PATIENTS

Polyp samples were collected from 60 patients taking part in the Nottingham colorectal cancer screening study between 1984 and 1991. Samples were formalin fixed and paraffin embedded. The histological type and size of polyp, confirmed by a consultant histopathologist, are shown in table 1. Median age of the patients was 67 years (interquartile range (IQR) 63–70), and there were 37 males and 23 females. Gastrin and CCKBR expression was assessed by both reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry.

REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT-PCR)

Sections of 10 μm thickness were cut from the tissue block and two 10 μm sections were placed in an Eppendorf tube. Xylene 1 ml was added, the sample was vortexed for five minutes, and placed in a warming cabinet at 50°C for at least 30 minutes. The sample was then homogenised, followed by five minutes of centrifugation at 13 000g. Xylene was eluted and the process was repeated four times. Ethanol (100%) 1 ml was added to the sample, vortexed, and centrifuged at 13 000 g. Ethanol was eluted and the process was repeated. The Eppendorf tube was then placed in a heated block at 50°C to allow evaporation. Once dry, RNA extraction was undertaken.25

RNAzol-B (Biogenesis, Poole, UK) 1 ml was added to the sample and homogenised. Chloroform 100 μl (Sigma, Poole, UK) was added and the Eppendorf tube shaken vigorously for 15 seconds. The Eppendorf tube was placed on ice for five minutes, followed by 30 minutes centrifugation at 13 000 g at 4°C. The upper colourless aqueous layer, composed of approximately 400 μl, was carefully removed to a clean Eppendorf. An equal volume of isopropanol was added to the aqueous phase to precipitate RNA from the eluate, and mixed and kept on ice for at least 30 minutes. The sample was centrifuged at 13 000 g for 30 minutes. The supernatant was decanted, the pellet washed with 1 ml of 75% ethanol, and centrifuged at 8000 g at 4°C for seven minutes. Following the final wash, ethanol was allowed to evaporate from the tube and the pellet was resuspended in 50 μl of 0.1% diethyl pyrocarbonate (DEPC) treated water.

Total RNA preparations were divided into two 25 μl aliquots, one of which became the RT negative control by omission of the RT enzyme. Random hexamer primer (90 U/ml) 3 μl was then added and heated to 70°C for 10 minutes.

The tubes were cooled on ice, after which the RT reaction buffer was added, which was composed of 5 μl 10× PCR buffer (Flowgen, Litchfield, UK); 1.5 μl of 5 mM deoxynucleoside triphosphate (dNTP) (Pharmacia, Little Chalfont, UK); 5 μl of 0.1 M deoxythymidine triphosphate (GibcoBRL, Irvine, UK); 1 μl of 200 U Superscript RT (GibcoBRL); and DEPC treated H₂O to make the reaction mixture volume 50 μl. In the negative control, the RT enzyme was replaced with water, vortexed, incubated for 10 minutes at room temperature and then for one hour at 37°C and 95°C.

Details of all primers and probes have been described previously.26 PCR, gel electrophoresis, and Southern blotting were performed using protocols described previously.26 In this study different cDNA volumes were used for individual reactions. Reactions for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and gastrin mRNA detection (50 μl) were prepared using the following: 1.0 U of Dynazyme (Flowgen) in 1× Dynazyme PCR buffer (Flowgen) with 40 μmol of dNTPs (Pharmacia) and 10 μmol of upper and lower primers in a 50 μl final volume. The volumes of cDNA preparations were 1 μl for GAPDH and 5 μl for gastrin. The reaction for total CCKBR mRNA (the primer pairs were designed to detect long/short and truncated isoforms) detection was as follows: 2.5 U Amplitaq Gold (PE Applied Biosystems, Warrington, UK) in 1×PCR buffer (PE Applied Biosystems) and 1.5 mM MgCl₂ (PE Applied Biosystems) with 40 μmol of dNTPs (Pharmacia) and 10 μmol of upper and lower primers, to which 10 μl of CCKB cDNA were added.

IMMUNOHISTOCHEMISTRY: GASTRIN PEPTIDES AND GASTRIN/CCKB RECEPTOR

Paraffin embedded sections (5 μm) were adhered to glass coated slides, dewaxed, deparaffinised, and rehydrated in alcohol. The sections were then incubated in 10% hydrogen peroxide in methanol for 15 minutes at room temperature to quench endogenous peroxidase followed by two further alcohol washes and a one minute water soak.27

The sections were then blocked in swine serum (DakoPatts, Cambridge, UK) for 20 minutes at room temperature, excess serum was removed, and sections were incubated with either a 1/100 dilution of rabbit antiserum directed against carboxy amidated gastrin, 1/80 diluted rabbit antiserum against glycine extended gastrin, rabbit 1/40 diluted antiserum against progastrin (provided by Andrea Varro), or 1/40 rabbit anti-CCKBR (GRP1) antiserum (directed against the amino terminal domain of the human CCKBR) with and without pre-absorption (Aphton Corporation, California, USA) for 60 minutes. The antisera against the different gastrin species have previously been characterised in studies by immunoprecipitation and high pressure liquid chromatography.28 The anti-CCKBR antiserum has been characterised by western blotting.29

The methodology for primary and secondary antibody staining has been described previously.27 For assessment of the presence, degree, and intensity of staining for gastrin peptides in colonic samples, image analysis was performed by the use of the Leica Qwin Image processing and analysis system run on a Leica Q5001W PC. A program was used enabling cross sectional area of DAB staining to be specifically measured and expressed as a function of total cross sectional area of tumour tissue. The positively stained areas appear black/brown and were selected by density and wavelength of staining and expressed as a percentage of total adenoma tissue on the section. Six to 10 fields were assessed per section. Interobserver variation was found to be 5.5% and intraobserver variation 7.2%. This was performed by assessing the variation in the scoring of a complete section either twice by the same individual or by two observers. Identical fields were therefore not used for this analysis.

REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION

GAPDH was obtained from 42/60 cDNA samples analysed. The absence of GAPDH implied failure either to extract messenger RNA from the sample or of RT. Normal human gastric antrum cDNA samples gave an expected gastrin PCR product of 125 bp which was detected on Southern blots by a specific oligo probe. There was no expression of gastrin in the normal mucosa from non-malignant cases (5/5).

Gastrin was expressed in 29/37 polyps where GAPDH was obtained. Gastrin PCR products from the products showed a two band pattern that has been reported previously in colonic adenocarcinoma.30 The expected product, derived from a mature mRNA, of 215 bp was coexpressed with a higher molecular weight band of 345 bp. Negative control RT-PCR lanes showed no hybridisation on Southern blots, confirming that the 345 bp band was derived from a gastrin RNA species. The gastrin PCR primers bind to regions on exons 2 and 3 of the cDNA and therefore the extra band is derived from retention of the intron as previously reported.30

The primers used for CCKBR RT-PCR detected both CCKB and the truncated CCKB isoforms. The expected CCKBR RT-PCR products were detected and verified by specific hybridisation with an oligo probe on Southern blots in 30/37 samples in which GAPDH was identified. The receptor was coexpressed with gastrin in 29/30 cases.

IMMUNOHISTOCHEMISTRY

The distribution of staining for gastrin and CCKBR in the control gastric antrum and fundus has previously been reported.27

Immunohistochemistry results for normal mucosa and polyps are presented in table 1. Positive staining is expressed as a percentage of total adenoma tissue on the section. A value of 10% staining was used as the minimum to indicate the presence of the peptide. In the normal control colonic mucosa there was no staining of gastrin peptides or CCKBR. Progastrin staining was seen in the cytoplasm in 91% of polyps (fig1A), with a mean of 41% (SD 11.2%) coverage. Cytoplasmic staining of Gly-G17 was seen in 80% of polyps (fig 1B) (mean 23% (SD 7.9%) coverage), and amidated gastrin 17 only in 47% (13.2% (SD 6.8) coverage). CCKBR was present in 96% (fig 1C) with 43% (SD13.3) coverage. Staining for CCKBR was located within the cytoplasm but the intensity was greatest at the nucleus.

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Figure 1

(A) Progastrin staining was seen in the cytoplasm in 91% of polyps. (B) Cytoplasmic staining of glycine extended gastrin 17 was seen in 80% of polyps. (C) Gastrin/cholecystokinin type B receptor was present in 96% of polyps.

Expression of gastrin peptides and CCKBR occurred in small tubular adenomas <1 cm. Expression of the peptides was seen in all histological types of polyp; there was no difference in the proportion and level of expression between the histological types.