[CANCER RESEARCH 61, 625–631, January 15, 2001] Hypergastrinemia Promotes Adenoma Progression in the APCMin؊/؉ Mouse Model of Familial Adenomatous Polyposis1 Sue A. Watson2 and Andrew M. Smith Academic Unit of Cancer Studies, Department of Surgery, University of Nottingham, Nottingham NG7 2UH, United Kingdom ABSTRACT nary findings of Smith and Watson (18) that human adenomas of all sizes and grades express an isoform of the cholecystokinin B/gastrin Serum hypergastrinemia promotes the growth of colorectal adenocar- receptor. cinoma. Some colorectal adenomas express cholecystokinin B/gastrin re- Thus, the aim of the present study was to evaluate the effect of ceptor mRNA, and thus hypergastrinemia may increase progression through the adenoma-carcinoma sequence. This was investigated in the serum hypergastrinemia induced by omeprazole, a proton pump in- multiple intestinal neoplasia APCMin؊/؉ mouse. Serum gastrin levels in hibitor that increases serum gastrin levels 2–4-fold (11, 12, 19), on APCMin؊/؉ mice were elevated 5–6-fold by oral administration of ome- colonic neoplastic progression in the multiple intestinal neoplasia MinϪ/ϩ prazole (75 mg/kg). Terminal tumor burden was monitored by onset of APC mouse model of APC. anemia. A labeling index was generated by immunohistochemical detec- tion of bromodeoxyuridine incorporation. Serum gastrin was neutralized MATERIALS AND METHODS by antigastrin antibodies raised in situ by use of a gastrin immunogen, Gastrimmune. Hypergastrinemia resulted in reduced survival of the APC- APCMin؊/؉ Mice. APCϪ/ϩ mice were bred within the Cancer Studies Unit ؊ ؉ Min / mice from a median survival of 13 weeks in the controls to 10 weeks at the University of Nottingham under Home Office Project License No. PPL following omeprazole treatment (P < 0.00001, log-rank test). The labeling 4011454. Heterozygous male APCMinϪ/ϩ mice were bred with wild-type indices of adenomas from the small and large intestines of omeprazole- C57/Bl6 female mice. The offspring were characterized for the Min genotype treated mice were increased 35 and 29%, respectively (P < 0.05 and P < by PCR as described previously (20). 0.025, respectively). Gastrimmune immunization reversed both the sur- Entry of Mice into Therapeutic Studies. After results from the PCR vival effect and the increased proliferation resulting from serum hyper- analysis were obtained, APCMinϪ/ϩ-positive mice were randomized into each gastrinemia. Hypergastrinemia may promote the progression of existing experimental group on an ongoing basis because of the limited number of mice premalignant colonic lesions by increasing proliferation. Clinical investi- positive for the Min mutation that are generated from established breeding gations should determine whether this occurs in the human scenario, colonies. Mice were randomized into groups on a litter basis with unequal considering the widespread use of proton pump inhibitors. numbers being a result of the distribution of Min positive and negative mice. Experiment 1: Effect of Omeprazole-induced Hypergastrinemia on Adenoma Number and Size and Animal Survival. An initial study was INTRODUCTION performed to determine the effect of hypergastrinemia on tumor burden and survival of APCMinϪ/ϩ mice. APCMinϪ/ϩ mice 4 weeks of age were recruited Gastrin has been described as a growth factor for colorectal malig- to the following groups: group 1, oral vehicle for omeprazole [PBS (pH 7.2)]; nancy for well over a decade, and it is known to mediate proliferation group 2, omeprazole (75 mg/kg in a single oral dose; n ϭ 10). via both endocrine and autocrine/paracrine mechanisms (1–3). Tumor size was measured by image analysis by use of Leica Qwinn image Elevated serum gastrin levels can be a feature of a number of processing and analysis system run on a Leica Q5001W PC. clinical conditions, including ZES3 (4–7) and PA (8), but possibly Experiment 2: Effect of Omeprazole-induced Hypergastrinemia on more importantly as a result of infection with Helicobacter pylori (9, Survival with or without Immunization with the Gastrin Immunogen, Gastrimmune. APCMinϪ/ϩ mice 4 weeks of age were recruited to the fol- 10) and after the administration of proton pump inhibitors (11, 12). lowing groups: group 1, oral vehicle for omeprazole ϩ control immunogen In humans, increased proliferation of colonic crypts has been con- (n ϭ 20); group 2, omeprazole (75 mg/kg in a single oral dose) ϩ control firmed in patients with PA and ZES (13). However, the majority of immunogen (n ϭ 20); group 3, omeprazole ϩ Gastrimmune (n ϭ 16). epidemiological studies performed to date in patients with sustained Gastrimmune is a gastrin immunogen composed of the NH2-terminal nine hypergastrinemia have failed to show an enhanced risk of colorectal amino acids of gastrin-17 linked via a peptide spacer to diphtheria toxoid. cancer. In patients with PA, although conflicting studies have been Treatment with Gastrimmune generates antibodies that neutralize the amidated reported, the majority show that colorectal cancer incidence is not and glycine-extended forms of gastrin-17 (Aphton Corporation, CA; Ref. 21). increased (14, 15). This has also been shown in patients with ZES A rat-specific form of the immunogen was formulated at 2 mg/ml and injected (16). This failure may be attributed to lack of control of confounding s.c. into mice in 100-␮l volumes at week 4, and at three-week intervals factors, such as H. pylori in the control groups. A recent study, in thereafter. The rat sequence used within the immunogen differs only by one amino acid from the mouse sequence, and antibodies raised by rat Gastrim- which H. pylori infection was controlled for, showed that hypergas- mune previously have been shown to neutralize mouse gastrin (data not trinemia is associated with a 3.3-fold increase in the relative risk of shown). Control mice receiving immunogen constituents only were given the developing colorectal cancer (17). It is conceivable, therefore, that formulation without the active peptide. hypergastrinemia may promote progression through the adenoma- Experiment 3: Effect of Preimmunization with Gastrimmune Immuno- carcinoma sequence. This hypothesis is strengthened by the prelimi- gen on the Survival Effect Observed with Omeprazole Treatment. APC- MinϪ/ϩ mice 4 weeks of age were recruited to the following groups: group 1, ϩ ϭ Received 3/27/00; accepted 11/6/00. vehicle control immunogen (n 22 mice); group 2, omeprazole (75 The costs of publication of this article were defrayed in part by the payment of page mg/kg) ϩ control immunogen (n ϭ 30 mice); group 3, omeprazole ϩ Gastrim- charges. This article must therefore be hereby marked advertisement in accordance with mune (n ϭ 30 mice); group 4, vehicle ϩ Gastrimmune (n ϭ 18 mice). 18 U.S.C. Section 1734 solely to indicate this fact. Gastrimmune was injected 2 weeks prior to omeprazole. (The latter was 1 This work was supported by the Aphton Corporation, Woodland, CA. 2 To whom requests for reprints should be addressed, at Cancer Studies Unit, D Floor, administered from week 6 when antibodies against Gastrimmune were known West Block, QMC University Hospital, Nottingham NG7 2UH, United Kingdom. Phone: to be detectable.) Fax: 44-115-9709902; E-mail: [email protected]. Termination of APCMin؊/؉ Mouse Experiments. At the end-stage of ;44-115-9709248 3 The abbreviations used are: ZES, Zollinger Ellison syndrome; PA, pernicious ane- their disease, APCMinϪ/ϩ mice bleed from their multiple polyps, giving rise to mia; APC, adenomatous polyposis coli; Min, multiple intestinal neoplasia; GI, gastroin- testinal; BrdUrd, bromodeoxyuridine; CCKBR, cholecystokinin B receptor; GAPDH, anemia, which was initially confirmed by hematocrit measurements [median glyceraldehyde-3-phosphate dehydrogenase; LI, labeling index. hematocrit, 0.136 (range, 0.058–0.153) compared with 0.385 (range, 0.381– 625 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2001 American Association for Cancer Research. HYPERGASTRINEMIA AND ADENOMA PROGRESSION 0.391] in wild-type C57/Bl6 mice. Twelve days after the onset of anemia, mice triphosphates, 20 ␮M each primer, and 1 unit of AmpliTaq Gold (PE Applied were terminally anesthetized by a 300-␮l injection containing 0.315 ng/ml Biosystems). The PCR cycle consisted of a 95°C activation stage for 10 min Hypnorm and 5 ng/ml Hypnovel (Jannsen-Wickham, Bucks, United Kingdom, followed by 40 cycles of 94°C for 30 s, 60°C for 60 s, and 72°C for 30 s. After and Roche-Lewis, East Sussex, United Kingdom) by an experienced, inde- a final extension stage of 72°C for 10 min, the PCR products were separated pendent observer who was blinded to the treatment groups. Anemia previously on a 2% (w/v) agarose gel and stained with ethidium bromide. The mouse has been validated as a consistent end point for APCMinϪ/ϩ mouse termination colon cancer cell line MC26 was used as a positive control. A negative control (20). After termination, the GI tracts of the APCMinϪ/ϩ mice were removed, was also included that was not reverse transcribed and confirmed the absence and the numbers of visible tumor nodules in both the small and large bowel of genomic DNA. were counted with the aid of a dissecting microscope. The mucosa was fixed Real-Time PCR to Quantify CCKBR Expression. RNA was extracted in formal calcium (40% formaldehyde, 10% CaCl2 in distilled water) prior to from tissue and reverse transcribed from random hexamer primers (Pharmacia) embedding in paraffin wax for histological analysis. using Supercript reverse transcriptase (Life Technologies, United Kingdom). The United Kingdom Coordinating Committee for Cancer Research Guide- Real-time PCR was performed using the 5700 Sequence Detection System (PE lines were adhered to throughout all animal experimentation. Applied Biosystems, Warrington, United Kingdom). Each PCR was performed Measurement of the Proliferation Index of the GI Mucosa. One h before according to manufacturer’s instructions using 1 ␮l of sample cDNA in a 25-␮l termination, mice received i.p.