SHORT TERM INFUSION OF GLYCINE-EXTENDED GASTRIN17 STIMULATESBOTH PROLIFERATION AND FORMATION OF ABERRANT CRYPT FOCI INRAT COLONIC MUCOSAAhmad ALY, Arthur SHULKES and Graham S. BALDWIN*

University of Melbourne Department of Surgery, Austin Campus, A&RMC, Melbourne, Victoria, Australia

Evidence is accumulating that gastrin precursors may actas growth factors for the colonic mucosa in vivo and forcolorectal carcinoma cell lines in vitro. The effect of shortterm administration of synthetic gastrins on the colonic mu-cosa in vivo, however, has not been reported. The aim of ourstudy was to determine whether continuous systemic infu-sion of glycine-extended gastrin17 stimulated proliferationand accelerated carcinogenesis in the colorectal mucosa. Asignificant increase in colonic mucosal proliferation as as-sessed by metaphase index was seen in the caecum (23%, p <0.02) and distal colon (27%, p < 0.001), but not the rectum,after treatment of intact rats with glycine-extended gastrin17for 1 week using implanted miniosmotic pumps. Defunction-ing of the rectum reduced both the proliferative index andcrypt height of the rectal mucosa of untreated rats. Treat-ment of rectally defunctioned animals with glycine-extendedgastrin17 for either 1 or 4 weeks resulted in a significantincrease in both the proliferative index (40% and 93%, respec-tively) and crypt height (11% and 19%, respectively) of therectal mucosa. The total number of aberrant crypt foci inintact rats treated with the procarcinogen azoxymethaneplus glycine-extended gastrin17 was increased by 48% com-pared to the value in controls treated with azoxymethaneonly (p 5 0.01). We conclude that short term administrationof glycine-extended gastrin17 to mature rats not only has aproliferative effect upon colonic mucosa, but also increasesthe number of aberrant crypt foci formed in the colorectalmucosa after treatment with azoxymethane. Glycine-ex-tended gastrin17 could thus potentially act as a promoter ofcarcinogenesis.© 2001 Wiley-Liss, Inc.

Key words: aberrant crypt foci; azoxymethane; colorectal carci-noma; gastrin17gly; proliferation

Gastrin is aclassical gut peptide hormone, which was identifiedoriginally as astimulant of gastric acid secretion. Like many otherpeptide hormones, gastrin is synthesized as a large precursormolecule (101 amino acids), which is converted to progastrin (80amino acids) by cleavage of the N-terminal signal peptide. Pro-gastrin isprocessed further by endo- and carboxypeptidasesand byC-terminal amidation to yield either glycine-extended gastrin17 oramidated gastrin17.1 Although amidated gastrin17 was thoughtoriginally to be the only form of the hormone with biologicalactivity, glycine-extended gastrin17 has been shown to stimulatethe proliferation of several cell lines, including some of colonicorigin.2–6 Non-amidated gastrinsalso seem to act asgrowth factorsfor normal colonic mucosa, as transgenic mice overexpressingeither progastrin7 or glycine-extended gastrin17

8 have increasedserum concentrations of the appropriate progastrin-derived pep-tides and a hyperplastic colonic mucosa.

Progastrin-derived peptides may also stimulate the developmentor growth of colorectal carcinoma.9–11 Transgenic mice overex-pressing progastrin have 2–3-fold more aberrant crypt foci thanwildtype controls after treatment with azoxymethane (a colonspecific carcinogen),9 but no such increase was observed in trans-genic miceoverexpressing amidated gastrin17.9 Furthermorecross-ing of Min mice, which are heterozygous for mutation in theAdenomatous Polyposis Coli gene, with mice overexpressing gly-cine-extended gastrin17 increases thenumber of aberrant crypt fociin the progeny.10 Conversely crossing Min mice with gastrinknockout mice increases the survival time of the progeny.10 The

survival time of Min mice may also be increased by reduction ofthe levels of progastrin-derived peptides by immunization againstgastrin.11

In the currently available transgenic mouse models the durationof exposure to progastrin-derived peptides and the serum concen-trations of progastrin-derived peptides are not controlled. Forexample, the transgene utilized by Singh et al.9 was not inducible.As a result, the transgenic mice were exposed to increased con-centrations of progastrin-derived peptides both in utero andthroughout the 5–7-week period between birth and the commence-ment of the treatment.9. The observed increase in aberrant cryptfoci may therefore be at least partly a consequence of progastrinacting on the immature colon. In addition the concentrations ofprogastrin varied widely (means of 1675–8640 pM). Furthermore,in some treatment animals, concentrations of amidated gastrinwere more than 2 orders of magnitude higher than the concentra-tions found in wildtype mice (means in wildtype mice of 41–81pM).9

To avoid these confounding variables, we investigated the shortterm effect of continuous systemic infusion of glycine-extendedgastrin17 on proliferation of the colonic mucosa of mature rats.Because the normal colonic mucosa proliferates rapidly, any ad-ditional effect a putative growth factor may have on the colono-cytesmay bemasked. Someworkershaveused starvation and totalparenteral nutrition to induce hypoplasia of the gut epithelium in abid to lower the proliferative rate and thus create amore sensitiveassay system for testing of putative gut growth factors.12 Alterna-tively animals with a defunctioned colorectum are suitable fortesting the effect of putative growth factors on colonic epithelium,because defunctioning reliably induces hypoplasia of the rectalmucosa and is simple to maintain.13 A further advantage of thelatter model is that the topical effect of test substances can beexamined by direct instillation into the lumen of the defunctionedsegment. We therefore studied the effect of glycine-extended gas-trin17 upon both the intact colon and the defunctioned colon invivo.

We also investigated the short term effect of glycine-extendedgastrin17 in a widely accepted murine model of colonic carcino-genesis. Aberrant crypt foci (ACF) are considered to be preneo-plastic lesions inducible in rat colons by exposure to azoxymeth-ane, a colon specific carcinogen.14 The risk of malignancy iscorrelated to the number of foci induced and the degree of aber-rancy asmeasured by thenumber of cryptsper focus. Wethereforestudied the effect of glycine-extended gastrin17 upon both the

Grant sponsor: Austin Hospital Medical Research Foundation; Grantsponsor: Royal Australian College of Surgeons; Grant sponsor: NationalHealth and Medical Research Council of Australia; Grant numbers:114123, 980625.

*Correspondence to: University of Melbourne Department of Surgery,Austin Campus, A&RMC, Studley Rd., Heidelberg, Victoria 3084, Aus-tralia. Fax: 1613-9458-1650. E-mail: grahamsb@unimelb.edu.au

Received 2 April 2001; Revised 29 May 2001; Accepted 4 June 2001

Published online 18 August 2001; DOI 10.1002/ijc.1483

Int. J. Cancer: 94, 307–313 (2001)© 2001 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

number of ACFs and the number of crypts per focus in the colonicmucosa of rats after azoxymethane treatment.

MATERIAL AND METHODS

ChemicalsGlycine-extended gastrin17 was custom synthesized by Auspep

(Melbourne, Australia). Azoxymethane was obtained from Sigma(St. Louis, MO).

Animals and housingYoung adult male Sprague–Dawley rats were used in all exper-

iments. The animals were approximately 250–350 g in weight atthe commencement of experiments. The rats were caged in pairswith a 12 hr light-dark cycle and were fed standard rat chow withfree access to tap water. In experiments involving the administra-tion of carcinogens, rats were housed as above but in an isolatedcytotoxic room. For 48 hr post administration of azoxymethane,the rats were kept under an evacuation fume hood as a safetyprecaution against inhalation of exhaled carcinogenic by-productsof azoxymethane.15

Study 1: intact and defunctioned colon studyGlycine-extended gastrin17 was administered systemically for 1

week via an implanted mini-osmotic pump (Alzet miniosmoticpumps, models 2ML4 and 2ML1, Alza, Mountain View, CA) tointact rats and rats with defunctioned colons. The theoretical dosebased on the initial concentration and specified delivery rate was10 nmol/kg/hr. The following 2 additional experiments were per-formed in the rats with defunctioned colons: first, glycine-extendedgastrin17 was administered directly into the lumen of the defunc-tioned rectum (10 nmol/kg/hr for 1 week) and second, glycine-extended gastrin17 was infused systemically (2.5 nmol/kg/hr for 4weeks). For all experiments, 5 rats were allocated to a controlgroup and 5 to a treatment group. Plasma concentrations of gly-cine-extended gastrin17 were measured in all rats. At the end of theexperimental period, colons were harvested and specimens pre-pared for analysis. Mucosal proliferation was assessed by thestathmokinetic technique of counting mean metaphases per cryptat 3 hr (herein referred to as the metaphase index) and by mea-surement of crypt height.

Operative procedures for insertion of minipumps aloneRats underwent inhalational anesthesia with enflurane. A small

(1 cm) incision was made in the midline to enter the peritonealcavity. The pre-primed minipump was then placed into the peri-toneal cavity and the wound closed.

Operative procedures for rectal defunctioning and insertion ofminipumps

Rats were anesthetized using intraperitoneal phenobarbitone at adose of 40–50 mg/kg. A lower abdominal midline incision wasmade to enter the peritoneal cavity. The descending colon wasidentified, mobilized by division of peritoneal attachments to thepancreas and small bowel and delivered into the wound. A lym-phoid follicle (very constant in position) on the antimesentericborder of the rectum corresponding to the level of branching of theinferior mesenteric vessels was identified and selected for the pointof division. The mesentery was displayed to allow vascular divi-sion between ligaclips and the rectum divided at this point. Therectal stump was flushed clear of feces using 0.9% saline and wasclosed with ligaclips. A descending colon end colostomy wasfashioned in the left lower quadrant of the abdomen. Whereintraluminal delivery was required, the rectal stump was closedaround the pump catheter with a holding stitch. The pre-primedpumps were placed in the peritoneal cavity and the wound wasclosed.

Glycine-extended gastrin17 deliverySolutions of glycine-extended gastrin17 (250mM in phosphate-

buffered saline [PBS] containing 0.1% bovine serum albumin

[BSA]) were filtered through 0.4mm Millipore filters (Millipore,North Ryde, Australia). The miniosmotic pumps were filled withglycine-extended gastrin17 solution and placed in saline at 37°C 6hr before insertion. Treatment rats received glycine-extended gas-trin17 in PBS containing 0.1% BSA at 10 nmol/kg/hr for 1 week.Control rats received PBS with 0.1% BSA alone.

Preparation of specimens for measurement of proliferativeindices

Colon harvests took place at the same time of day for all groups(12:00–13:00) to avoid diurnal variability in mucosal prolifera-tion.16 All rats were injected with vincristine (1 mg/kg) via theintraperitoneal route, 3 hr before sacrifice, to arrest cycling cells inmetaphase. Euthanasia was performed with 100% CO2. Colonswere then harvested from caecum to anus and opened along theantimesenteric border. Specimens were washed clean with 0.9%saline and pinned flat to be divided into separate specimens.Sections were prepared from caecum, descending colon and rec-tum. Care was taken to avoid including mucosa from the stomalend in any of the specimens. The rectal specimen was sent wholeafter excision of the distal 5 mm (to avoid anal skin) and proximal8–10 mm (to avoid non-specific changes due to over-sewing of thestump). Specimens were embedded in paraffin and multiple sec-tions taken 30mm apart (to avoid inclusion of the same crypt at thesame axial level in separate sections). Sections were stained withhaematoxylin and eosin and mounted on glass slides by the De-partment of Anatomical Pathology, Austin & Repatriation MedicalCentre, Melbourne, Australia.

Measurement of proliferative indicesStained sections were read using an Olympus BH-2 light mi-

croscope with a320 objective lens. The number of metaphase-arrested nuclei per crypt were counted and a mean value obtainedafter counting 20 crypts per rat for each section of colon. Cryptswere deemed suitable for assessment if the base of the crypt wasin close proximity to the muscularis mucosa and the full length ofthe crypt lumen could be seen in the section.

Crypt length was measured in pixels using an Olympus micro-scope at310 magnification connected to Leica Q500MC imageanalysis software. Ten to 15 suitable crypts per specimen weremeasured and representative crypts were chosen from all areas ofthe specimen. Criteria for suitable crypts were as above but inaddition care was taken to include only crypts with intact surfaceepithelium and no fixing artefact.

Study 2: aberrant crypt foci studyIn this study, 28 rats were allocated equally to control or

treatment groups. The preparation of the miniosmotic pumps con-taining glycine-extended gastrin17 was as for Study 1. The mini-osmotic pumps were placed in the peritoneal cavity through a 2 cmmidline incision under enflurane anesthesia 24 hr before the firstdose of azoxymethane. All rats received azoxymethane at 30mg/kg. Treatment rats received glycine-extended gastrin17 at 2.5nmol/kg/hr for 4 weeks. Plasma concentrations of glycine-ex-tended gastrin17 were measured weekly in all rats. At the end of 4weeks, animals were sacrificed and colons removed for examina-tion. The number and nature of aberrant crypt foci were docu-mented and compared between the control and treatment groups.

Azoxymethane administrationAzoxymethane (5 mg/ml in PBS) was injected subcutaneously

in 2 15 mg/kg doses, 1 week apart, with rats held in restrainers tominimize needle stick injuries.

Preparation of specimens for measurement of aberrant cryptfoci.

Colons were opened longitudinally, rinsed in PBS and pinnedflat. After fixation in 10% buffered formalin solution for 16–24 hr,each colon was cut into 5 sections (caecum, proximal colon, midcolon, distal colon and rectum). The junction of proximal and midcolon was taken as the point at which the heavily ridged mucosa of

308 ALY ET AL.

the proximal colon changed to the flat mid colonic mucosa. Themid colon/distal colon junction was taken as the point of transitionto the more vertically oriented folds of the distal colon and thebeginning of the rectum was marked by the lymphoid follicle onthe anti-mesenteric border of the rectum. The lengths of the fixedcolon and of each section were recorded. Specimens were coded toprevent observer bias before reading and stored in 50% ethanol inPBS until examination.

Measurement of aberrant crypt fociSections of colonic mucosa were stained in 0.1% methylene

blue solution for 3–6 min and examined under an Olympus BH-2light microscope at310 magnification. Crypts were consideredaberrant if they were at least twice the size of surrounding crypts.The total number of aberrant crypt foci and the number of cryptsper focus were recorded and foci grouped accordingly.

Measurement of glycine-extended gastrin17

Glycine-extended gastrin17 concentrations in the blood of therats were measured by radioimmunoassay.17 For experiments of 7days duration blood (1 mlvia tail vein) was sampled on Day 3, 5and 7. For experiments of 28 days duration, blood was sampledweekly.

StatisticsSignificance of difference was measured using the Student’s

t-test for normally distributed data and the Mann-WhitneyU-testfor non parametric data. Unless otherwise stated, data is expressedas the mean6 SEM.

RESULTS

Study 1: intact and defunctioned colon studyEffect of systemic glycine-extended gastrin17 on intact colon.A

significant increase in colonic mucosal proliferation as assessed bymetaphase index was seen in the caecum and distal colon after 1week of systemic treatment with glycine-extended gastrin17 at 10nmol/kg/hr (Table I). There was no significant change in meta-phase index in the rectum. Measurement of crypt height revealedno significant change in caecum, descending colon and rectum(103%, 105% and 102% of control, respectively).

The mean serum concentration of glycine-extended gastrin17 inthe control group was 576 6.1 pM (range 33–61 pM) and in thetreated group 7306 139 pM (range 442–977 pM) during thecourse of the experiment.

Effect of systemic glycine-extended gastrin17 on defunctionedcolon.Defunctioning the rectum reliably induced hypoplasia of therectal mucosa. The metaphase index was reduced to 40% of

control (Fig.1), whereas crypt height was reduced to 80% ofcontrol after defunctioning for either 1 or 4 weeks. Treatment withglycine-extended gastrin17 for either 1 or 4 weeks produced asignificant increase in both the metaphase index (Table II, Fig. 1)and crypt height (Table II) of the defunctioned rectal mucosa. Theincrease was more pronounced in the rats treated for 4 weeks, butthe metaphase index had still not returned completely to the valueobserved in the unoperated animals.

Plasma glycine-extended gastrin17 concentrations in the grouptreated for 1 week at 10 nmol/kg/hr averaged 6826 202 pM(203–1,372 pM) whereas in the group treated for 4 weeks at 2.5nmol/kg/hr the mean plasma glycine-extended gastrin17 concen-tration was 6556 79 pM (range 316–968 pM). Controls had amean glycine-extended gastrin17 concentration of 376 2 pM.

Effect of luminal glycine-extended gastrin17 on defunctionedcolon. Glycine-extended gastrin17 administered at a rate of 10nmol/kg/hr for 1 week directly into the lumen of the defunctionedrectum had no discernible effect on mucosal proliferative rate asmeasured by metaphase index or crypt height (data not shown).

Study 2: aberrant crypt foci studyEffect of glycine-extended gastrin17 upon induction of ACF by

azoxymethane.The total number of aberrant crypt foci per colon inthe rats treated with glycine-extended gastrin17 (150.6) was in-creased by 48% compared to the value in controls (101.6) (p 50.01). The mean number of aberrant crypt foci per cm of colon(crypt density) in the rats treated with glycine-extended gastrin17(8.06 0.8 crypts/cm) was also significantly increased compared tothe value in controls (5.16 0.4 crypts/cm) (p 5 0.006, Fig. 2).This increase in crypt density of 56% was spread uniformly acrossthe mid colon, distal colon and rectum, with no significant increasein the caecum or proximal colon.

The mean serum concentration of glycine-extended gastrin17 forWeeks 1–4 in the control rats was 436 4 pM (range 23–64 pM)

TABLE I – EFFECT OF GLYCINE-EXTENDED GASTRIN17 ON COLONICMUCOSAL PROLIFERATION IN INTACT RATS1

SectionMean metaphase index2

Increase(%) p-ValueControl

(n 5 5)Treated(n 5 5)

Caecum 4.76 0.2 5.86 0.3 23 0.02Descending colon 2.96 0.2 3.76 0.2 27 ,0.001Rectum 2.46 0.1 2.76 0.2 12 0.08Overall 3.36 0.2 4.16 0.2 22 0.081Rats were infused with glycine-extended gastrin17 (250mM in PBS

containing 0.1% BSA) or with PBS containing 0.1% BSA alone viaAlzet miniosmotic pumps. After 1 week animals were injected intra-peritoneally with vincristine and sacrificed 3 hr later. Sections from thecolorectal mucosa were prepared as described in the Material andMethods section, and examined microscopically in order to determinethe metaphase index (mean number of metaphase-arrested nuclei percrypt). Data are given as means6 SEM.p-values were determined byStudent’st-test. Infusion of glycine-extended gastrin17 significantlyincreased metaphase index in the caecum and descending colon, butnot in the rectum.–2Mean metaphase index, mean number of accumu-lated arrested metaphases per crypt 3 hr after vincristine injection.

FIGURE 1 – Effect of glycine-extended gastrin17 treatment on meta-phase index in defunctioned rectal mucosa. Rats that had been sub-jected to a colostomy to induce hypoplasia of the defunctioned rectalmucosa,13 were infused with glycine-extended gastrin17 (Ggly, 250mM in PBS containing 0.1% BSA) or with PBS alone (Defunct.) viaAlzet miniosmotic pumps. After 1 or 4 weeks animals were injectedintraperitoneally with vincristine and sacrificed 3 hr later. Sectionsfrom the colorectal mucosa were prepared as described in the Materialand Methods section and examined microscopically to determine themetaphase index (mean number of metaphase-arrested nuclei percrypt). Data are given as means and bars represent the standard errorof the mean. The significance of differences between treated colonsand colons defunctioned for the same period was assessed by Mann-Whitney U-test (*p , 0.01; **p , 0.001).

309GASTRIN17GLY STIMULATES COLONIC MUCOSAL PROLIFERATION

and in the treatment group was 3496 36 pM (range 260 pM–1.6nM). The increase in serum concentrations of glycine-extendedgastrin17 did not lead to any significant increase in the meanweight gain of the rats in each group during the experiment(control group 43.5 g, treatment group 42.1 g,p 5 0.38). Similarly,there was no significant difference in the mean length of the fixedcolons (control group 19.9 cm, treatment group 19.1 cm,p 50.11), or in the lengths of the various segments of colon.

Although the total number of aberrant crypt foci increased, theirdistribution in the different segments of the colon and rectum wasnot affected by the increase in serum concentration of glycine-extended gastrin17 (Table III). In both groups, aberrant crypt fociwere rare in the caecum and proximal colon, with the caecumcontributing only 2% and the proximal colon 3–5% to overallnumbers of aberrant crypt foci. The aberrant crypt foci in theproximal colon tended to occur at the distal end of that segment

and there was an abrupt increase in the number of aberrant cryptfoci once the mid colon was reached. The mid colon contributedapproximately 50% of the total number of aberrant crypt foci, thedistal colon 23% and the rectum 18–20%.

The number of aberrant crypts per focus differed between con-trol and treated rats (Table IV). Among control rats single cryptfoci predominated, contributing 60% of all crypts and the fre-quency of foci with multiple crypts decreased progressively withincreasing multiplicity (Table IV). The rats treated with glycine-extended gastrin17 had increased numbers of all types of foci, butthe percentage increase above control was more marked in the fociwith the greatest number of crypts (4 or more crypts per focus).Nevertheless the overall proportions of foci containing the differ-ent numbers of crypts remained similar between the 2 groupsbecause the more aberrant foci were still relatively few in number.

DISCUSSION

Gastrin17 amide is an important trophic factor for gastric epi-thelium and is known to stimulate proliferation of the enterochro-maffin-like cells of the stomach and proximal small intestine.Gastrin17 amide, however, does not seem to have a significant rolein proliferation of cells in other regions of the gastrointestinal tractincluding the colon.18 Until recently, non-amidated gastrin pep-tides were thought to have no physiological function, but a numberof in vitro studies have suggested that glycine-extended gastrin17may have growth effects upon cell lines of gastrointestinal origin.Sevaet al.2 reported that glycine-extended gastrin17 stimulatedproliferation of the pancreatic carcinoma cell line AR4-2J at apotency equal to that of gastrin amide whereas Hollandeet al.4reported a growth effect of glycine-extended gastrin17 on a mousecolon-derived cell line, YAMC. Both these reports suggested thereceptor responsible for mediating the growth effects was distinctfrom the classical gastrin/CCK-B and CCK-A receptors. Similarstudies have reported growth effects of glycine-extended gastrin17on the gastric carcinoma cell lines AGS and SIIA,19 and on theembryonic kidney line HEK.20 Stepanet al.5 demonstrated agrowth effect of glycine-extended gastrin17 in the human coloncarcinoma cell lines LoVo and HT29, again via a novel and yet tobe characterized, receptor. Xenografts in nude mice of the humancolon carcinoma cell line DLD-1 proliferated in response to gly-cine-extended gastrin17 and were inhibited by treatment withJMV1155, a gastrin receptor antagonist.6

The first reports that non-amidated forms of gastrin (progastrinand glycine-extended gastrin17) exert a growth effect directly uponthe colonic mucosain vivo were provided by Wanget al.7,8 Anincrease in BrdU labeling index of 86% and in crypt height of 43%was observed in the colonic mucosa of transgenic mice overex-pressing progastrin, with similar increases in mice overexpressingglycine-extended gastrin17. In relative terms, gastrin amide wasnot elevated in these mice because the animals failed to process thehuman gastrin precursors produced by the relevant transgene. It

TABLE III – PROPORTION OF ABERRANT CRYPT FOCI BY SITE

SiteProportion of aberrant crypt foci

(%)

Control Treated

Caecum 2.8 2.0Proximal colon 5.5 3.8Mid colon 50.3 52.5Distal colon 23.5 22.7Rectum 17.9 19.1Total 100 100

1Experimental procedures are described in the legend to Figure 2.The number of Type 1 ACF (Single Crypts) in the indicated regions ofthe large intestine after infusion with glycine-extended gastrin17 wasdetermined as described in the Material and Methods section. Thedifference in proportions between control and treated groups was notsignificant (p 5 0.96).

TABLE II – PROLIFERATIVE EFFECT OF GLYCINE-EXTENDED GASTRIN17ON DEFUNCTIONED RECTAL MUCOSA1

Mean metaphase index2

Increase(%) p-valueControl

(n 5 5)Treated(n 5 5)

After 1 weekMetaphase index 1.06 0.1 1.46 0.1 40 0.01Crypt height

(pixels)302.86 7.2 336.56 8.3 11 0.001

After 4 weeksMetaphase index 0.966 0.1 1.96 0.1 93 ,0.001Crypt height

(pixels)292.66 8.4 347.66 10.9 19 ,0.001

1Experimental procedures are described in the legend to Figure 1.Infusion of glycine-extended gastrin17 significantly increased meta-phase index in the defunctioned rectum.p-values were determined byMann-WhitneyU-test.–2Mean metaphase index, mean number of ac-cumulated arrested metaphases per crypt 3 hr after vincristine injec-tion.

FIGURE 2 – Effect of glycine-extended gastrin17 plus azoxymethanetreatment on the number of aberrant crypt foci. Rats were infused withglycine-extended gastrin17 (Ggly, 250 mM in PBS containing 0.1%BSA) or with PBS alone for 4 weeks, together with 2 doses ofazoxymethane (5 mg/ml in PBS) on Day 1 and Day 8. The animalswere sacrificed and the lengths of the indicated sections of their colonsmeasured. The colonic mucosa was then examined microscopically forthe presence of aberrant crypt foci. Data are given as means and bars5SEM. Significance was assessed by Student’st-test (*p , 0.02; **p ,0.01).

310 ALY ET AL.

must be noted, however, that although these findings are important,the non-inducible nature of the transgene meant that the progas-trin-derived peptides were able to act upon both developing andimmature colonic epithelium. Because the developing rodent colonexpresses higher levels of progastrin-derived peptides than theadult colon,21 the developing colon may be relatively more sensi-tive to the growth effects of these peptides than the mature colon.Furthermore, a long duration of exposure occurred because ani-mals were sacrificed at 1 year (progastrin study) and at 2 or 3months (glycine-extended gastrin17 study) of age. In contrast, thestudy reported here, which examined the effect of glycine-ex-tended gastrin17 upon the colon in mature animalsin vivo aftereither a short (1 week) or moderate (4 week) duration of exposure,is the first such study to demonstrate a proliferative effect underthese conditions.

In the first part of the study, the effect of glycine-extendedgastrin17 upon the intact colon was examined. After 1 week, themetaphase index of the colonic mucosa in the treatment groupincreased by 22% above controls. This effect was seen throughoutthe length of the colon, though it appeared least obvious in therectum (12%) where it did not reach significance. Crypt height didnot appreciably increase over this time (103–106% of control). Itis interesting to note that although the cecal mucosa had thehighest baseline metaphase index (4.7 metaphases/crypt/3 hr) itstill seemed to respond well to the influence of glycine-extendedgastrin17, suggesting either that the cecal mucosa may be moresensitive to the effect of glycine-extended gastrin17, or that itsimply has greater proliferative reserve. A similar result was foundin transgenic mice overexpressing progastrin,7 with progastrinaffecting the proximal colon more than the distal colon. No seg-mental analysis was reported for transgenic mice overexpressingglycine-extended gastrin17,8 where crypts were sampled through-out the colon to obtain a combined score.

In further experiments the rectum was defunctioned to induce astate of low baseline proliferation to enhance any proliferativeeffect of glycine-extended gastrin17 in this region. Metaphaseindex in the defunctioned rectum after 1 week exposure to glycine-extended gastrin17 increased to 40% above controls, confirming anenhanced effect from a low proliferative state. When exposed toglycine-extended gastrin17 for 4 weeks, the effect was evengreater, increasing to 90% above control. Crypt height also in-creased by 11% and 19% after exposure to glycine-extendedgastrin17 for 1 and 4 weeks, respectively. A similar effect on thehypoplastic colonic mucosa of gastrin-deficient mice has beenreported after infusion of glycine-extended gastrin17 for 2 weeks,though it is possible that the effect in this case is related toupregulation of gastrin receptors in response to the decreasedserum gastrin concentration.8

Glycine-extended gastrin17 had no proliferative effect whenadministered directly into the lumen of the defunctioned rectum.The absence of any effect after topical exposure suggests that anyreceptors (as yet uncharacterized) for glycine-extended gastrin17 incolonic epithelium are not at the luminal surface. A similar dis-tinction between luminal and systemic effects has been reported

previously for epidermal growth factor, which stimulated colonicproliferation when administered intravenously, but had no effectwhen administered via the lumen.13 Clearly, the fecal stream isimportant in maintaining a healthy colonic epithelium (hence thehypoplasia induced with defunctioning), but our results suggestthat progastrin-derived peptides secreted into the lumen of thegastrointestinal tract are unlikely to play any role in this process.In contrast the observation that gastrin-deficient mice have hypo-plastic mucosa suggests that systemic gastrins may be important inmaintaining the health of colonic epithelium.8

Growth factors are thought to act as promoters of carcinogenesisby increasing DNA turnover and thus the risk of mutagenesis.Once a neoplastic clonal cell line has been established, growthfactors, acting in an autocrine fashion, may aid the growth of acarcinoma. The induction of aberrant crypt foci22 in rodent colonafter treatment with the procarcinogen azoxymethane23 is widelyaccepted as a valid model of the early stages of colorectal carci-nogenesis,14,24,25 and has been used to test the role of growthfactors as potential promoters of carcinogenesis.

The effects of amidated gastrin on the development of colorectalcarcinoma in this and related animal models have been controver-sial. Rats with azoxymethane-induced colorectal carcinomas arehypergastrinemic,26 and the gastrin/CCK-B receptor antagonistCR2945 reduces the incidence of colorectal carcinoma in micetreated with 1,2-dimethylhydrazine.27 The following 3 observa-tions, however, argue against a role for amidated gastrin on thedevelopment of colorectal carcinoma. First, transgenic mice over-expressing amidated gastrin have no more aberrant crypt foci aftertreatment with azoxymethane than wildtype controls.9 Second,amidated gastrin did not influence cell division in explants ofazoxymethane-treated colonic mucosa.28 Third, elevation of serumconcentrations of amidated gastrin by treatment with proton pumpblockers did not enhance the growth of chemically induced coloncancers in rats.29

In contrastnon-amidatedprogastrin-derived peptides appear toaccelerate the development of colorectal carcinomas. After treat-ment with azoxymethane transgenic mice overexpressing progas-trin have 2–3-fold more aberrant crypt foci,9 and ultimately de-velop greater numbers of larger adenomas and adenocarcinomas ofthe colon,30 than wildtype controls. Similarly Kohet al. havereported in abstract form that crossingAPCminmice with miceoverexpressing glycine-extended gastrin17 increases the number ofaberrant crypt foci in the progeny and that crossingAPCminmicewith gastrin knockout mice increases the survival time of theprogeny.10 The survival time ofAPCmin mice may also be in-creased by reduction of the levels of progastrin-derived peptidesby immunization with gastrimmune.11

In our experiments treatment with glycine-extended gastrin17for only 4 weeks significantly increased the number of aberrantcrypt foci induced per cm of colon after azoxymethane exposure.This increase was observed in mid-colon, distal colon and rectum,but not in caecum or proximal colon (Fig. 2). The percentageincrease of 56% was in good agreement with the value of 60%

TABLE IV – INCREASED OCCURRENCE OF ABERRANT CRYPT FOCI AFTER TREATMENT WITH GLYCINE-EXTENDED GASTRIN171

Crypts per aberrant focusAberrant crypt foci density

(aberrant crypt foci/cm) Increase(%) Significance2

Proportion of total (%)3

Control Treated Control Treated

1 3.06 0.2 4.56 0.5 47 0.011 60 562 1.36 0.2 2.26 0.3 77 0.009 25 283 0.66 0.1 0.96 0.1 49 0.038 11.7 11.64 0.156 0.02 0.306 0.05 98 0.018 3 4

.4 0.0176 0.01 0.0346 0.02 99 NS 0.3 0.4Overall 5.16 0.4 8.06 0.8 56 0.006 100 1001Experimental procedures are described in the legend to Figure 2, except that the colonic mucosa was examined microscopically not only for

the presence of aberrant crypt foci, but also for the number of crypts per aberrant crypt focus. Data are given as means6 the SEM. NS, notsignificant.–2Significance was assessed by Student’st-test.–3The difference in proportions between treated and control groups was not significant(p 5 0.97).

311GASTRIN17GLY STIMULATES COLONIC MUCOSAL PROLIFERATION

reported by Singhet al.9 in transgenic mice overexpressing pro-gastrin. The absence of any detectable increase in the density offoci in the caecum or proximal colon may be due to the resistanceof these areas to the effect of azoxymethane. Indeed, some workershave reported that the caecum was entirely free of aberrant cryptfoci after azoxymethane treatment. Our results suggest that ani-mals treated with glycine-extended gastrin17 for times as short as1 month have an increased risk of malignant transformation be-cause the greater the number of aberrant crypt foci present, themore likely tumor development is to occur.31,32

Although the relative proportions of foci containing single,double, triple and quadruple crypts were unchanged, the percent-age increase seen in the more aberrant foci (4 or more crypts perfocus) was double that seen in the single crypt foci (47% vs. 98%increase). Previous work has demonstrated a correlation betweencrypt multiplicity and both degree of dysplasia31 and tumorigenic-ity32 in this model. Although our study lacks the power to discrim-inate whether or not this tendency toward greater proportionalincrease in the very aberrant subset of foci is real, a similarincrease in crypt multiplicity was observed in transgenic miceoverexpressing progastrin after azoxymethane treatment.9

The pattern of results obtained in our study suggests that gly-cine-extended gastrin17 acts as a proliferative factor that increasesthe amount of DNA available for mutagenesis and thus results ina greater number of aberrant crypt foci. These observations areconsistent with the findings in the defunctioned rectum model andwith previous data from transgenic mice overexpressing glycine-extended gastrin17.7 The colonic mucosa of the transgenic micewas hyperplastic, but no tumors were detected in the colons in1-year-old mice in the absence of carcinogen administration. Re-ports of altered numbers of aberrant crypt foci in response togrowth factors, with the exception of progastrin-derived pep-tides,9–11 are rare. Insulin injections have been shown to increasecrypt multiplicity without affecting total numbers of aberrant cryptfoci in rats treated with azoxymethane,33 and conversely controlledrelease of transforming growth factorb-1 inhibits formation ofaberrant crypt foci in rats treated with dimethylhydrazine.34 Anumber of reports studying the potential chemopreventative effectof non-steroidal anti-inflammatory agents have described a reduc-tion in aberrant crypt foci and subsequent tumour formation.35,36

Our study clearly demonstrates that glycine-extended gastrin17increases the number of aberrant crypt foci formed in the colorec-tal mucosa of rats after treatment with azoxymethane in a patternconsistent with its action as a proliferative agent in the colonicmucosa. Glycine-extended gastrin17 could thus potentially act as apromoter of carcinogenesis, but definitive evidence for this pro-posal would require a long term study to establish that tumorsoccur either earlier, or more abundantly, or both, in rats treatedwith glycine-extended gastrin17 compared to controls. Previouswork on the ACF model suggests that in general increases in ACFnumber are associated with a subsequent increase in tumour fre-

quency.32,35,36In the case of transgenic mice overexpressing pro-gastrin the increase in number of aberrant crypt foci after treatmentwith azoxymethane compared to wildtype controls9 is ultimatelyfollowed by the development of greater numbers of larger adeno-mas and adenocarcinomas of the colon.30

The implications of these findings for human disease are still tobe elucidated. The advent of acid suppression therapy and im-proved understanding of the pathology associated withHelicobac-ter pylori infection, have raised concern about the possible effectsof hypergastrinemia on the development of colorectal carcinoma.Studies of patients with classical causes of hypergastrinemia suchas Zollinger-Ellison syndrome or pernicious anemia have sug-gested that, although colonic mucosal proliferative rates are in-creased, tumors do not occur more commonly.37–40The possibilityshould also be considered that immature gastrins rather than gas-trin amide may be responsible for the hyperproliferative colonicmucosa in such hypergastrinemic states.41,42 Although the avail-able data is limited, serum concentrations of progastrin and gly-cine-extended intermediates are elevated in patients withZollinger-Ellison syndrome, in greater proportion than gastrinamide.41–43. None the less, the concentrations of glycine-extendedgastrin17 achieved in the present study were 6 times normal andsuch high levels have not been documented in patients with hy-pergastrinemia. It is in principle possible that local production andsecretion of glycine-extended gastrin17 is increased in patientspredisposed to colorectal cancer. Increased proportions of non-amidated forms of gastrin have been observed in colonic adenomasand carcinomas,44–49 and there is recent evidence to suggest thatgastrin gene activation occurs early in the adenoma-carcinomasequence.44 Thus the production of progastrin-derived peptidessuch as glycine-extended gastrin17 by an established carcinoma orindeed in the early stages of carcinoma development, perhaps evenin the premalignant stage of a benign adenoma, may be an impor-tant autocrine method of enhancing growth or progression tocancer. Our results support the possibility of this notion by dem-onstrating that short term administration of glycine-extended gas-trin17 to mature rats stimulates growth of the colonic mucosa andaccelerates the production of premalignant lesions in response toazoxymethane in the rodent ACF model. We conclude that gly-cine-extended gastrin17 has the potential to act as a promoter ofcolorectal carcinogenesis.

ACKNOWLEDGEMENTS

We gratefully thank Ms. J. Baker, Ms. L. Hogan and Ms. T.Volombello for radioimmunoassays and assistance with the animalsurgery. This work was supported in part by the Austin HospitalMedical Research Foundation, by the Royal Australian College ofSurgeons (Reg Worcester Fellowship to A.A.) and by grants114123 (to A.S.) and 980625 (to G.B.) from the National Healthand Medical Research Council of Australia.

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