1 Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, Texas 77555-1043; 2 Department of Digestive Diseases and Gastrointestinal Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030; 3 Physiology Department, University of Liverpool, Liverpool L69 3BX, United Kingdom; and 4 Gastrointestinal Unit, Massachusetts General Hospital, Boston, Massachusetts 02114
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ABSTRACT |
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Recent studies show that nonamidated gastrins (Gly-gastrin and progastrin) stimulate colonic proliferation. However, the role of nonamidated vs. amidated gastrins in colon carcinogenesis has not been defined. We measured intermediate markers of carcinogenesis in transgenic mice overexpressing either progastrin (hGAS) or amidated gastrin (INS-GAS) in response to azoxymethane (AOM). The hGAS mice showed significantly higher numbers of aberrant crypt foci (140-200% increase) compared with that in wild-type (WT) and INS-GAS mice (P < 0.05) after AOM treatment. The bromodeoxyuridine-labeling index of colonic crypts also was significantly elevated in hGAS mice vs. that in WT and INS-GAS mice. The results therefore provide evidence for a mitogenic and cocarcinogenic role of nonamidated gastrins (progastrin), which is apparently not shared by the amidated gastrins. Although nonamidated gastrins are now believed to mediate mitogenic effects via novel receptors, amidated gastrins mediate biological effects via different receptor subtypes, which may explain the difference in the cocarcinogenic potential of nonamidated vs. amidated gastrins. In conclusion, our results provide strong support for a cocarcinogenic role for nonamidated gastrins in colon carcinogenesis.
transgenic mice
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INTRODUCTION |
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COLORECTAL CANCER REPRESENTS one of the leading causes of cancer-related mortality in the United States (1). Studies by Vogelstein and associates (see Ref. 13) have defined the sequence of many of the genetic events involved in the multistep progression from normal colonic mucosa to adenomatous polyp to malignant tumor in humans. In addition, a number of familial syndromes have been defined in which colon cancer develops at a high incidence because of germline mutations in known tumor suppressor genes (12). However, the majority of colorectal cancers develop in patients with as yet no clearly identified genetic predisposition, and the factors responsible in these individuals have remained poorly defined. Hyperproliferation of the colonic epithelium is recognized as the most common alteration in high-risk conditions for colorectal cancer development and is believed to be the first step in a sequence of events leading to adenocarcinoma formation, growth, and malignant transformation (16). Several dietary and hormonal factors have been identified as possibly important in modulating the proliferative and tumorigenic potential in the colon (3, 31, 40). Recently, incompletely processed gastrins have emerged as a possible regulator of colonic growth and proliferation (6, 35, 38, 41).
Studies published in the early 1990s, from our laboratory and those of others, have indicated that glycine-extended gastrin exerts significant mitogenic effects in vitro on several cell types, including human and mouse colon cancer cells (6, 10, 35, 38, 41). Many primary colon cancers have been shown to express Gly-extended gastrin, but recent studies indicate that progastrin may represent the predominant form of gastrin produced by most human colonic tumors (reviewed in Ref. 2). Recent studies from our laboratory (39) and that of others (8) indicate a critical role of autocrine gastrins (largely nonamidated gastrins) in the proliferative potential of human colon cancer (39) and mouse colonic (8) cells. Transgenic mice with increased plasma progastrin levels (hGAS) have been shown to exhibit markedly increased colonic proliferation, consistent with a mitogenic role for progastrin in the colon (49).
Cocarcinogens are agents that do not initiate tumorigenesis but simulate cell proliferation and expand the pool of transformed cells. Thus gastrinlike peptides may function as cocarcinogens for transformed cells and accelerate tumorigenesis induced by other agents or genetic events. To investigate a possible cocarcinogenic role of nonamidated gastrins, we used a colon carcinogenesis model (18, 22, 51) and examined the effect of either elevated progastrin levels (hGAS transgenic mice) or elevated amidated gastrin levels (INS-GAS transgenic mice); wild-type (WT) FVB/N mice were used as controls. Azoxymethane (AOM) was used as the chemical carcinogen. Carcinogens such as AOM and dimethylhydrazine are methalating agents (15), which form various DNA adducts including O- to 6-methylquinone, resulting in G to A point mutations. This mutation can potentially activate specific oncogenes such as k-ras and inactivate tumor suppressor genes (52). AOM shows particular specificity for the colon (19), and treatment of mice with AOM has been shown to lead to development of single to multiple aggregates of atypical crypts known as aberrant crypt foci (ACFs), followed later by colonic adenocarcinomas (19, 30, 32). Mutated k-ras and APC genes are similarly detected in the ACFs and colonic adenomas of both humans (11, 26, 42) and AOM-treated mice (17, 26, 52). ACFs are therefore considered reliable intermediate markers for colon tumorigenesis and are likely precursors of colonic adenomas in human patients as well (45).
A number of previous studies have examined the effects of gastrin on carcinogen-induced tumor formation in rodents, but these earlier studies focused solely on amidated gastrins, and the results of these studies have been mixed. Some studies have shown that amidated gastrin enhances the growth of colon cancer induced by chemical carcinogens in rats (reviewed in Ref. 47), whereas other studies have shown that endogenous hypergastrinemia induced by acid-suppressing drugs does not enhance colon cancer induced by chemical carcinogens (29). However, the role of progastrin in enhancing colonic carcinogenesis has not been examined previously. The results of the present studies indicate that in contrast to amidated gastrin, elevated levels of progastrin significantly enhance the risk of developing preneoplastic lesions in the colon.
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MATERIALS AND METHODS |
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Animals. Transgenic hGAS and INS-GAS (both in an FVB/N background) and control FVB/N mice, as described previously (49), were used in this study. Transgenic and WT mice were age and sex matched, had similar body weights, and were raised in a common facility at Massachusetts General Hospital (MGH). The hGAS transgenic mice contain a human gastrin minigene, resulting in overexpression of gastrin in mouse hepatic tissue and markedly elevated (1-100 nM) serum levels of human progastrin (49). The INS-GAS mice contain a chimeric rat insulin-human gastrin transgene, resulting in overexpression of gastrin in pancreatic beta cells and elevated (~150 pM) serum levels of human amidated gastrin (49). Specific pathogen-free mice were bred in the MGH OLAR Facility. At 3-5 wk of age the mice were shipped to the Environmental Containment Facilities (ECL) at The University of Texas Medical Branch (UTMB; Galveston, TX). On arrival at UTMB, mice were quarantined and tested for pinworms twice, over a period of 2 wk. Only pathogen- and pinworm-free animals were used. Animals were housed in a microisolator in solid-bottomed polycarbonate cages, fed a commercial diet (M/R rodent sterile diet 7012; Harlan Tech Labs; Indianapolis, IN), and given autoclaved water ad libitum. The animals were maintained on a 12:12-h light-dark cycle, and all experiments were conducted during the daytime. All experiments were approved by the Subcommittee on Research and Animal Care at MGH and the Committee on Animal Care at UTMB.
Induction of ACFs in animals with chemical carcinogen AOM. Male and female mice, housed in separate cages, were placed in separate laminar flow hoods within the ECL facility. We first established optimal conditions for inducing ACFs in FVB/N mice in response to AOM. Male and female mice were injected with various doses of AOM (5-20 mg/kg body wt), and animals were monitored for the induction of an optimal number of ACFs per colon within 2 wk of the last injection. A dose of 15 mg/kg body wt and higher was determined to be toxic to >50% of the male mice but was less toxic to the female mice. More than 80% of the male and female mice (hGAS, INS-GAS, and WT) survived a dose of 12-13 mg/kg body wt per week. At lower doses all animals survived. Both male and female mice, in each group of animals (hGAS, INS-GAS, and WT), developed a significant number of ACFs per animal (>4 per colon) on treatment with 12 mg AOM/kg body wt per week ×2 within 2 wk of the last injection. At lower doses of 10 mg/kg body wt, only a few INS-GAS and WT mice developed significant number of ACFs per animal (<2 per animal). Based on these results, we chose 12 mg of AOM/kg body wt per week ×2, as an optimal dose for inducing ACFs in male and female mice of all animals groups, and animals were killed ~2 wk after the last injection. In experiments 1 and 2, an equal number of animals per group were treated with AOM or with the vehicle control (saline). None of the control, saline-injected animals, from all groups of animals, developed aberrant crypts within the time frame of the studies. Therefore, in experiments 3-5 only two to four animals per group were used as control (non-AOM treated). In all cases, AOM (Sigma Chemical; St. Louis, MO) was purchased within a week of the start of the experiment and diluted to the required dose with sterile saline within 30 min of the injections. Animals were injected with either AOM or saline intraperitoneally in 0.1 ml saline/10 g body wt. Animals were weighed once a week and at the time of death. In experiments 3 and 4, each animal was injected with 10 mg of bromodeoxyuridine (BrdU; Boehringer Mannheim; Indianapolis, IN) per kg body wt in ~0.2 ml saline intraperitoneally 1 h before being killed. Animals were killed either by cervical dislocation or decapitation (for purposes of blood collection). Within 3 min after the animals were killed the full-length colon (from the anal to cecal end) was removed and flushed with saline. The colons were slightly distended by filling them with the saline solution under mild pressure, slit open by midline incision, and fixed flat in 70% ethanol on Whatman filter papers (with the mucosal side facing up), as described previously (51). Twenty-four hours after fixation, the colonic specimens were placed in 70% ethanol in tightly sealed scintillation vials until further analysis for intermediate end points.
Analysis of colons for intermediate end points. At the time of
ACF analysis (within 1 wk of collection), colons were removed from 70%
ethanol and briefly stained with methylene blue (0.3%) for enumeration
of ACFs as described by McLellan and Bird (19). The total number of
ACFs per colon, their location, and the number of aberrant crypts per
foci were recorded using light microscopy (magnification
×10-12.5). ACFs that involved only one crypt were termed
singlets and those that involved multiple crypts per lesion were termed
doublets (2 crypts) or multiplets (triplets, quadruplets), depending on
the number of crypts involved per foci. Representative ACFs from the
hGAS mice are shown in Fig. 1A. In
all experiments, the majority of the ACFs were singlets, whereas a
significant number of doublets and triplets were also observed.
Multiplets, involving more than three crypts per foci, were rare.
Representative ACFs from each colon were marked with india ink, punched
out with a skin biopsy punch (2.5-mm diam), embedded in paraffin
blocks, and processed for tissue sectioning by published procedures
(26, 51). Simultaneously, uninvolved crypts (UCs) that were at a distance from the aberrant crypts (ACs) were similarly punched out and
processed for tissue sectioning. Control, normal crypts (NCs) from each
group of nontreated animals were also processed for tissue sectioning.
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Labeling index. Serial sections of longitudinal ACs and UCs from AOM-treated animals and NCs from control animals were prepared, and representative sections were stained with hematoxylin and eosin. The histological data obtained from the hematoxylin and eosin-stained crypts were used to confirm the aberrant-nonaberrant nature of the crypts as in the definition of McLellan and Bird (18, 19), as described recently by Olivo and Wargovich (26). In all cases, lesions or foci, identified by us as containing ACs, were determined to display typical features of dysplasia (Fig. 1B). A representative NC from untreated WT animals is shown in Fig. 1B for comparison. Sections of ACs, UCs, and NCs were immunocytochemically stained to determine the presence of BrdU-labeled cells, as a function of cells in the S phase of the cell cycle, used as a marker of the proliferative index of epithelial cells within a crypt. Cells positive for the presence of BrdU were stained using the In Situ Cell Proliferation Kit (Boehringer Mannheim) as published previously (37). Briefly, sections were deparaffinized, digested with trypsin, treated with 4 N HCl, exposed to the monoclonal antibody to BrdU conjugated to alkaline phosphatase, followed by a red chromagen, and lightly counterstained with hematoxylin. The immunostained sections were examined by bright-field microscopy, and the labeling index (LI) of individual ACs, UCs, and NCs was determined. To determine the LI, images were digitally captured, nuclei were counted, and the percentage of nuclei stained for the presence of BrdU in the crypts was calculated using Image Tool for Windows (version 1.28) image analysis software (University of Texas Health Science Center; San Antonio, TX). The angular transformation (arcsin %) was performed, and ANOVA with Newman-Keuls analysis of groups was used to determine statistical significance (P < 0.05).
Analysis of plasma for measuring molecular forms of gastrin.
Blood was collected from representative animals per group in experiments 4 and 5, in lithium heparin-coated 4-ml
tubes (Becton Dickinson; Franklin Lakes, NY), and left undisturbed at
4°C for 24 h to separate the cellular fraction from the noncellular
plasma. The tubes were centrifuged for 20 min at 4°C at 2,000 g in a Beckman J6B centrifuge, and the supernatant (plasma) was
carefully removed with Pasteur pipettes into tightly capped microfuge
tubes. The plasma samples thus collected were frozen at 80°C
and kept frozen until RIA was performed for human progastrin and
amidated gastrin as described previously (24). Antibody L289 was used
to measure progastrin, which is specific for human progastrin, whereas
the antibody L2 was used to measure amidated gastrins, which measures both human and mouse gastrins and reacts equally with all molecular forms of amidated gastrins (G-34, G-17). The concentrations of progastrin and amidated gastrins (where applicable) were plotted against the total number of ACFs measured in the animal, for each group
of animals, and the data were analyzed by linear regression analysis.
Statistical analysis. The incidence of ACs and number of crypts per foci were analyzed for significant differences by one-way ANOVA using SigmaStat Software. Significance was assigned when P < 0.05.
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RESULTS |
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Effects of AOM treatment on number of ACFs in hGAS, INS-GAS, and WT
mice. AOM was given at a dose of 12 mg/kg body wt in a series of
five sets of experiments 1-5, involving hGAS and
control mice, as described in MATERIALS AND METHODS.
Control mice received saline injections alone. In experiments 4 and 5, AOM was additionally given to INS-GAS mice, at the same
dose and schedule. In experiments 1 and 4 female mice
were used, and in experiments 2, 3, and 5 male
mice were used. The administration of AOM had no significant effect on
the body weight of the animals during the observation period. The body
weight of male and female mice for all three groups of animals (hGAS,
INS-GAS, and WT) increased similarly by 10-20% during the 4-wk
experimental period (data not shown). In all groups examined, ACFs
(Fig. 1) were more prevalent in the distal one-third of the colon, with
fewer numbers present in the middle and proximal thirds of the colon.
ACFs were found in all groups of AOM-treated mice but were absent in
all groups of saline-treated mice. In all five experiments involving
the hGAS mice, the total number of ACFs per colon was greater in the
hGAS mice compared with that in the WT mice, and this difference was
significant in almost all cases (P < 0.05, Table
1).
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In contrast, the total number of ACFs per colon was similar in the
INS-GAS mice compared with that in the WT mice, in the experiments
4 and 5 in which INS-GAS mice were used (Table 1). On an
average, the percent increase in total number of ACFs per colon
compared with that in the corresponding WT mice (irrespective of the
sex of the animal) was similar in all experiments (Table 1). We
therefore combined the data from all experiments, and the percent
change in the total number of ACFs per colon in the transgenic (hGAS,
INS-GAS) vs. that measured in the WT mice is presented in Fig.
2. As can be seen from Fig. 2, on average
the total number of ACFs per animal was significantly increased by ~61 ± 12.6% in the hGAS vs. WT mice. The percent change in the total number of ACFs per animal in the INS-GAS vs. WT mice was insignificant (Fig. 2).
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The multiplicity of the crypts per foci (i.e., singlets vs. doublets vs. multiplets) was analyzed for each group of mice. The results are presented in Table 1. The majority of the foci contained single ACs (singlets). A significant number of ACFs also contained two (doublets) or more than two (multiplets) ACs per foci. In all experiments other than experiment 3, the total number of singlets was significantly higher in the hGAS vs. that in the WT mice (Table 1). The total number of doublets and multiplets per colon was on average higher in the hGAS vs. WT mice, achieving statistical significance in experiments 1, 3, and 5. On the other hand, the multiplicity of crypts per foci in the colons of INS-GAS mice was similar to that in the WT mice (Table 1).
Effect of AOM on BrdU-labeling index of colonic crypts in hGAS,
INS-GAS, and WT mice. BrdU staining was carried out in both AOM-treated and -untreated mice, and the LI was calculated as the
percent cells labeled within a whole crypt. In the AOM-treated animals,
UC and AC foci were analyzed separately for BrdU staining and
calculation of the LI. BrdU labeling in the NCs of untreated WT mice
was generally present at very low levels in the lower one-third of the
crypts (Fig. 3a). On AOM treatment,
the BrdU labeling increased markedly both in the UCs and ACs of the WT mice (Fig. 3, b and c). The UCs in AOM-treated WT mice
were labeled more heavily than the NCs of the WT mice, but the labeling
was confined to the lower one-third of the crypts (Fig. 3b) and
was significantly lower than the BrdU labeling of ACs in the WT mice (Fig. 3c). The BrdU labeling of NCs from saline-injected hGAS mice on the other hand was already high, being markedly higher than the
labeling of NCs of WT animals (Fig. 3, a and d). The NCs of hGAS mice were labeled for BrdU mostly in the lower one-third of
the crypts (Fig. 3d). In comparison, the ACs of the AOM-treated hGAS mice were labeled for BrdU in the lower as well as the upper compartments of the crypts (Fig. 3f). The labeling of
UCs and ACs from AOM-treated hGAS mice could not be distinguished and appeared to be similar in its pattern of labeling within the lower two-thirds of crypts (Fig. 3, e and f). Once
again in the case of INS-GAS mice, the BrdU labeling of NCs, UCs, and
ACs was very similar to that of the corresponding crypts from the WT
mice (data not shown).
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To evaluate the BrdU LI, we initially counted representative crypts
(NCs, UCs, and ACs) from all the WT, hGAS, and INS-GAS mice from
experiments 3 and 4 in which we had injected the
animals with BrdU (as described in MATERIALS AND METHODS).
The data from WT and hGAS mice is presented in Fig.
4. The LI of NCs from hGAS mice was almost
twice that of NCs from WT mice (Fig. 4) and confirms our previous
findings (49). The LI of UCs and ACs from AOM-treated WT mice increased
linearly and significantly compared with the LI of NCs from the WT
control mice (Fig. 4). In the case of hGAS mice, however, because the
LI of NCs in untreated mice was already quite high, it was increased by
only ~40% in UCs and by ~50% in ACs on AOM treatment (Fig. 4).
The LI of ACs in hGAS vs. that in WT mice was therefore not
significantly different and may represent a maximizing-plateauing
response of colonic mucosa to AOM. As determined with all other
parameters, the LI of NCs, UCs, and ACs from INS-GAS mice was similar
to that measured for the corresponding crypts in the WT mice (data not
shown).
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To confirm whether the LI measured for representative crypts from each
animal was in fact a true reflection of the LI for the specific crypts
from a particular group of animals, we further analyzed ~10-20
crypts per animal (NCs and UCs) from all animals from the
representative experiment 4. All available midline sections of
aberrant crypts from this experiment were counted for this purpose, and
the data thus obtained from a much larger sample number are presented
in Table 2. The data in Table 2 are almost identical to the data presented in Fig. 4, further confirming that the
LI of NCs and UCs from hGAS mice were indeed significantly higher than
those from the corresponding crypts from WT and INS-GAS mice. Once
again the LI of ACs was similar for all three groups of animals (Table
2 and Fig. 4).
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A significant increase in the total number of ACFs in AOM-treated hGAS mice vs. that in the WT mice (Table 1) likely reflects the higher LI activity of NCs and UCs in hGAS mice (that may have translated into an increased conversion of NCs to ACs on AOM treatment).
Correlation between ACF formation and levels of progastrin and
gastrin. To examine a possible relationship between gastrin levels
and ACF formation, plasma was obtained from AOM-treated animals when
they were killed and analyzed for the concentration of progastrin and
amidated gastrins. The results are shown in Table
3. In the case of WT mice, amidated gastrin
levels were ~40-80 pM, whereas progastrin levels were undetected
(assay not sensitive at <40 pM). The levels of progastrin were
variable but markedly elevated in the hGAS mice (~1,000-8,000
pM), whereas the levels of amidated gastrin were similar to those of WT
mice. In contrast, the INS-GAS mice showed approximately threefold
elevations in serum levels of amidated gastrin (~150 pM) but only
slight increases in progastrin levels. The peak level of gastrins in INS-GAS mice, in response to food stimulation at night, however, is not
known.
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In animals in which gastrin determinations were obtained, attempts were
made to correlate the total number of ACFs per animal with the
concentration of progastrin and amidated gastrins. With respect to
progastrin, no correlation could be discerned in the WT mice (no
measurable progastrin) or the hGAS mice (where progastrin levels were
>1,000 pM). However, in the INS-GAS mice, where progastrin levels
ranged between 0 and 100 pM, there was a clear, positive correlation
between progastrin levels and the number of ACFs per mouse (Fig.
5). In contrast, there appeared to be a
negative correlation between the level of amidated gastrins and the
number of ACFs per hGAS mouse (Fig. 5).
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DISCUSSION |
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The possibility that processing intermediates of gastrin (mainly Gly-gastrin and progastrin) may function as autocrine-endocrine growth factors has evolved as a novel concept in the last few years (2, 6, 10, 35, 38, 39, 41, 49). Our study for the first time suggests a role for incompletely processed progastrins in determining the risk of colon carcinogenesis, possibly through effects on stimulation of colonic proliferation. Previous studies from our group showed that progastrin-expressing hGAS mice had a twofold increase in colonic proliferation at 10-12 mo of age (49). The present study demonstrates that this twofold increase in proliferation occurs as early as 2-3 mo of age. Published reports from a number of groups have suggested that an increased rate of colonic proliferation enhances the risk of progression to colon cancer (16).
A striking finding of the present study was that the intermediate markers of colon carcinogenesis were significantly increased in hGAS mice vs. those in WT mice in response to AOM. The progastrin-expressing hGAS mice had significantly increased numbers of ACFs, a surrogate marker for progression to colon cancer, compared with WT mice. The hGAS mice also showed a trend toward increased multiplicity of their ACFs, which has also been recognized to correlate with increased colon cancer risk (reviewed in Refs. 26 and 45). Because the total number and multiplicity of ACFs is now believed to correlate strongly with the risk for developing adenomas and adenocarcinomas (18, 22, 26, 45), and because ACFs display genetic mutations known to be associated with colon carcinogenesis in humans and rodents (as recently reviewed in Refs. 26 and 45), it is reasonable to speculate that the significantly higher number and multiplicity of ACFs measured in the hGAS mice will translate into the formation of higher numbers of adenomas and adenocarcinomas in hGAS mice vs. WT animals; our preliminary studies confirm this possibility (48).
Interestingly, the INS-GAS mice, which over-express amidated gastrin, showed no increase in ACFs compared with control mice. There was in fact a negative correlation between plasma levels of amidated gastrins and number of ACFs (Fig. 5), suggesting that amidated gastrins might even be protective. In contrast, there was a positive correlation between progastrin levels and the number of ACFs in the INS-GAS mice, consistent with a cancer-promoting role for these incompletely processed forms of gastrin. The number of ACFs in hGAS mice did not strongly correlate with progastrin levels in the mice (which may be explained by the extremely high levels in most of these mice) possibly beyond the dose-dependent range, which may have resulted in maximal stimulation of the proliferative pathways. It remains to be seen whether a >100-fold increase in levels of amidated gastrins can similarly result in increasing predisposition to preneoplastic changes in colonic mucosa in response to AOM. Based on the results presented in Fig. 5, however, that seems less likely. The present studies with hGAS mice thus provide the first evidence that high levels of circulating nonamidated gastrins can potentially function as cocarcinogens and significantly augment the carcinogenic potential of agents such as AOM.
The possible relationship between hypergastrinemia and colon cancer has been a confusing and controversial area (21, 25, 27, 28, 34, 43, 44). In a majority of these studies only amidated gastrin was measured, which may have contributed to the discrepancy in results. Whereas most human colon cancers express gastrin mRNA (reviewed in Ref. 2), gastrin peptides are poorly processed in colon tumors and colon cancer cell lines (2, 6, 41) and are largely secreted as progastrin and Gly-extended gastrin (2, 6, 41). In a recent study, circulating forms of both amidated and nonamidated gastrins were measured (4). Nonamidated gastrins were significantly higher (by almost 2- to 5-fold) in patients with colorectal carcinomas, compared with levels in control patients (4). More recently, a good correlation between ras mutations and gastrin gene expression was demonstrated, suggesting that gastrin gene may be an important downstream target of ras (23). Based on these recent reports (4, 23) it can be speculated that colon tumors positive for ras mutations are likely to express and secrete nonamidated gastrins.
In a recent prospective study, amidated gastrin and Gly-gastrin were measured in the serum of a large number of patients, some of whom eventually developed colorectal carcinomas (46). The results suggested that in a subpopulation of patients (~8.6%), high levels of serum gastrin may have contributed to the development of colorectal carcinomas (46); serum levels of progastrin were not measured in this study. However, other studies (21, 25, 27, 28) have not shown a clear association between levels of amidated gastrin and colorectal cancer. We now know that amidated gastrin and nonamidated gastrins can potentially exert proliferative effects on several cell types, including gastrointestinal cancer cells in vitro (6, 10, 35, 38, 40, 41), suggesting that the difference in the cocarcinogenic potential of amidated gastrins (INS-GAS mice) and nonamidated gastrins (hGAS mice), as observed in the present in vivo studies, probably reflects other contributing factors. We speculate that the receptor subtypes that mediate the mitogenic actions of amidated and nonamidated gastrins have an important role in the cocarcinogenic potential of gastrinlike peptides, as discussed below.
The CCK-B receptor (CCKB-K) represents the major receptor for amidated gastrins (50) and has been variously reported to be either expressed or not expressed in the colons of many species (5, 9, 20). The CCKB-R does not bind progastrin or Gly-extended forms of gastrin (50) and is unlikely to be responsible for proliferative effects of progastrin observed in the colon of hGAS mice. Several candidate receptors have been described, including a gastrin-binding protein (CCKC-R) (2), a gastrin preferring receptor (GP-R) (38), and a Gly-gastrin binding receptor (GG-R)(35); the latter two receptors have not been cloned. So, whereas any one or more of these receptors can potentially mediate mitogenic effects of nonamidated gastrins, the mitogenic effects of amidated gastrins can potentially be mediated via CCKB-R and GP-R (since both these receptor subtypes bind amidated gastrins with high affinity) (38, 50). However, besides mediating mitogenic effects, CCKB-R can also mediate growth inhibitory- (7) and morphogenic-differentiation effects (7, 14, 36). Thus the significant difference in the cocarcinogenic potential of nonamidated gastrins and amidated gastrins (as observed in the present study) may reflect the conflicting growth-inhibitory vs. growth-stimulatory effects of amidated gastrins (mediated via different receptor subtypes).
The present studies with hGAS mice thus provide the first evidence that high levels of circulating progastrin can potentially function as cocarcinogens and significantly augment the carcinogenic potential of methylating agents such as AOM. We believe that the increase in the proliferative index of colonic crypts in the hGAS mice most likely rendered the colonic cells more susceptible to AOM, resulting in conversion of a significantly higher number of colonic crypts into aberrantly proliferating crypts (demonstrating typical features of dysplasia). Thus, on the basis of our results in transgenic mice and previous human studies (4, 46), we speculate that high levels of incompletely processed gastrins may potentially increase the risk for developing colon cancer in human patients.
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ACKNOWLEDGEMENTS |
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The secretarial help of Carla Pairett and the technical support of Azar Owlia are gratefully acknowledged.
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FOOTNOTES |
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This study was supported by National Cancer Institute Grant CA-72992 and Grants 449150 and 440700 from the John Sealy Memorial Foundation to P. Singh; National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52778 to T. C. Wang; and Grant CA-16772 from the Cancer Center Support at MD Anderson Cancer Center to M. Wargovich.
Part of this study was published in its preliminary form as an abstract (Gastroenterology 114: A4134, 1998).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. Singh, Dept. of Anatomy and Neurosciences, Univ. of Texas Medical Branch, 301 Univ. Boulevard, Galveston, TX 77555-1043 (E-mail: posingh{at}utmb.edu).
Received 27 July 1999; accepted in final form 17 November 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
American Cancer Society.
Cancer Facts and Figures-1999. Atlanta, GA: American Cancer Society, 1999, p. 1-36.
2.
Baldwin, G. S.,
and
A. Shulkes.
Gastrin, gastrin receptors and colorectal carcinoma.
Gut
42:
581-584,
1998
3.
Brattain, M. G.,
G. Howell,
L. Z. Sun,
and
J. K. Willson.
Growth factor balance and tumor progression.
Curr. Opin. Oncol.
6:
77-81,
1994[Medline].
4.
Ciccotosto, G. D.,
A. McLeish,
K. J. Hardy,
and
A. Shulkes.
Expression, processing, and secretion of gastrin in patients with colorectal carcinoma.
Gastroenterology
109:
1142-1153,
1995[ISI][Medline].
5.
Czerwinski, G.,
N. I. Tarasova,
and
C. J. Michejda.
Cytotoxic agents directed to peptide hormone receptors-defining the requirements for a successful drug.
Proc. Natl. Acad. Sci. USA
95:
11520-11525,
1998
6.
Dai, B.,
Z. Xu,
B. Dhruva,
R. Espejo,
and
P. Singh.
Gastrin gene expression in human colon cancer cells: autocrine role of gastrin (Abstract).
Gastroenterology
102:
A352,
1992.
7.
Detjen, K.,
M. C. Fenrich,
and
C. D. Logsdon.
Transfected CCK receptors mediate growth inhibitory effects on human pancreatic cancer cell lines.
Gastroenterology
112:
952-959,
1997[ISI][Medline].
8.
Hollande, F.,
A. Imdahl,
T. Mantamadiotis,
G. Ciccotosto,
D. Shulkes,
and
G. S. Baldwin.
Glycine-extended gastrin acts as an autocrine growth factor in a nontransformed colon cell line.
Gastroenterology
113:
1576-1588,
1997[ISI][Medline].
9.
Imdahl, A.,
T. Mantamadiotis,
S. Eggstein,
E. H. Farthmann,
and
G. S. Baldwin.
Expression of gastrin, gastrin/CCK-B and gastrin/CCK-C receptors in human colorectal carcinomas.
J. Cancer Res. Clin. Oncol.
121:
661-666,
1995[ISI][Medline].
10.
Iwase, K.,
B. M. Evers,
M. R. Hellmich,
Y. S. Guo,
S. Higashide,
H. J. Kim,
and
C. M. Townsend, Jr.
Regulation of growth of human gastric cancer by gastrin and glycine-extended progastrin.
Gastroenterology
113:
782-790,
1997[ISI][Medline].
11.
Jen, J.,
S. M. Powell,
N. Papadopoulos,
K. J. Smith,
S. R. Hamilton,
B. Vogelstein,
and
K. W. Kinzler.
Molecular determinants of dysplasia in colorectal lesions.
Cancer Res.
54:
5523-5526,
1994[Abstract].
12.
Kennedy, E. P.,
and
S. R. Hamilton.
Genetics of colorectal cancer.
Semin. Surg. Oncol.
15:
126-130,
1998[ISI][Medline].
13.
Kinzler, K. W.,
and
B. Vogelstein.
Lessons from hereditary colorectal cancer.
Cell
87:
159-170,
1996[ISI][Medline].
14.
Langhans, N.,
G. Rindi,
M. Chiu,
J. F. Rehfeld,
B. Ardman,
M. Beinborn,
and
A. S. Kopin.
Abnormal gastric histology and decreased acid production in cholecystokinin-B/gastrin receptor-deficient mice.
Gastroenterology
112:
280-286,
1997[ISI][Medline].
15.
Lawley, P. D.
Carcinogenesis by alkylating agents.
In: Chemical Carcinogens, edited by C. D. Searle. Washington, DC: American Chemical Society, 1984, p. 325-484 (ACS Monograph 182).
16.
Lipkin, M.
Biomarkers of increased susceptibility to gastrointestinal cancer: new application to studies of cancer prevention in human subjects.
Cancer Res.
48:
235-245,
1988[ISI][Medline].
17.
Maltzman, T.,
J. Whittington,
L. Driggers,
J. Stephens,
and
D. Ahnen.
AOM-induced mouse colon tumors do not express full-length APC protein.
Carcinogenesis
18:
2435-2439,
1997[Abstract].
18.
McLellan, A.,
and
R. P. Bird.
Aberrant crypts: potential preneoplastic lesions in the murine colon.
Cancer Res.
48:
6187-6192,
1988[Abstract].
19.
McLellan, E. A.,
and
R. P. Bird.
Specificity study to evaluate induction of aberrant crypts in murine colons.
Cancer Res.
48:
6182-6186,
1988.
20.
McWilliams, D. F.,
S. A. Watson,
D. M. Crosbee,
D. Michaeli,
and
R. Seth.
Coexpression of gastrin and gastrin receptors (CCK-B and delta CCK-B) in gastrointestinal tumor cell lines.
Gut
42:
795-798,
1998
21.
Mihas, A. A.,
R. J. Maliakkal,
M. Shueke,
and
J. L. Achord.
Serum gastrin levels in patients with colorectal cancer and adenomatous polyps-a prospective study.
Res. Commun. Mol. Pathol. Pharmacol.
90:
301-304,
1995[ISI][Medline].
22.
Moen, C. J.,
M. A. van der Valk,
R. P. Bird,
A. A. Hart,
and
P. Demanded.
Different genetic susceptibility to aberrant crypts and colon adenomas in mice.
Cancer Res.
56:
2382-2386,
1997[Abstract].
23.
Nakata, H.,
S. Wang,
D. C. Chung,
J. K. Westwick,
and
L. G. Tillotson.
Oncogenic ras induces gastrin gene expression in colon cancer.
Gastroenterology
115:
1144-1153,
1998[ISI][Medline].
24.
Nemeth, J.,
B. Taylor,
S. Pauwels,
A. Varro,
and
G. J. Dockray.
Identification of progastrin derived peptides in colorectal carcinoma extracts.
Gut
34:
90-95,
1993[Abstract].
25.
Niv, Y.,
M. Heizelracht,
S. A. Lamprecht,
A. D. Sperber,
G. M. Fraser,
and
B. Schwartz.
Gastrin levels in colorectal cancer.
Isr. J. Med. Sci.
33:
186-189,
1997[ISI][Medline].
26.
Olivo, S.,
and
M. J. Wargovich.
Inhibition of aberrant crypt foci by chemo preventive agents.
In Vivo
12:
159-166,
1998[ISI][Medline].
27.
Orbuch, M.,
D. J. Venzon,
I. A. Lubensky,
H. C. Weber,
F. Gibril,
and
R. T. Jensen.
Prolonged hypergastrinemia does not increase the frequency of colonic neoplasia in patients with Zollinger-Ellison syndrome.
Dig. Dis. Sci.
41:
604-613,
1996[ISI][Medline].
28.
Penman, I. D.,
E. El-Omar,
J. E. S. Ardill,
J. R. McGregor,
D. J. Galloway,
and
P. J. O'Dwyer.
Plasma gastrin concentrations are normal in patients with colorectal neoplasia and unaltered following tumor resection.
Gastroenterology
100:
1264-1270,
1994.
29.
Pinson, D. M.,
N. Havu,
M. I. Sztern,
H. Mattsson,
G. A. Looney,
B. F. Kimler,
and
A. Hurwitz.
Drug-induced hypergastrinemia: absence of trophic effects on colonic carcinoma in rats.
Gastroenterology
108:
1068-1074,
1995[ISI][Medline].
30.
Pretlow, T. P.,
B. J. Barrow,
W. S. Ashton,
M. A. O'Riodan,
T. G. Pretlow,
J. A. Jurcisek,
and
T. A. Stellato.
Aberrant crypts: putative preneoplastic foci in human colon mucosa.
Cancer Res.
51:
1564-1567,
1991[Abstract].
31.
Reddy, B. S.
Nutritional factors and colon cancer.
Crit. Rev. Food Sci. Nutr.
35:
175-190,
1995[ISI][Medline].
32.
Roncucci, L.,
D. Stamp,
A. Medline,
J. B. Cullen,
and
W. R. Bruce.
Identification and quantification of aberrant crypt foci and microadenomas in the human colon.
Hum. Pathol.
22:
287-294,
1991[ISI][Medline].
33.
Rubin, N. H.,
P. Singh,
G. Alinder,
G. H. Greeley, Jr.,
P. L. Rayford,
W. J. Rietveld,
and
J. C. Thompson.
Circadian rhythms in gastrin receptors in the rat fundic stomach.
Dig. Dis. Sci.
33:
931-937,
1988[ISI][Medline].
34.
Seitz, J. F.,
M. Giovannini,
J. Gouvernet,
and
A. P. Gauthier.
Elevated serum gastrin levels in patients with colorectal neoplasia.
J. Clin. Gastroenterol.
13:
541-545,
1991[ISI][Medline].
35.
Seva, C.,
C. Dickinson,
and
T. Yamada.
Growth-promoting effects of glycine-extended progastrin.
Science
265:
410-412,
1994[ISI][Medline].
36.
Simon, B.,
J. L. Merchant,
R. Eissele,
K. Kohler,
and
R. Arnold.
Transient transcriptional activation of gastrin during sodium butyrate-induced differentiation of islet cells.
Regul. Pept.
70:
143-148,
1997[ISI][Medline].
37.
Singh, P.,
B. Dai,
R. L. Given,
X. Lu,
and
P. E. Holthuizen.
Differential activation of IGF-II promoters P3 and P4 in CaCo2 cells during growth and differentiation.
Gastroenterology
114:
1221-1229,
1998[ISI][Medline].
38.
Singh, P.,
A. Owlia,
R. Espeijo,
and
B. Dai.
Novel gastrin receptors mediate mitogenic effects of gastrin processing intermediates of gastrin on Swiss 3T3 fibroblasts.
J. Biol. Chem.
270:
8429-8438,
1995
39.
Singh, P.,
A. Owlia,
A. Varro,
B. Dai,
S. Rajaraman,
and
T. Wood.
Gastrin gene expression is required for the proliferation and tumorigenicity of human colon cancer cells.
Cancer Res.
56:
4111-4115,
1996[Abstract].
40.
Singh, P.,
C. M. Townsend, Jr.,
J. C. Thompson,
S. Narayan,
and
Y.-S. Guo.
Hormones in colon cancer; past and prospective studies.
Cancer J.
3:
28-33,
1990.
41.
Singh, P.,
Z. Xu,
B. Dai,
S. Rajaraman,
and
B. Dhruva.
Incomplete processing of progastrin expressed by colon cancer cells: role of non-carboxyamidated gastrin in proliferation.
Am. J. Physiol. Gastrointest. Liver Physiol.
266:
G459-G468,
1994
42.
Smith, A. J.,
H. S. Stern,
M. Penner,
K. Hay,
A. Mitri,
B. V. Bapat,
and
S. Gallinger.
Somatic APC and K-ras condon 12 mutations in aberrant crypt foci from human colons.
Cancer Res.
54:
5527-5530,
1994[Abstract].
43.
Smith, J. P.,
J. G. Wood,
and
T. E. Solomon.
Elevated gastrin levels in patients with colon cancer or adenomatous polyps.
Dig. Dis. Sci.
34:
171-174,
1989[ISI][Medline].
44.
Sobhani, I.,
T. Lehy,
P. Laurent-Puig,
G. Cadiot,
P. Ruszniewski,
and
M. Mignon.
Chronic endogenous hypergastrinemia in humans: evidence for a mitogenic effect on the colonic mucosa.
Gastroenterology
105:
22-30,
1993[ISI][Medline].
45.
Takayama, T.,
S. Katsuki,
Y. Takahashi,
M. Ohi,
S. Nojiri,
S. Sakamaki,
J. Kato,
K. Kogawa,
and
H. Miyake.
Aberrant crypt foci of the colon as precursors of adenoma and cancer.
N. Engl. J. Med.
339:
1277-1284,
1998
46.
Thorburn, C. M.,
G. D. Friedman,
C. J. Dickinson,
J. H. Vogelman,
N. Orentreich,
and
J. Parsonnet.
Gastrin and colorectal cancer: a prospective study.
Gastroenterology
115:
275-280,
1998[ISI][Medline].
47.
Townsend, C. M., Jr.,
P. Singh,
and
J. C. Thompson.
Gastrointestinal hormones and gastrointestinal and pancreatic carcinomas.
Gastroenterology
91:
1002-1006,
1986[ISI][Medline].
48.
Velasco M. , T. Wang, R. Given, M. Wargovich, and P. Singh.
Non-amidated gastrins, but not amidated gastrins function as
co-carcinogens in an azoxymethane (AOM) induced colon cancer model
(Abstract). Proc. Dig. Dis. Week (Orlando, FL), 1999, p.
640.
49.
Wang, T. C.,
T. J. Koh,
A. Varro,
R. Cahill,
C. A. Dangler,
J. A. Fox,
and
G. J. Dockray.
Processing and proliferative effects of human progastrin in transgenic mice.
J. Clin. Invest.
98:
1918-1929,
1996
50.
Wank, S. A.
G-protein-coupled receptors in gastrointestinal physiology. I. CCK receptor: an exemplary family.
Am. J. Physiol. Gastrointest. Liver Physiol.
274:
G607-G613,
1998
51.
Wargovich, M. J.,
C. D. Chen,
C. Harris,
E. Yang,
and
M. Velasco.
Inhibition of aberrant crypt growth by non-steroidal anti-inflammatory agents and differentiation agents in the rat colon.
Int. J. Cancer
60:
515-519,
1995[ISI][Medline].
52.
Zaidi, N. H.,
T. P. Pretlow,
M. A. O'Riordan,
L. L. Dumenco,
E. Allay,
and
S. L. Gerson.
Transgenic expression of human MGMT protects against azoxymethane-induced aberrant crypt foci and G to A mutations in the K-ras oncogene of mouse colon.
Carcinogenesis
16:
451-456,
1995[Abstract].