Growth control factors in the gastrointestinal tract

Although there is abundant evidence that gastrin-releasing peptide acts as a mitogen in various carcinoma cell lines, the effect of administration of gastrin-releasing peptide on the colorectal mucosa in vivo has not been reported. The aims of this study were to determine whether continuous infusion of gastrin-releasing peptide stimulated proliferation or accelerated carcinogenesis in the rat gastrointestinal tract and other organs. The possible requirement for C-terminal amidation for mitogenic activity in vivo was also investigated. Proliferation was measured in the colon by metaphase index and by immunostaining for the proliferation marker Ki-67, and in other tissues by immunostaining alone. Acceleration of colorectal carcinogenesis was assessed by counting aberrant crypt foci after treatment with the carcinogen azoxymethane. Defunctioning of the rectum reduced both the proliferative index and the crypt height of the rectal mucosa of untreated rats. Treatment with amidated or glycine-extended gastrin-releasing peptide for 4 weeks using implanted mini-osmotic pumps resulted in a two- to three-fold increase in proliferation, and an increase in crypt height, in the defunctioned rectal mucosa (p < 0.001), with smaller but significant increases in the caecum and distal colon. No changes in proliferation were detected in lung, pancreas or gastric mucosa. The numbers of aberrant crypt foci in the mid-colon, distal colon and rectum following treatment with azoxymethane were also significantly increased by infusion with amidated or glycine-extended gastrin-releasing peptide. We conclude that administration of gastrin-releasing peptide to mature rats stimulates proliferation and accelerates carcinogenesis in the colorectal mucosa, and that C-terminal amidation is not essential for either effect. Gastrin-releasing peptides could thus potentially act as promoters of colorectal carcinogenesis.

The small bowel has a remarkable ability to adapt after injury, inflammation or resection. It has long been suggested that humoral factors, particularly enteroglucagon, epidermal growth factor, neurotensin and growth hormone/insulin-like growth factor I, might stimulate bowel growth. Of particular interest is the recent finding that glucagon-like peptide 2 (GLP-2), a product of the gene encoding proglucagon, exerts a trophic effect on the intestinal epithelium via a specific G-protein-coupled receptor. GLP-2 and/or these other trophic peptides might prove to have a role in the treatment of bowel diseases associated with structural or functional loss of the small bowel.

One of the first steps in multistage colonic carcinogenesis is increased cell proliferation and an upward shift of the proliferation zone of colonic crypts. In the present study, progression in cell kinetics was followed up at all sequential stages of colonic carcinogenesis, starting with aberrant crypt foci (ACF), the earliest putative preneoplastic lesions, hyperplastic and dysplastic polyps, and invasive carcinomas.

Colonic tissue and tumor specimens were prospectively obtained from 65 patients treated at our hospital for adenocarcinoma or malignant polyps. For identification of ACFs, dissected mucosal strips obtained from patients with colorectal cancer were stained with 0.1% methylene blue and scanned under dissecting microscope. Paraffin-embedded ACFs and macroscopic lesions were serially sectioned, deparaffinized, and stained with a monoclonal antiproliferating cell nuclear antigen (PCNA) antibody. The PCNA-labelling index (PCNA-LI), expressed as a ratio of positively stained nuclei to total nuclei counted, was calculated separately for basal, middle, and upper colonic crypt compartments. A comparison of the PCNA-LI was made for each compartment in normal mucosa, and hyperplastic and dysplastic lesions.

A stepwise increase in the PCNA-LI was observed during neoplastic progression of colonic lesions. The two most important variables of increased cell proliferation, expressed as PCNA-LI per crypt compartment, were the presence of dysplasia and the size of dysplastic lesions.

In colorectal carcinogenesis, hyperproliferation with upward expansion of proliferative compartment is a characteristic feature at all stages of malignant progression.

Despite the wealth of information concerning EGF and its related peptides, its precise role in the control of gastrointestinal functions is still not fully resolved. However, there is no doubt that it can have some very potent effects on the gastrointestinal tract. These may be related to the control of growth and development and to the regular control of cell renewal. Nevertheless, in the adult, EGF may only be active in response to luminal damage and repair, and furthermore this may also only occur if the luminal EGF is protected from proteolytic degradation. Notwithstanding this, ‘EGF’-like responses may be evoked in the gut by intestinal TGF-α. The possible therapeutic use of EGF and members of its family in ulcer therapy will be discussed in later Chapters of this volume, other potential uses are in the control of necrotising enteritis and in the alleviation of the mucositis associated with cancer treatment.

After the loss of small bowel through disease or surgery the residual bowel adapts by increasing its functional capacity. This process of adaptation involves dilatation, hypertrophy and mucosal hyperplasia, particularly distal to the area of bowel loss or disease. The response of the residual bowel is mediated by a complex interplay of factors including luminal nutrition, pancreaticobiliary secretions, luminal or local growth factors and also humoral or endocrine factors.

The experimental model commonly used to characterize the adaptive response, massive small bowel resection (MSBR), involves 80% resection of the small bowel in the rat. Of the various putative humoral factors, most work has focused on the products of the ileal L cells: enteroglucagon and peptide YY. Plasma levels of both hormones are increased after MSBR and indeed their mRNA levels are also increased as a result of an increase in the amount of message per L cell. Whilst PYY probably serves as an ‘ileal brake’ to slow the movement of the luminal contents and hence increase their mucosal contact time, the role of the enteroglucagon is unresolved. The molecular cloning of the proglucagon gene has revealed, firstly, that there are a number of biologically active peptides which derive from the propeptide and, secondly, that tissue-specific differential processing occurs. Most studies do not clearly define which of these products of proglucagon is being measured and is termed as glucagon-like or enteroglucagon immunoreactivity.

The insulin-like growth factors (IGF) have a potent mitogenic action on the bowel. Their role after MSBR is likely to be largely paracrine. Though IGF-I mRNA levels do not increase after MSBR, the precipitous and early fall in ileal IGF-binding protein-3 (IGFBP-3) mRNA levels suggests a fall in IGFBP-3 levels may increase local IGF-I bioactivity.

Polyamine synthesis is a critical component of the adaptive response, although the stimulus to their dramatic increase in synthesis after MSBR remains to be elucidated. Other humoral factors such as cholecystokinin, neurotensin and bombesin probably have minor indirect roles in the adaptive response. Components of the epidermal growth factor/transforming growth factor α response pathway family of growth factors may be involved as paracrine regulators.

There is thus strong evidence that humoral factors play an important role in intestinal adaptation; characterization of the nature of the humoral factors and their relationship with other influences such as luminal nutrition and pancreatic biliary secretions may facilitate the development of new therapeutic strategies for the short bowel syndromes.

There is now clear-cut evidence that polypeptide growth factors control the proliferation of the normal gastrointestinal mucosa. Epidermal growth factor (EGF) stimulates normal growth throughout the gastrointestinal tract, and accelerates the healing of ulcerated epithelium. While the effects of gastrin were at first thought to be similarly widespread, the gastrin target now appears to be restricted to the enterochromaffin-like cells in the stomach. Isolated reports suggest that several other hormones, including fibroblast growth factor and the insulin-like growth factors, have similar proliferative effects. In contrast, indirect evidence suggests that somatostatin and transforming growth factor-β inhibit the growth of the gastrointestinal mucosa.

The same growth factors profoundly affect the growth of some gastrointestinal carcinomas. Prolonged hypergastrinaemia increases the risk of development of gastric endocrine tumours, but has no effect on the incidence of gastric adenocarcinoma. Gastrin also stimulates the in vivo growth of 50% of gastric and colorectal carcinoma xenografts, but has no consistent effect on the growth of carcinoma cell lines in vitro. EGF, on the other hand, significantly stimulates proliferation of many gastrointestinal cell lines in culture.

Interest has recently focused on autocrine stimulation of gastrointestinal carcinoma growth. Elevated levels of EGF receptor, and of EGF or related mRNAs, have been demonstrated in gastric carcinomas, and the growth of some gastrointestinal cell lines is inhibited by antibodies against EGF, and by antisense oligonucleotides based on EGF mRNA. Similarly gastrin/cholecystokinin antagonists inhibit the growth of several colon carcinoma cell lines, although the spectrum of antagonist potencies suggests that classical gastrin and cholecystokinin receptors are not necessarily involved. Continued research on antagonists may therefore lead to novel therapies for gastrointestinal cancers.