Materials and methods

TISSUES

Normal human tissues were collected after investigative endoscopic biopsy (n = 10) or resection of colonic carcinomas (n = 10) at the Mercy Hospital, Cork. Normal specimens from patients undergoing surgical resection of colonic carcinoma were taken from the disease free resection margins and on routine haematoxylin and eosin staining were entirely normal morphologically, with no metaplasia or dysplasia evident. Serrated adenomas (n = 3) (table 1), hyperplastic polyps (n = 8) (table 2), and colonic adenomas (n = 38) (table 3) were collected after investigative endoscopic biopsy. Areas of peritumoral epithelium were examined from resected colonic carcinomas not arising in adenomas (n = 32). All specimens were collected according to a protocol approved by the ethics committee of the Cork University teaching hospitals.

immunohistochemical detection of fasl and fas

Paraffin wax embedded sections of normal colon and colonic adenomas were dewaxed in xylene and rehydrated before analysis. Slides were washed twice for five minutes in a wash buffer containing 50mM Tris/HCl (pH 7.6), 50mM NaCl, and 0.001% saponin, and endogenous peroxidase was quenched with 3.0% hydrogen peroxide in methanol for five minutes. Slides were washed as before except that the wash buffer for this and all subsequent steps included 1% normal goat serum. Slides were then blocked for one hour in wash buffer containing 5% normal goat serum. Slides were washed and incubated overnight at 4°C with affinity purified, rabbit polyclonal antihuman FasL or Fas specific IgG (Santa Cruz Biotechnology, Santa Cruz, California, USA) at 0.1 g/ml in wash buffer. Antibody binding was localised using a biotinylated secondary antibody, avidin conjugated horseradish peroxidase (HRP), and diaminobenzidine (DAB) substrate, contained within the Vectastain ABC detection kit (Vector Laboratories, Burlingame, California, USA). The appropriate immunising peptide to which the antibody was raised (FasL, amino acids 260–279; Fas, amino acids 316–335; Santa Cruz Biotechnology) was included at 1 g/ml during primary antibody incubation as a direct, internal competitive control for antibody specificity. In a separate control for specificity of the Santa Cruz FasL specific polyclonal antibody, we found previously that specific antisense oligonucleotide mediated inhibition of FasL expression²³ converted SW620 cells from positive to negative by immunohistochemistry with this antibody. The FasL specificity of the Santa Cruz antibody has also been verified by others: it stained FasL mRNA positive but not FasL mRNA negative cell lines,²⁴ and produced a similar staining pattern to other FasL specific monoclonal²⁵ and polyclonal²⁶ antibodies. FasL detection was confirmed using immunohistochemistry with a FasL specific monoclonal antibody (clone G247-4; Pharmingen, San Diego, California, USA), as described above. The monoclonal antibody was used at a concentration of 5 g/ml, and an isotype matched control antibody was also used. Slides were counterstained with haematoxylin.

immunophosphatase detection with fasl specific monoclonal antibody

After dewaxing and rehydration as above, sections were pretreated by microwave irradiation in 0.01M citrate buffer for five minutes at 370 W. The sections were cooled rapidly by immersing in 0.1M phophate buffered saline (PBS). The slides were then incubated with a mouse antihuman monoclonal FasL specific monoclonal antibody (clone G247-4; Pharmingen) at a concentration of 5 g/ml for one hour. The slides were then washed for five minutes, using 0.1M Tris buffered saline (TBS), which was used for all subsequent washes. After incubation with a secondary rabbit antimouse immunoglobulin (Dako Corp, Ely, Cambridgeshire, UK) at a dilution of 1/25 for 30 minutes, washing was repeated. APAAP (alkaline phosphatase conjugated anti-alkaline phosphatase) complex (DO651; Dako Corp), at a dilution of 1/50, was added to the slides for 30 minutes, after which they were washed again. To enhance staining, the secondary antibody incubation and APAAP complex incubation were repeated once as above, but the duration of the incubation steps was reduced to 10 minutes each. Alkaline phosphatase substrate solution was then added to the slides for 10 minutes. The phosphatase substrate used was Fast Red (Sigma Chemical Co, St Louis, Missouri, USA). All incubations were carried out at room temperature. Slides were counterstained with haematoxylin and viewed by light microscopy.

TUNEL IN SITU CELL DEATH DETECTION

Cell death was detected in situ in resected tissues by enzymatic labelling of DNA strand breaks using terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labelling (TUNEL; Boehringer Mannheim, GmbH, Mannheim, Germany), according to the manufacturer's instructions. Paraffin wax embedded sections were dewaxed in xylene and rehydrated before analysis. After pretreatment by microwave irradiation in 0.01M citrate buffer for five minutes at 370 W, sections were washed in PBS and endogenous peroxidase activity was blocked by 3% hydrogen peroxide in methanol for 30 minutes. The slides were then washed in PBS, which was used for all subsequent washes. The sections were treated with permeabilisation solution (0.1% Triton X-100 in 0.1% sodium citrate) for 15 minutes. After washing, the labelling reaction was performed using a solution containing TdT, its buffer, and fluorescein dUTP. During this step, slides were coverslipped and incubated at 37°C for 60 minutes in a humidity chamber. TdT was omitted from negative control slides, which were included in each run. To localise cells containing labelled DNA strand breaks, sections were washed and incubated with a sheep antifluorescein antibody Fab fragment conjugated with HRP at 37°C in a humidity chamber for 30 minutes. After washing, colour development was performed by incubating sections with a DAB substrate solution for 15 minutes. After a final wash, sections were viewed by light microscopy. Because the TUNEL technique can falsely label DNA strand breaks in proliferating or necrotic cells, only those cells that were both morphologically apoptotic and TUNEL positive were considered to be undergoing apoptosis.²⁷

Results

fasl is expressed by colonic adenomas

All colonic adenomas examined expressed FasL (fig 1A), irrespective of the degree of dysplasia, adenoma size, or the histological type. FasL was expressed throughout more than 70% of neoplastic cells in all adenomas, and Fas was also expressed by all the adenomas examined (fig 1B). The intensity and extent of Fas staining was more uniform than that of FasL throughout the adenomas. By staining consecutive sections, FasL and Fas were seen to be coexpressed in large areas of most adenomas. Carcinomas arising within adenomas all showed a degree of Fas and FasL staining equivalent to the surrounding adenomas (n = 12 of 38).

Using the TUNEL in situ cell death detection assay, positive cells were distributed throughout the adenomas. However, most cells in areas of adenomas coexpressing FasL and Fas were negative for apoptosis (fig 1C). Hence, coexpression of FasL and Fas failed to result in pronounced Fas mediated autocrine cell suicide or paracrine/juxtacrine cell death. In addition, peritumoral epithelium from 30 of the 32 colonic carcinomas examined showed increased FasL expression (fig 2). In these areas, FasL expression extended into the base of the crypts, in contrast to normal colonic epithelium, where FasL expression was restricted to the luminal surface. Most cells in these areas were non-apoptotic morphologically and by TUNEL.

normal colonic epithelium and hyperplastic polyps express fasl at the luminal surface

A distinct pattern of FasL expression was detected in normal colon epithelium. All normal colonic specimens immunohistochemically stained for FasL were positive (n = 20) (fig 3A–E). The FasL specificity of the polyclonal antibody used (Santa Cruz Biotechnology) was confirmed—staining was inhibited by the inclusion of the immunising peptide as a competitive inhibitor before incubation with the primary antibody. Positive staining of epithelium in normal colonic specimens was restricted to the luminal surface epithelial cells. The maximal intensity of epithelial staining was equivalent to that of FasL positive lymphoid cells observed in all specimens. Crypt epithelial cells were negative for FasL. To confirm the expression of FasL by normal surface colonic epithelial cells, staining was also performed with a different FasL specific antibody (monoclonal antibody clone 247-4; Pharmingen). Results using this FasL specific monoclonal antibody were similar to those obtained with the polyclonal antibody. In addition, FasL staining was independent of secondary detection systems, because similar results were found using either APAAP or immunoperoxidase assays.

Positive TUNEL staining and characteristic apoptotic morphology confirmed the occurrence of apoptotic cell death in the luminal surface epithelium. TUNEL staining was largely nuclear, as is typical of true apoptotic detection. This suggests that in contrast to adenoma cells, pronounced apoptosis of normal colon epithelial cells coincides with coexpression of Fas and FasL.

Hyperplastic polyps retained the same pattern of FasL expression as the normal epithelium (n = 8); the luminal epithelium stained positive for FasL whereas crypt epithelial cells were negative (fig 4).

serrated adenomas showed a mixed pattern of fasl expression

Serrated adenomas showed a mixed pattern of FasL expression, varying locally between the expression pattern of normal colonic epithelium and that of adenomatous polyps. FasL was restricted to the luminal surface throughout much of the adenoma, with smaller areas of more widespread expression representing less than one third of the total area of the polyp in all cases (n = 3). The areas of more widespread FasL expression were well demarcated and coincided with areas of dysplasia within the polyps. The pattern of FasL expression within these areas was identical to that of adenomatous polyps, with more than 70% of epithelial cells expressing FasL.

Discussion

Our results show that FasL expression is upregulated at the adenoma stage of colonic carcinogenesis. Colonic adenomas expressed FasL irrespective of adenoma size, histological type, or the degree of dysplasia. Most cells in colonic adenomas coexpressed Fas and FasL without undergoing apoptosis. FasL expression by normal colonic epithelium and hyperplastic polyps was restricted to the luminal surface, where expression coincides with epithelial apoptosis. Serrated adenomas have a mixed pattern of FasL expression. Throughout most of their extent, FasL is expressed at the luminal surface, as in normal colonic epithelium and hyperplastic polyps. In areas of dysplasia within serrated adenomas, FasL expression is upregulated and expressed throughout these areas in the same pattern as is found in adenomatous polyps.

Apoptosis is a central feature of the regulation of colonic epithelial cell numbers.²⁸ Currently, the mechanisms and control of apoptosis in colonic epithelial cell turnover are poorly understood. Although FasL expression was initially thought to be restricted to lymphoid cells, non-lymphoid FasL expressing cells include oesophageal epithelial cells,⁷ keratinocytes,⁸ Sertoli cells,⁹ ocular cells,¹⁰ cytotrophoblasts,¹¹ and neurones.¹² Fas is widely expressed in most cell types, including epithelial cells, and Fas mediated apoptosis might be involved in cell turnover in many tissues,¹⁴ including oesophageal epithelium,⁷ vaginal epithelium,¹⁵ the liver,¹⁶ and the testis.¹⁷ Our finding that terminally differentiated colonic epithelial cells at the luminal surface express FasL suggests a possible role for autocrine Fas mediated apoptosis in cell turnover.

Acquisition of resistance to apoptosis is thought to be crucial in the transformation of cells from normal to neoplastic in many tissues. A functional apoptotic pathway appears to be essential to maintain low numbers of mutated cells.²⁹ Cells that have acquired resistance to apoptosis either before or as a result of mutational changes fail to be eliminated by apoptosis, and are thus able to continue the progression towards a neoplastic phenotype. Failure of apoptosis may be of particular importance in the development of colorectal cancer.³⁰ There is in vitro²³ and in vivo²² evidence that colon cancer cells are resistant to apoptosis mediated by Fas. Despite intracellular defects in Fas signalling in colon cancer cells,³¹ the Fas receptor is downregulated in many colonic carcinomas. Fas downregulation might be a consequence of p53 mutation at later stages of colon tumour development because Fas is a target gene for transcriptional activation by p53.³² Transfection of wild-type p53 into various tumour cell lines has been shown to restore/upregulate Fas expression.³²

Our results demonstrate that Fas–FasL coexpression occurs in the absence of apoptosis in most epithelial cells in mildly dysplastic adenomas, a very early stage in the adenoma–carcinoma sequence. Fas–FasL coexpression without apoptosis was also seen in peritumoral epithelium. This suggests that colonic epithelial cells acquire resistance to Fas mediated apoptosis early in the adenoma–carcinoma sequence. Hyperplastic polyps retained the same pattern of FasL expression as the normal colonic epithelium, which is in keeping with their lack of well defined premalignant potential.

Finally, we previously demonstrated in vitro that FasL expression by colonic carcinoma cell lines may induce apoptosis of Fas sensitive immune effector cells.²³ Evidence from human and animal studies supports this “Fas counterattack” mechanism of tumour immune escape in vivo.^{22, 33} Our finding that FasL is upregulated at the adenoma stage of colon carcinogenesis suggests that colonocytes acquire mechanisms of immune escape early during transformation.