). Antibodies to the set of six modified proteins listed above are commercially available, known to be effective in archived paraffin-embedded tissue, and thought to recognize neo-epitopes (or altered location) frequently present within, and specific to, apoptotic cells. We assessed the usefulness and specificity of these antibodies in comparison to morphological measurements of apoptotic cells using multiple colonic tissue samples from nine individuals, treated ex vivo with deoxycholate.
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Materials and Methods |
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For the seven patients undergoing colonic resection, one to four larger samples were taken for assessing apoptosis by immunohistochemistry in ethanol-fixed tissue. These mucosal samples were either immediately fixed in 70% alcohol and embedded in paraffin, incubated in tissue culture media alone, or incubated in tissue culture medium with 1 mM NaDOC under standard apoptosis-inducing conditions (3 hr at 37C, 5% CO2) (Bernstein et al. 1999,2002
) and then fixed in 70% alcohol and embedded in paraffin. There were one to four samples each for assessment at time zero (no stress), for incubation for 3 hr in tissue culture media, and for incubation for 3 hr in media plus 1.0 mM NaDOC. In addition, we wished to compare results in formalin-fixed tissue with results in ethanol-fixed tissue. Thus, one to four samples were taken from the colon resections from two patients, incubated in media plus 1.0 mM NaDOC for 3 hr, fixed in formalin, embedded in paraffin, and then evaluated for apoptosis by immunohistochemistry with cleaved cytokeratin 18 and cleaved caspase 3.
All patients gave written informed consent under protocols approved by the Human Subjects Committee (the Institutional Review Board) of the University of Arizona. Three of the patients who had a colon resection had a colonic adenocarcinoma, three had diverticulitis, and one had no evidence of colonic disease. The patient with the normal colon, but a resection, had a recurrent liposarcoma external to, but not involving, the colonic wall. The colon was resected as part of the surgical procedure to remove the liposarcoma (and surrounding tissues).
We previously determined that stressing normal colonic tissue ex vivo with 1 mM NaDOC under standard conditions generates an average of 57.6% apoptosis (±13.0%, SD) among goblet cells (Bernstein et al. 1999). This high level of apoptosis was helpful for the quantitative evaluation of the immunohistochemical apoptotic markers tested here.
Expression of antibodies to c-CK18, c-cas-3, c-lam-A, H2AX, c-PARP, and AIF were evaluated using a modified immunohistochemical method described previously (Payne et al. 1998
). Briefly, the paraffin-embedded tissues were cut into 4-µm sections, deparaffinized, and rehydrated. Endogenous peroxidase activity was blocked by incubation in 1% hydrogen peroxide in methanol for 30 min, and the sections were then rinsed with phosphate-buffered saline (PBS). To prevent nonspecific binding, the slides were incubated with 1.5% appropriate normal serum (Vector Laboratories; Burlingame, CA). The primary antibodies used were mouse monoclonal antibody M30 specific for c-CK18 (Roche Molecular Biochemicals; Indianapolis, IN), rabbit polyclonal antibody for c-cas-3 (Cell Signaling Technology, Inc.; Beverly, MA), rabbit polyclonal antibody for c-lamin-A (Cell Signaling Technology, Inc.), rabbit polyclonal antibody for
H2AX (Upstate Biotechnology, Inc.; Lake Placid, NY), mouse monoclonal antibody for c-PARP (Cell Signaling Technology, Inc.), and rabbit polyclonal antibody for AIF (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA). After rinsing with PBS, biotinylated rabbit anti-mouse IgG F(ab')2 (DAKO; Carpinteria, CA) and goat anti-rabbit IgG (Vector Laboratories) were used for the mouse monoclonal and rabbit polyclonal primary antibodies, respectively. Immunocontrol slides were prepared by replacing the primary antibody with mouse IgG2b (for c-CK18 and c-PARP) or rabbit IgG (for c-cas-3, c-lam-A,
H2AX, and AIF) at the same protein concentration as the primary antibody. After rinsing in PBS, the Vectastain Elite ABC kit (Avidin, Biotin Enzyme Complex; Vector Laboratories) was used according to the manufacturer's instructions. Color was developed by applying diaminobenzidine tetrahydrochloride. (Sigma; St Louis, MO) supplemented with 0.04% hydrogen peroxide. Sections were counterstained with hematoxylin (Sigma) and mounted using Cytoseal. Observation of immunohistochemical staining of crypts was performed by one individual.
Apoptosis was determined by morphology in 1-µm epoxy resin sections as described previously (Bernstein et al. 1999, 2002
). Briefly, deoxycholate-stressed tissue was removed from its incubation medium and immersed in 2 ml of cold, half-strength Karnovsky's fixative (pH 7.2) overnight at 4C and then transferred to 0.1 M phosphate buffer (pH 7.2). The tissue was then post-osmicated, dehydrated in a graded series of ethanols, and embedded in Spurr's epoxy resin. Epoxy sections (1 µm) were prepared using glass knives, and the sections were heat-attached to slides for 5 min on a hot plate maintained at 80C. The sections were then stained with methylene blueazure IIbasic fuschin (polychrome stain) and rinsed with distilled water. The proportion of apoptotic goblet cells (ratio of darkly stained apoptotic cells to lightly stained non-apoptotic cells) was ascertained by light microscopy under a x100 oil-immersion lens. Only nuclei that clearly belonged with the mucin droplet-containing cytoplasm of the goblet cells were scored when obtaining an apoptotic index (AI). A mean AI was obtained as the ratio of apoptotic goblet cells in a tissue sample to the total number of clearly identified goblet cells in that tissue. At least 100 goblet cells obtained from more than 10 different crypts were scored. Goblet cells throughout entire crypts were evaluated in obtaining the AI. Inter-observer variability was previously determined for this method of evaluating AI, and the correlation between observers was found to be 0.89 (p<0.001), with 1.0 representing complete agreement (Bernstein et al. 1999
).
In addition, a rapid crypt apoptotic index (CAI) was measured, using the same polychrome-stained 1-µm sections as used for AI. Here, only the basal one-fourth of well-oriented crypts (with the crypt lumen visible) was examined. The strictly morphologic CAI was measured as the percent of these crypt basal regions with at least one cell recognized as apoptotic by morphologic features, whether a goblet cell or non-goblet cell. An immunohistochemical CAI was also measured on the 4-µm immunohistochemically stained, paraffin-embedded tissue sections. In this case, CAI was the percent of crypt basal regions with at least one immunohistochemically stained epithelial cell. CAI was determined by conjoint observation by two individuals.
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Results |
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Apoptosis after stress by incubation in media with NaDOC, quantitated by strictly morphologic criteria and by four of the immunohistochemical markers (c-CK18, c-cas-3, c-lam-A, and H2AX), yielded fairly high values, and these were quantitated, along with immunohistochemical values obtained at time zero, as controls for background (Table 1). On the other hand, the antibody to c-PARP was considerably less useful for marking deoxycholate-induced apoptotic cells in colonic mucosa, and the antibody to AIF was fairly nonspecific under our conditions. Therefore, these latter two markers were not quantitated in this study. Table 1 gives, for all seven patients, the strictly morphologic AIs and CAIs (using 1-µm epoxy sections), plus the immunohistochemical CAIs (using 4-µm sections) for the four most reactive apoptosis markers.
The time zero values for all four of the immunohistochemical markers quantitated were very low, varying from 0% to 3.9%. The immunohistochemical CAIs obtained after tissues were incubated for 3 hr in media with 1.0 mM NaDOC were generally lower than the strictly morphologic CAIs for such stressed tissues and were much above the background values obtained at time zero (Table 1). The values were close to the same whether tissues were fixed in ethanol or in formalin (Table 1). However, the CAIs for c-CK18 came, on average, closest to the strictly morphologic CAIs. The strictly morphologic CAIs averaged 95% and the c-CK18 CAIs averaged 79%. For c-cas-3, c-lam-A, and -H2AX, the average CAI values were 59%, 51%, and 54%, respectively. Figures 2C2F show images indicating the presence of c-CK18, c-cas-3, c-lam-A, and
-H2AX, respectively, in typical 4-µm paraffin-embedded samples of deoxycholate-stressed colonic tissue.
The c-CK18 CAI of individuals without colonic neoplasia varied from 86% to 100%, while the CAI of individuals with colonic neoplasia varied, in this study, from 33% to 78%.
In one of the patients (AdCa 3 in Table 1), the majority of apoptotic cells seen were non-goblet cells. Such non-goblet cells undergoing apoptosis were also observed in colonic mucosal tissue from the other two patients with adenocarcinoma (see Figure 2B, where one apoptotic non-goblet cell is shown), as well as in colonic mucosa of patients without colonic neoplasia but at lower frequencies.
For c-cas-3, c-lam-A, and -H2AX, the CAI values were variable, falling between 26% and 100% for individuals without colonic neoplasia and between 6% and 65% for individuals with a colonic adenocarcinoma (Table 1).
For each of the antibodies specific for c-CK18, c-cas-3, c-lam-A, and HAX, only morphologically identifiable apoptotic cells showed a positive reaction, verifying that these antibodies are specific for apoptosis. Each of these antibodies reacted positively with both goblet cells and non-goblet cells which were apoptotic by morphology in the 4-µm paraffin-embedded sections. Figures 2C2F show crypt basal regions immunohistochemically stained for c-CK18, c-cas-3, c-lam-A, and
H2AX, respectively. The c-CK18 antibody reacted positively with nearly all observed apoptotic cells. However, each of the antibodies against c-cas-3, c-lam-A, and
H2AX failed to react positively with a significant fraction of cells that were clearly identified as apoptotic using strictly morphologic criteria. To clearly identify apoptotic cells by strictly morphologic criteria, it was necessary to focus up and down through the planes of focus in the 4-µm immunohistochemically stained sections. When examined microscopically, it is clear that in deoxycholate-treated colonic tissue, antibodies against c-cas-3, c-lam-A, and
H2AX are less useful for detection of apoptosis than either the c-CK18 antibody or morphology alone.
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Discussion |
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The antibody to c-cas-3 identified apoptotic cells at a frequency lower than the antibody to c-CK18. It is possible that the epitope for c-cas-3 is affected more adversely than the epitope of c-CK18 by the alcohol and heat treatments used. This explanation is likely because we (and others) have shown that caspase-3 is cleaved in deoxycholate-treated cells of colonic origin (Schlottman et al. 2000; Washo-Stultz et al. 2002
). On the other hand, while caspase-3 is a major protease involved in the execution phase of apoptosis (Slee et al. 2001
), c-cas-3 may not be activated in some ex vivo deoxycholate-treated cells from the colonic mucosa in which CK18 is cleaved, or it may initially be present at lower levels than CK18. Activated c-cas-3 cleaves many substrates, including CK18 [although CK18 is also cleaved by caspases-6 and -7 (Caulin et al. 1997
), as well as by the DEDD-procaspase-3 complex (Lee et al. 2002
)]. Cleaved caspase-3 antibody detects the activated form of caspase-3 by specifically recognizing the large fragment (1720 kD) that results from cleavage after Asp175. The antibody to c-cas-3 has been reported to be useful for detecting apoptotic cells in archival paraffin sections (Gown and Willingham 2002
). In the present study, we evaluated the specificity and usefulness of the c-cas-3 antibody for distinguishing the normal mucosa of patients without colonic neoplasia from that of patients with colon cancer. The mean CAI in the mucosa of the four individuals without neoplasia [67.8% (±16.0% SEM)] was higher than the mean for the mucosa of the three individuals with adenocarcinoma [47.7% (±15.7% SEM)], but the difference was not statistically significant.
Lamin A is an intermediate filament protein and a major component of the nuclear lamina (Moir and Spann 2001). The nuclear lamins polymerize to form the nuclear lamina, a fibrous structure on the inner face of the nuclear membrane. The lamins also form structures within the nucleoplasm that help maintain the shape of the nucleus and participate in various nuclear processes (Moir and Spann 2001
). C-lam-A antibody detects the large (4045 kD) fragment of lamin A after c-cas-6 cleaves the protein at Asp230. The mean CAI for c-lam-A in the mucosa of the individuals without neoplasia was 63.5% (±8.7% SEM), higher than the mean for the mucosa of the individuals with adenocarcinoma [35.0% (±15.3% SEM)], but the difference was, again, not significant.
H2AX is a member of the H2A histone family that becomes rapidly phosphorylated (H2AX) by ATM at Ser139 in response to DNA double-strand breaks (Rogakou et al. 2000
; Burma et al. 2001
). Extensive phosphorylation of H2AX appears to be an early chromatin modification following initiation of DNA fragmentation (Rogakou et al. 2000
; Burma et al. 2001
). The antibody used here recognizes
H2AX phosphorylated at Ser139 in the carboxy terminus. The mean CAI for
H2AX-positive cells in the mucosa of the four individuals without neoplasia was 68.8% (±15.2% SEM), higher than the mean for the mucosa of individuals with adenocarcinoma [34.0% (±12.8% SEM)], but the difference, again, was not significant.
In conclusion, strictly morphologic assessment was the most useful method for detecting colonic crypt apoptotic cells in deoxycholate-stressed tissue. c-CK18 was specific for apoptosis and nearly as useful as morphologic measurements when tissue from patients without neoplasia was evaluated but showed a lower level of identifying apoptosis in patients with a colonic neoplasia. This is interesting as a possible biomarker in stressed tissue because the cancer cases that were prospectively obtained for this apoptosis marker study were "normal" using our standard apoptosis goblet cell AI score (Bernstein et al. 1999). Reduced c-CK18 reactivity may prove useful as an intermediate biomarker for cancer risk; however, a larger study is necessary to validate this point. Antibodies to c-cas-3, c-lam-A, and
H2AX were specific for identification of apoptotic cells of the colonic epithelium but were less useful than morphologic assessment or c-CK18 for identifying apoptotic cells. The specific antibodies to detect c-PARP and AIF appear to be inefficient for assessing apoptosis in alcohol-fixed, paraffin-embedded colonic epithelium.
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Acknowledgments |
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Footnotes |
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