PATIENTS AND SPECIMENS

Histologically normal tissue was obtained from colectomy specimens from patients with colorectal neoplasia from a site distant from the lesion (n=12) and surgical specimens were obtained from a similar site in patients undergoing surgery for refractory ulcerative (n=12) and Crohn's (n=6) colitis. Tissue was immediately frozen and stored at −70°C and post fixed as required. Characteristics of the patient groups are shown in table 1.

REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT-PCR)

Tissue samples were collected as discussed, and immediately frozen in liquid nitrogen. Total RNA was purified from 200 mg of tissue according to the method of Chomczynski and Sacchi.17 This entailed homogenisation in guanidine thiocyanate solution, acid phenol/chloroform extraction, and isopropanol precipitation. RNA pellets were washed in 70% ethanol, dissolved in sterile water, and stored at −70°C. Aliquots of each RNA sample (5 μg) were reverse transcribed to produce single stranded cDNA with a kit (Invitrogen, USA) using random primers and avian myeloblastosis virus reverse transcriptase. Samples of cDNA (50 ng) were then analysed by PCR amplification using pairs of oligonucleotide primers specific for COX-2. Primer sequences were: 5′ gTT CCA CCC gCA gTA CAg 3′ sense, and 5′ ggA gCg ggA AgA ACT TgC 3′antisense primer, amplifying a 483 bp cDNA fragment.

Each PCR reaction contained 1× NH₄ CL PCR buffer, 1.875 mM MgCl₂ , 0.1 μM of each primer, 0.5 mM dNTPs, 1 μCi³²P-dCTP, and 2.5 units of BioTaq DNA polymerase (Bioline, UK). Following five minutes of denaturation at 93°C, hot start reactions were initiated (annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, and denaturation at 93°C for 30 seconds) and run for up to 35 cycles. Semiquantitative PCR data were generated by kinetic analysis and comparison with levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. Reaction products were analysed by 6% polyacrylamide gel electrophoresis and film autoradiography using radiolabelled DNA size markers. Amplification products separated in this way were excised and eluted from the gel into 0.5 M ammonium acetate and ethanol precipitated. The identity of the product was confirmed by direct cycle sequencing using a kit (Circumvent, New England Biolabs, USA).

IN SITU HYBRIDISATION

Fresh tissue samples were fixed by immersion in 1% paraformaldehyde in phosphate buffered saline. Samples were then frozen in cryostat embedding compound for preparation of 10 μm sections on 3-aminopropyltriethoxysilane coated slides. Sense and antisense³²P labelled single stranded DNA probes were generated by PCR. Riboprobes were generated using T7 RNA polymerase after making templates by PCR with an appropriate T7 promoter tagged primer. Probes were purified by phenol/chloroform extraction and ethanol precipitation and were washed six times with 70% ethanol to remove unincorporated nucleotides. The probes were dissolved in water, denatured at 90°C for one minute, cooled on ice, and serially diluted in hybridisation buffer (50% formamide, 6× standard saline citrate (SSC), 5× Denhardt's solution (containing bovine serum albumin, ficoll, and polyvinyl pyrrolidone), 100 μg/ml sheared and denatured herring sperm DNA, and 100 μg total RNA from baker's yeast.) Sections were hybridised under siliconised coverslips at 42°C overnight in humidified chambers. Coverslips were removed in 5× SSC at room temperature and racks of slides were washed at 42°C in 2× SSC, 1× SSC, and 0.5× SSC for 20 minutes in each solution. Washed slides were allowed to dry before performing film autoradiography (with Amersham Hyperfilm B-max, 24–72 hour exposure) followed by emulsion autoradiography (2–5 days with Ilford K5 liquid emulsion). Silver grains were developed using Kodak D-19 developer and fixed in dilute Amfix (Champion, UK). Sections were counterstained, coverslipped, viewed, and photographed.

IMMUNOHISTOCHEMISTRY

COX-2 protein expression was examined in tissue using a standard ABC Vectastain technique. Tissue sections were post fixed in 1% paraformaldehyde. Briefly, endogenous peroxidase activity was blocked in tissue sections (10 μm sections on 3-aminopropyltriethoxysilane coated slides) using endogenous peroxidase suppressor “Immunopure” (Pierce Laboratories) for 22 minutes followed by application of normal goat serum (1:20) to block non-specific staining for 30 minutes. The primary antibody, a rabbit polyclonal raised against COX-2, was obtained from Cayman Chemical (Ann Arbor, Michigan, USA). This was applied to serial sections at a concentration 1:1000 and incubated for one hour at room temperature. Following this the second antibody was applied, a goat antirabbit biotinylated antibody (applied at 5 μg/ml, obtained from Vecta Laboratories, California, USA). This was further probed with AB Vectastain and developed with diaminobenzadine tetrahydrochloride dihydrate for 3–5 minutes, with resultant brown staining indicating sites of COX-2 immunolocalisation. Normal rabbit serum (concentration up to 2 μg/ml) was applied on serial sections as a negative control. Sections were counterstained with haematoxylin (1:20). Localisation of COX-2 was compared with that of the neural marker protein G peptide (PGP9.5) and COX-1 (the constitutive isoform of cyclo-oxygenase) using appropriate antibodies against these proteins.