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An Early Event in the Adenoma-Carcinoma Sequence Gut: First Published As 10.1136/Gut.47.6.820 on 1 December 2000

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An Early Event in the Adenoma-Carcinoma Sequence Gut: First Published As 10.1136/Gut.47.6.820 on 1 December 2000 820 Gut 2000;47:820–824 Gastrin and gastrin receptor activation: an early event in the adenoma-carcinoma sequence Gut: first published as 10.1136/gut.47.6.820 on 1 December 2000. Downloaded from A M Smith, S A Watson Abstract naemia on in vitro colonic cancer cell lines3–6 Background and aims—Gastrin and the and in tumours in vivo.7–9 The action of gastrin cholecystokinin type B/gastrin receptor in colorectal cancer is potentially mediated by (CCKBR) have been shown to be ex- several receptor subtypes. The presence of a pressed in colorectal adenocarcinoma. high aYnity binding site has been seen in 57% Both exogenous and autocrine gastrin of samples,10 although gastrin/cholecystokinin have been demonstrated to stimulate type B receptor (CCKBR) mRNA has only growth of colorectal cancer but it is not been demonstrated in less than 20% of known if gastrin aVects the growth of samples.11 12 However, other receptor isoforms colonic polyps. The purpose of this study may be responsible for the proliferative action was to determine if gastrin and CCKBR of gastrins, including the cholecystokinin type are expressed in human colonic polyps C receptor,11 the glycine extended gastrin 17 and to determine at which stage of (Gly-G17) receptor,13 or potentially truncated progression this occurs. isoforms of CCKBR.14 15 Methods—A range of human colonic Following malignant transformation of the polyps was assessed for gastrin and colonic epithelial cell, there is activation of the CCKBR gene and protein expression. gastrin gene16 and the cell associated gastrin Results—Normal colonic mucosa did not can act in an autocrine/paracrine manner.17 express gastrin or CCKBR. Gastrin and The presence of tumour associated gastrin has CCKBR reverse transcription- been demonstrated in several human tumour 18–23 polymerase chain reaction products were series. These series showed that the non- detected and verified by specific hybridi- amidated precursors progastrin and Gly-G17 sation with an oligo probe on Southern are present in the majority of specimens, with a blots. Gastrin and CCKBR were expressed wide variation in the proportion demonstrating in 78% and 81% of polyps, respectively. complete processing of gastrin. The precursor Both genes were coexpressed in 97% of peptides have assumed greater importance cases. Immunohistochemistry identified with recent reports demonstrating that Gly- http://gut.bmj.com/ progastrin in 91%, glycine extended gas- G17 and progastrin have proliferative ef- 13 24 trin 17 in 80%, and amidated gastrin 17 in fects. Ciccotosto et al demonstrated that non-amidated and total gastrin levels are only 47% of polyps. CCKBR was present in 20 96% of polyps. Expression of gastrin and elevated in patients with colorectal cancer and CCKBR was seen in all histological types this excess systemic non-amidated gastrin may and sizes of polyps. arise from the tumour. The point in the adenoma-carcinoma se- Conclusions—This study is the first to on September 29, 2021 by guest. Protected copyright. show widespread expression of both gas- quence when the gastrin autocrine pathway is trin and its receptor in colorectal polyps. activated has not been determined. In normal colonic mucosa, gastrin production is confined Their activation occurs early in the 18 adenoma-carcinoma sequence. Gastrin to neuroendocrine cells. Therefore, the gas- may promote progression through the trin autocrine pathway must be activated adenoma-carcinoma sequence. during the adenoma-carcinoma sequence. If (Gut 2000;47:820–824) autocrine gastrin activation is an early event, its presence may promote polyp progression, and Keywords: gastrin; colon; adenomas; polyps; autocrine furthermore, expression of CCKBR may allow the adenoma to be aVected by circulating gas- trin concentrations. Colorectal carcinoma is one of the most Only one limited study has examined common cancers and is the second leading expression of gastrin mRNA in six adenoma- Academic Unit of cause of cancer mortality in the USA.1 The tous polyps by northern blotting.18 The aim of Cancer Studies, work of Muto and colleagues2 revealed that the the present study, using a range of colorectal Department of majority of colorectal carcinomas develop from Surgery, University polyps, was to determine if colonic adenomas Hospital, Nottingham an adenomatous polyp. Progression from express either gastrin, CCKBR, or both, in a NG7 2UH, UK adenoma to carcinoma requires a combination larger series of polyps. We have determined the A M Smith of genetic mutations which are either heredi- S A Watson tary and/or occur as a result of environmental risk factors. Gastrin has been extensively stud- Abbreviations used in this paper: Gly-G17, glycine Correspondence to: ied as a growth factor for colorectal adenocar- extended gastrin 17; CCKBR, gastrin/cholecystokinin Mr A M Smith. type B receptor; RT-PCR,reverse transcription- andrew.smith@ cinoma but few studies have investigated its nottingham.ac.uk polymerase chain reaction; DEPC, diethyl role in the adenoma-carcinoma sequence. pyrocarbonate; dNTP, deoxynucleoside triphosphate; Accepted for publication Numerous studies have demonstrated the APC, adenomatous polyposis coli; GAPDH, 22 June 2000 proliferative eVect of exogenous hypergastri- glyceraldehyde-3-phosphate dehydrogenase. www.gutjnl.com Gastrin and colonic polyps 821 point in the adenoma-carcinoma sequence triphosphate (GibcoBRL, Irvine, UK); 1 µl of when gastrin and CCKBR gene activation 200 U Superscript RT (GibcoBRL); and Gut: first published as 10.1136/gut.47.6.820 on 1 December 2000. Downloaded from occur. DEPC treated H2O to make the reaction mix- ture volume 50 µl. In the negative control, the Materials and methods RT enzyme was replaced with water, vortexed, PATIENTS incubated for 10 minutes at room temperature Polyp samples were collected from 60 patients and then for one hour at 37°C and 95°C. taking part in the Nottingham colorectal Details of all primers and probes have been cancer screening study between 1984 and described previously.26 PCR, gel electrophor- 1991. Samples were formalin fixed and paraYn esis, and Southern blotting were performed embedded. The histological type and size of using protocols described previously.26 In this polyp, confirmed by a consultant histopatholo- study diVerent cDNA volumes were used for gist, are shown in table 1. Median age of the individual reactions. Reactions for patients was 67 years (interquartile range glyceraldehyde-3-phosphate dehydrogenase (IQR) 63–70), and there were 37 males and 23 (GAPDH) and gastrin mRNA detection (50 females. Gastrin and CCKBR expression was µl) were prepared using the following: 1.0 U of assessed by both reverse transcription- Dynazyme (Flowgen) in 1× Dynazyme PCR polymerase chain reaction (RT-PCR) and buVer (Flowgen) with 40 µmol of dNTPs immunocytochemistry. (Pharmacia) and 10 µmol of upper and lower primers in a 50 µl final volume. The volumes of REVERSE TRANSCRIPTION-POLYMERASE CHAIN cDNA preparations were 1 µl for GAPDH and REACTION (RT-PCR) 5 µl for gastrin. The reaction for total CCKBR Sections of 10 µm thickness were cut from the mRNA (the primer pairs were designed to tissue block and two 10 µm sections were detect long/short and truncated isoforms) placed in an Eppendorf tube. Xylene 1 ml was detection was as follows: 2.5 U Amplitaq Gold added, the sample was vortexed for five (PE Applied Biosystems, Warrington, UK) in minutes, and placed in a warming cabinet at 1×PCR buVer (PE Applied Biosystems) and ° 50 C for at least 30 minutes. The sample was 1.5 mM MgCl2 (PE Applied Biosystems) with then homogenised, followed by five minutes of 40 µmol of dNTPs (Pharmacia) and 10 µmol of centrifugation at 13 000 g. Xylene was eluted upper and lower primers, to which 10 µl of and the process was repeated four times. Etha- CCKB cDNA were added. nol (100%) 1 ml was added to the sample, vor- texed, and centrifuged at 13 000 g. Ethanol was IMMUNOHISTOCHEMISTRY: GASTRIN PEPTIDES eluted and the process was repeated. The AND GASTRIN/CCKB RECEPTOR Eppendorf tube was then placed in a heated ParaYn embedded sections (5 µm) were block at 50°C to allow evaporation. Once dry, adhered to glass coated slides, dewaxed, depar- 25 RNA extraction was undertaken. aYnised, and rehydrated in alcohol. The http://gut.bmj.com/ RNAzol-B (Biogenesis, Poole, UK) 1 ml was sections were then incubated in 10% hydrogen added to the sample and homogenised. Chloro- peroxide in methanol for 15 minutes at room form 100 µl (Sigma, Poole, UK) was added temperature to quench endogenous peroxidase and the Eppendorf tube shaken vigorously for followed by two further alcohol washes and a 15 seconds. The Eppendorf tube was placed on one minute water soak.27 ice for five minutes, followed by 30 minutes The sections were then blocked in swine centrifugation at 13 000 g at 4°C. The upper serum (DakoPatts, Cambridge, UK) for 20 colourless aqueous layer, composed of approxi- minutes at room temperature, excess serum on September 29, 2021 by guest. Protected copyright. mately 400 µl, was carefully removed to a clean was removed, and sections were incubated with Eppendorf. An equal volume of isopropanol either a 1/100 dilution of rabbit antiserum was added to the aqueous phase to precipitate directed against carboxy amidated gastrin, 1/80 RNA from the eluate, and mixed and kept on diluted rabbit antiserum against glycine ex- ice for at least 30 minutes. The sample was tended gastrin, rabbit 1/40 diluted antiserum centrifuged at 13 000 g for 30 minutes. The against progastrin (provided by Andrea Varro), supernatant was decanted, the pellet washed or 1/40 rabbit anti-CCKBR (GRP1) antiserum with 1 ml of 75% ethanol, and centrifuged at (directed against the amino terminal domain of 8000 g at 4°C for seven minutes. Following the the human CCKBR) with and without pre- final wash, ethanol was allowed to evaporate absorption (Aphton Corporation, California, from the tube and the pellet was resuspended USA) for 60 minutes.
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