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Identification of Progastrin Derived Peptides In 90 Gut 1993; 34: 90-95 Identification of progastrin derived peptides in colorectal carcinoma extracts Gut: first published as 10.1136/gut.34.1.90 on 1 January 1993. Downloaded from J Nemeth,* B Taylor, S Pauwels, A Varro, G J Dockray Abstract certain colorectal cancer cell lines in vitro and in The possible production of gastrin by transplantable tumours in vivo.'-' Gastrin colorectal carcinomas has been studied. receptor blocking agents (either antagonists or Extracts of 44 tumours and adjacent macro- antibodies) have been shown to bring about scopically normal tissue were examined in significant inhibition of growth in several cell radioimmunoassay using the following anti- lines.8'12 bodies: (i) L289 raised to a C-terminal frag- Together, the observations outlined above ment of progastrin which shows specificity raise the possibility that gastrin might be acting for intact progastrin, but not the extreme as an autocrine growth factor in some colorectal C-terminal tryptic peptide; (ii) LW60 raised to cancers. It is well established that peptides a C-terminal fragment of progastrin which derived from the gastrin precursor, progastrin, reacts with progastrin and its C-terminal are produced in a number of tumours of endo- tryptic peptide; (iii) 109-21 which was raised crine origin, and it seems possible that some to, and reacts with, Gly-extended forms of primary gastric and colorectal tumours may also heptadecapeptide gastrin - that is, biosyn- contain gastrin.2 The latter point, however, has thetic intermediates on the pathway producing not yet been intensively studied. In the present active gastrin; and (iv) L2 which reacts with study we have examined extracts prepared from amidated, biologically active gastrins. AU 44 colorectal tumours, and from 'normal' samples contained detectable material in mucosa, with a range of antibodies which assays using LW60; in general, concentrations together allow us to identify progastrin, its measured with this antibody were higher than C-terminal fragment produced by tryptic with the other antibodies, and in particular cleavage, a biosynthetic intermediate (glycine there were higher concentrations in tumour extended gastrin), as well as biologically active compared with normal tissue extracts. (amidated) gastrins (Fig 1). The results show Tumour extracts also contained higher con- that progastrin derived peptides are commonly http://gut.bmj.com/ centrations of immunoreactivity compared present in colorectal cancers, and suggest that with normal tissue, in assays using antibodies they may be manufactured locally. L289 and 109-21. In contrast, amidated gastrins were found in similar concentrations in tumour and normal tissue, and concentra- Methods tions were the lowest of those recorded in the four assays. Separation on Sephadex G50 PEPTIDES on September 27, 2021 by guest. Protected copyright. revealed peaks compatible with progastrin and The C-terminal pentadecapeptide ofhuman pro- its C-terminal flanking peptide, and two other gastriniepreprogastrin 87-101 wasobtainedfrom peaks that are so far unidentified. In conclu- UCB Products (YGWMDFGRRSAEDEN, in sion most colorectal carcinomas contain the single letter notation); the C-terminal Medical Research peptides derived from the gastrin precursor, decapeptide of progastrin with Tyr in the first Council Secretory progastrin, but for the most part these tumours position - that is, (Tyr92) preprogastrin 92-101 Control Research Group and Physiological do not convert progastrin into biologically (YGRRSAEDEN), was obtained from Multiple Laboratory, active products. Peptide Systems (San Diego, CA, USA); Gly- J Nemeth (Gut 1993; 34: 90-95) extended C-terminal octapeptide fragment of A Varro G17 ie preprogastrin 86-93 (AYGWMDFG), G J Dockray was a gift from Dr J Walsh. Other human Department of Surgery, In addition to their acute effects on gastro- progastrin fragments were obtained from University of Liverpool, intestinal motility and Peninsula Ltd. Liverpool secretion, regulatory Human G17 was a gift from the B Taylor peptides of the gut may also have mitogenic late Professor R A Gregory, and natural human actions on gastrointestinal and other cells. In progastrin was isolated from a gastrinoma as Department of Nuclear recent years it has become clear that certain previously described. 13-15 Medicine, University of tumour Louvain, Brussels, derived cell lines can be associated with Belgium the production of gut peptides, and that some of S Pauwels these peptides are mitogenic to the same cells.' In PATIENTS AND TUMOURS Correspondence to: the case of gastrin, cell lines derived from An unselected, but approximately consecutive, Professor G J Dockray, Physiological Laboratory, colorectal carcinomas have recently been shown series of 44 patients was studied presenting with University of Liverpool, to contain gastrin mRNA,2 and some human colorectal cancers between June 1989 and Crown Street, PO Box 147, Liverpool L69 3BX. colorectal cancers in short term tissue culture November 1990 to the University Department of Accepted for publication release a peptide into the culture medium which Surgery in Liverpool. All patients had sporadic 12 June 1992 appears to have the immunological character- tumours which were managed electively. *Present address: istics of gastrin.3 Colorectal tumour cell lines Patients who presented as emergencies were Department of Medicine, Medical University of Szeged, may also express receptors specific for gastrin,4 excluded, as were patients treated during the Szeged, Hungary. and gastrin has been shown to be mitogenic to same period of time with cancers arising in a Identification ofprogastrin derivedpeptides in colorectal carcinoma extracts 91 NA ",-A ",-A I I I I I -1- Signal 4 I Gut: first published as 10.1136/gut.34.1.90 on 1 January 1993. Downloaded from .. Tyr-Gly-Trp-Met-Asp-Phe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn I I L289 *v Tyr-Gly-Trp-Met-Asp-Phe-Gly Ser-Ala-Glu-Asp-Glu-Asn 109-21 LW60 Tyr-Gly-Trp-Met-Asp-Phe-NH2 L2 Figure 1: Schematic representation ofhuman preprogastrin and its majorproducts showing the specificity ofantibodies used in the present study. The C-terminal region is shown in an expandedform. Cleavage at the Arg residues by endopeptidase and carboxypeptidase generates Gly-extended gastrin intermediates and the C-terminal tryptic peptidefragment. The Gly-extended intermediate is converted to amidated active products. Antibody L289 reacts with C-terminalfragments ofprogastrin longer than eight residues, these include intact progastrins, but exclude the C-terminal tryptic peptide. The latter reacts with LW60 which also reveals intact progastrin, MAb 109-21, and L2 react with Gly extended and amidated peptides respectively. background of familial Polyposis coli or long EXTRACTS standing ulcerative colitis. Deeply frozen specimens were quickly weighed, There was an equal sex distribution in the boiled in water, homogenised and centrifuged as study group, with the majority of tumours (in previously described.'5 This extraction method both sexes) being located distally, in the rectum gives good recovery of progastrin and its major (35%) or sigmoid colon (30%). Other sites were products. Extracts were stored at -20°C before involved less frequently: left colon, 5%; trans- assay or fractionation by gel filtration. verse colon, 10%; right colon and caecum, 20%. The types of operative procedures undertaken are shown in Table I. ANTIBODIES Upon removal from the patient, intestinal The specificity of the various antibodies used in resection were specimens immediately opened this study is shown schematically in Figure 1, http://gut.bmj.com/ along the antimesenteric border, and small and the antibody characteristics are listed in pieces (c 2-5 g each) harvested under direct Table II. Antibody L2 has been shown to vision, both from the tumour itself and from be specific for the C-terminus of amidated normal looking mucosa as far distant from gastrins,'6 109-21 for the C-terminus of Gly- the tumour as was possible. Specimens were extended gastrins,'7 and, antibody L289 for the immediately frozen on dry ice until extracted C-terminus of progastrin.'8 The latter antibody (see has for below). high affinity C-terminal fragments on September 27, 2021 by guest. Protected copyright. The majority (88%) of the tumours studied greater than eight residues and no appreciable were described histologically as moderately affinity for the C-terminal tryptic hexapeptide. differentiated, with only a single tumour (2%) The rabbit antiserum LW60 was raised by described as well differentiated and the immunisation with preprogastrin 87-101 remainder (10%), poorly differentiated. In terms of the stage of the disease, the majority were Dukes' stage B (35%) or C (33%), with only a few TABLE II Characteristics ofantibodies used in in Dukes' stage A (12%). The remaining patients radioimmunoassay in this study (20%) had advanced disease, usually with liver Detection metastases - that is, stage D, at the time of their Antibody Antigen Specificity limit initial surgery. In eight patients, there were L289 h prog 87-101* C-terminus progastrin 0 4 pmol/g other benign tumours within the resection speci- >8 residues LW60 h prog 87-101* C-terminus progastrin 1 0 pmol/g men - that is, tubular adenomas or tubulovillous >5 residues adenomas; these tumours were not sampled 109-21 Gly-extended G6 G-gly 0-1 pmol/g L2 G17 C-terminal amidated 0 1 pmol/g during the present study, while one patient had gastrins two separate, synchronous primary tumours (in the left colon and transverse colon). One patient *Human progastrin C-terminal pentadecapeptide. also had a distal cancer which was gastric TABLE III Specificity ofantibody L W60* resected at the same time as the colonic primary. Relative immunochemical Peptide potency TABLE I Operative procedures Preprogastrin 22-101 1-0 YGWMDFGRRSAEDEN 1-0 Operation % YGRRSAEDEN 0-77 RSAEDEN 0-46 Abdominoperineal excision of rectum 12 5 SAEDEN 0-53 Anterior resection of rectum 22-5 Hartman's resection of rectum 7 5 Sigmoid colectomy 22 5 *The relative potency of various peptides in inhibiting binding of Left hemicolectomy 5 0 label to antibody LW60 is shown.
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