Mutagenesis, Vol. 16, No. 4, 309-315,
July 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Intestinal tumours induced by the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in multiple intestinal neoplasia mice have truncation mutations as well as loss of the wild-type Apc+ allele
Department of Environmental Medicine, National Institute of Public Health, PO Box 4404 Nydalen, N-0403, Oslo, Norway
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C57BL/6J-Min/+ (multiple intestinal neoplasia) is a murine model for familial adenomatous polyposis (FAP), where the mice are heterozygous for a nonsense ApcMin (adenomatous polyposis coli) mutation, and therefore develop numerous spontaneous adenomas in the small intestine and colon. Neonatal exposure of Min/+ mice to the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) (eight subcutaneous injections of 25 or 50 mg/kg PhIP to pups or 50 mg/kg PhIP to lactating dams) markedly increased (2–9-fold) the number of intestinal tumours, especially in the small intestine. We examined whether the Apc gene was affected in small intestinal and colonic tumours induced by PhIP. In spontaneous tumours formed in these mice, the main mechanism for tumour induction is loss of the wild-type Apc+ allele, i.e. loss of heterozygosity (LOH). Also in the PhIP-induced tumours, this is a major mechanism, since large fractions of PhIP-induced tumours had LOH in Apc. However, mechanisms other than LOH must also prevail, since a lower frequency of LOH was found in the small intestinal tumours from male mice exposed to PhIP either via breast milk (65%) or by direct injection (68%), compared with the untreated controls (92%). Tumours that had retained the wild-type Apc+ allele were further analysed for presence of truncated Apc proteins with in vitro synthesized protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in 20% (8 of 40) of the tumours not showing LOH from the small intestine after PhIP exposure, all in segment 2 (codons 686–1217). Seventeen percent (2 of 12) of the colonic tumours had a truncated Apc protein in segment 3 (codons 1099–1693). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc+ allele. These results show that PhIP induces intestinal tumours in the Min/+ mice both by causing LOH and truncation mutations in the wild-type Apc+ allele.
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Introduction |
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A high consumption of red meat (Willett, 1995
), especially when cooked well-done (Gerhardsson de Verdier et al., 1991; Sinha et al., 1999) appears to be associated with colorectal cancer. Heterocyclic amines formed in the crust of meat during cooking are multipotent carcinogens in rodents and non-human primates (Adamson et al., 1996). The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which was first isolated from fried ground beef (Felton et al., 1986), is the most abundant heterocyclic amine in various cooked fish and meat (Felton et al., 1986; Wakabayashi et al., 1992). PhIP induces cancer in the colon, breast and prostate in rats (Adamson et al., 1996; Shirai et al., 1997). In mice, however, PhIP induces lymphomas and no intestinal tumours (Esumi et al., 1989), although PhIP-induced aberrant crypt foci (ACF) have been reported (Tudek et al., 1989; Steffensen et al., 1997).
There is an association between dominant inheritance and predisposition to colorectal adenomas and cancers with a gene frequency of 13–19% (Cannon-Albright et al., 1988; Houlston et al., 1992). In the human disease familial adenomatous polyposis (FAP), a dominant inherited autosomal disorder, the affected individuals show an early development of multiple colorectal adenomas (Miyaki et al., 1995). Subsequent studies have demonstrated that germ-line mutations in the adenomatous polyposis coli (APC) gene account for the disease in the majority of FAP patients (Cottrell et al., 1992; Fodde et al., 1992; Miyoshi et al., 1992; Nagase et al., 1992; Groden et al., 1993; Olschwang et al., 1993; Powell et al., 1993; Varesco et al., 1993). Mutations in the APC gene, which occur in more than 85–95% of FAP tumours and in ~80% of the sporadic cancers in humans, are regarded as an early event in the carcinogenic process (Kinzler and Vogelstein, 1996; Laken et al., 1999). Like in human FAP, various germ-line mutations in the murine Apc gene, which is homologous to the human APC gene, cause a spontaneous development of multiple intestinal adenomas, of which some progress to adenocarcinomas in older mice (Moser et al., 1990; Su et al., 1992; Fodde et al., 1994; Oshima et al., 1995). In contrast to the human FAP syndrome where numerous polyps develop in the colon, the majority of tumours develops in the small intestine in the murine FAP models. One of these mouse strains, C57BL/6J-Min/+ (multiple intestinal neoplasia), is heterozygous for a nonsense mutation in the Apc gene at codon 850, changing a leucine (TTG) to a stop (TAG) codon, thereby producing a truncated non-functional Apc protein (Moser et al., 1990; Su et al., 1992). Loss of the remaining wild-type allele of the Apc gene is found in 78–100% of the spontaneously formed intestinal adenomas from Min/+ mice (Levy et al., 1994; Luongo et al., 1994; Laird et al., 1995; Ritland and Gendler, 1999).
The Min/+ mouse and analogous strains provide good carcinogenesis models for both sporadic and inherited forms of colorectal cancer, and they represent an opportunity to study the pathogenesis of a neoplasm in which the same gene is affected in both human and mouse. It appears that intestinal tumour development both in humans (Levy et al., 1994; Miyaki et al., 1995) and in murine FAP models (Levy et al., 1994; Luongo et al., 1994; Oshima et al., 1995; Smits et al., 1997) is associated with the loss of function of both APC/Apc alleles either by two mutations or by mutation and loss of heterozygosity (LOH) (Miyaki et al., 1995). However, the truncation mutations in humans and mice are located in different areas within the APC/Apc gene (Miyaki et al., 1995). In both familial and sporadic colorectal cancer mutations are distributed with a bias towards the 5'-end of the gene and with hotspot mutations at codon 1061 and 1309 in FAP and within the mutation cluster region (MCR) in the sporadic form of the disease, while the various murine models have a truncation mutation either at codon 716, 850 or 1638.
We have shown in a previous study (Steffensen et al., 1997) that adult C57BL/6J-Min/+ mice were more susceptible than the wild-type mice to the effects of PhIP on numbers of tumours in the small intestine and ACF in the colon. In a subsequent study (Paulsen et al., 1999), we demonstrated that Min/+ mice exposed neonatally to PhIP directly or via breast milk were particularly susceptible. A substantial increase in number of tumours in the small intestine and colon was observed. It is known that PhIP induces mutations in the Dolichos biflorus-1 locus in the small intestinal epithelium of mice (Brooks et al., 1994). Furthermore, some tumours from PhIP-exposed rats display a sequence-specific deletion mutation of a guanine base in the 5'-GGGA-3' sequence in the Apc gene (Kakiuchi et al., 1995; Okochi et al., 1999). In addition, studies on other genes are showing a mutational fingerprint of PhIP exposure, demonstrating that PhIP predominantly induces point mutations (G to T transversions) and single base deletions (predominantly involving G) (Okonogi et al., 1997; Lynch et al., 1998; Okochi et al., 1999; Glaab et al., 2000).
The mechanisms by which PhIP induces intestinal tumours in the Min/+ mice are not known. Since Min/+ mice are particularly susceptible to PhIP, our main hypothesis is that PhIP induces tumours primarily by affecting the remaining wild-type Apc+ allele. The objective of the present study was therefore to examine whether intestinal tumours from PhIP-exposed Min/+ mice had lost the wild-type Apc+ allele, i.e. showed LOH, or in those cases where the wild-type allele was retained, had truncation mutations in the Apc gene.
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Materials and methods |
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Mice
All mice were bred at the National Institute of Public Health (Oslo, Norway) from mice originally purchased from The Jackson Laboratory (Bar Harbor, ME). The Min/+ pedigree was maintained by mating C57BL/6J-+/+ (wild-type) females and C57BL/6J-Min/+ males. The Apc+/ApcMin offspring were identified by an allele-specific polymerase chain reaction (PCR) assay described in detail previously (Steffensen et al., 1997
). The experiments were approved and supervised by the National Experimental Animal Board in Norway. The mice were treated and housed in accordance with the laws and regulations controlling experiments with live animals in Norway, and with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes, March 18, 1986. The mice were housed in plastic cages in a room with 12 h light/dark cycle and controlled humidity (55 ± 5%) and temperature (20–24°C). Water and food were given ad libitum. The mice were fed a breeding diet, SDS RM3 (E), from Special Diets Services (Witham, UK) during gestation and until 5 weeks of age, thereafter they were given a standard maintenance diet from B&K Universal (Grimston, UK). The mice were killed by cervical dislocation at 11 weeks of age.
PhIP treatment
PhIP of >98% purity purchased from Toronto Research Chemicals (North York, Ontario, Canada) was dissolved in concentrated HCl, which thereafter was evaporated. PhIP–HCl was dissolved in 0.9% saline and adjusted to pH 3.5 with NaOH. C57BL/6J-Min/+ pups of both sexes were given eight subcutaneous injections of 25 or 50 mg/kg b.w. of PhIP, three times a week. Other Min/+ pups were exposed through breast milk from their dams given eight s.c. injections of 50 mg/kg PhIP. All mice were injected 3–6 days after being born or giving birth. Untreated pups were used as negative controls.
Scoring of tumours
The colon and small intestine were removed separately, rinsed in ice-cold phosphate buffered saline and slit open along the longitudinal axis. The small intestine was divided into proximal, middle and distal sections. The intestinal tissues were then spread flat between sheets of filter paper and fixed overnight in absolute ethanol. The number and location of adenomas in the colon and small intestine were scored by transillumination in an inverse light microscope at a magnification of x20. The size of the tumours scored was in the range of 0.2 to 4.5 mm.
Collection of tumour samples
From a subgroup of 47 C57BL/6J-Min/+ mice with a total of 4063 tumours in the small intestine and colon reported in our previous study (Paulsen et al., 1999), we sampled all small intestinal tumours with a diameter 
0.75 mm found in the region 21–30 cm from the ventricle, being the area in the small intestine where the effect of PhIP on tumour number was most pronounced (Figure 1). The few colonic tumours present in these mice with a diameter 0.75 mm were all sampled. Intestinal samples from 120 tumours from 7 mice exposed to 50 mg/kg PhIP directly, 60 tumours from 5 mice exposed to 25 mg/kg PhIP directly, 80 tumours from 9 mice exposed to PhIP via breast milk and 110 tumours from 26 unexposed control mice (n = 370) were collected from the centre of the tumours by puncture with a cannula (diameter 0.5 mm). Later, one sample from the 50 mg/kg PhIP group and two from the control group were removed from further analysis of LOH frequencies, making final n = 367 (see explanation further down).
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DNA isolation and assessment of the Apc allele status
DNA was isolated by grinding the samples with a pestle before boiling for 10 min at 100°C in 30 µl 10 mM Tris–HCl pH 8.0, 1 mM EDTA and 0.05% SDS. After incubation with 1/10 vol proteinase K (6.5 µg/µl) for 1.5 h at 55°C, the reaction was stopped by boiling the samples for 10 min at 100°C. In order to assess the Apc allele status, PCR on the Apc gene was performed as described by Luongo et al. (1994). In short, the wild-type Apc+ allele contains a HindIII restriction site that is not present in the ApcMin allele. Following enzymatic cutting with HindIII (Gibco BRL, Bethesda, MA), the two alleles were separated on a 15% TBE–PAGE Ready gel (Bio-Rad, Hercules, CA). The fluorescence ratio between the mutated and wild-type bands was scored semi-quantitatively by Gel-ProTM Analyzer (Media Cybernetics, Silver Spring, MD).
Apc+/ApcMin band ratios in normal tissues of the Min/+ mouse
In order to determine the cut-off value that defines the retaining of the wild-type Apc+ allele, the distribution of the measured Apc+/ApcMin band ratios in 43 samples from normal intestine, liver and ear cartilage of Min/+ mice were analysed by two independent amplification reactions. The measured band ratios varied from 0.625 to 1.0 with a mean value of 0.781. The cut-off value was set at 0.649 [mean – (2x SD)], and tumour samples with measured Apc+/ApcMin band ratio 
0.645 were classified as having lost the wild-type Apc+ allele, i.e. having LOH in the Apc gene (Figure 2A). This cut-off value is in agreement with the value set previously by others (Shoemaker et al., 1998).
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Presence of normal tissue in a tumour sample would increase the measured Apc+/ApcMin band ratio. Tumours with loss of the Apc+ allele, but with some normal tissue present could, therefore, be misclassified as not having LOH in the Apc gene. Based on histological examination of sliced tumour samples, the relative number of nuclei from normal interstitial cells and tumour cells was determined. We estimated that with our sampling method the tumour samples contained <15% normal tissue (data not shown). All tumours initially classified as not having LOH, i.e. with a measured Apc+/ApcMin band ratio above 0.645, were corrected for a 15% maximal normal tissue contamination. Three samples had a corrected ratio of 0.629–0.641 and may have been misclassified. These samples were therefore removed from the data set.
Analysis of truncated Apc proteins
All samples with LOH, i.e. with a Apc+/ApcMin band ratio >0.645, having retained the wild-type Apc+ allele, were analysed for truncated Apc proteins by a slightly modified in vitro synthesized protein (IVSP) assay (Levy et al., 1994; Shoemaker et al., 1997). Two overlapping segments, segment 2 (codons 686–1217 ) and segment 3 (codons 1099–1693), in exon 15 of the Apc gene, which covers the human `hot spot' mutation region called mutation cluster region (codons 1286–1513), were analysed. PCR fragments were produced by amplification of DNA prepared from ethanol-fixed tumour samples as described above. Each PCR was done in a 24 µl reaction volume with 12 µl 1:200 DNA sample and 0.2 mM each dNTP, 10 mM Tris–HCl, pH 8.3 at 25°C (segment 2) or 15 mM Tris–HCl, pH 8.0 (segment 3), 50 mM KCl, 0.1% Triton X-100, 0.5 µl protein mixture (0.5 µg single strand binding protein, 0.5 µg acetylated bovine serum albumin in 100 µl 100 mM Tris–HCl pH 8.3 and 100 mM KCl) and 1.25 U Ampli Taq Gold polymerase (Perkin Elmer, CA). For segment 2, each PCR reaction contained in addition 0.39 µM forward primer, 1.2 µM reverse primer and 2.0 mM MgCl2. For segment 3, each PCR contained 0.39 µM forward primer, 0.87 µM reverse primer and 2.0 mM MgCl2. Both PCR reactions used the same program of a 10 min denaturation step at 95°C followed by 37 cycles of 30 s at 95°C, 1 min 30 s at 65°C and 2 min at 70°C, followed by one cycle of 7 min at 70°C.
The quality and quantity of all PCR products were assessed by running 10 µl of each reaction product on 1% agarose gels. Products of successful PCR amplifications were used in a 10 µl coupled in vitro transcription–translation reaction following the manufacturer's protocol (Promega, Madison, WI). [35S]Methionine-labelled polypeptides from these reactions were analysed by 15% Tris–HCl PAGE Ready gel (Bio-Rad, Hercules, CA). The gels were fixed, dried and the films were exposed by autoradiography (Figure 2B
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Statistical analysis
For analyses of tumour number we used one-way ANOVA, or one-way ANOVA on ranks for non-parametric data, followed by the appropriate multiple comparison procedure (SigmaStat software, Jandel Scientific, Erkrath, Germany). For analyses of differences in the frequency of presence or absence of LOH we used 
2 analysis or Fischer's exact probability test, as appropriate. Fischer's exact probability test was used to evaluate incidence data. A value of P < 0.05 was considered significant.
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Results |
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Tumour numbers
The incidence of small intestinal tumours was 100% in all groups, including the controls (Table I
). Min/+ mice exposed through breast milk from dams injected with 50 mg/kg PhIP showed a 2–4-fold increase in the number of small intestinal tumours compared with untreated control mice (Table I). This effect was highly significant in the females (P < 0.001). An even greater increase was seen in the pups exposed directly to 25 (4–6-fold) or 50 (7–9-fold) mg/kg PhIP (Table I). However, the difference was only statistically significant between the unexposed controls and the highest dose of PhIP in the males (P < 0.05), but with both doses of PhIP in the females (P < 0.001). The small additional increase in the number of small intestinal tumours after direct exposure to 50 compared with 25 mg/kg PhIP was only statistically significant in the females (P < 0.001). No significant differences in gender were seen in the various PhIP-treated groups.
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The distribution of tumours along the small intestine was plotted separately for each experimental group on pooled data from males and females (mean number of tumours/cm intestine/mouse). The graphs clearly show an accumulation of tumours in the distal part of the small intestine in both the PhIP-treated Min/+ mice and in the controls (Figure 1
). In this area of the small intestine PhIP induces tumours in a dose-dependent manner.
In the colon, 50 mg/kg PhIP administered directly to male Min/+ mice increased the number of colonic tumours significantly relative to the controls (P < 0.001) (Table II). This was the only group with 100% tumour incidence. Significantly more colonic tumours were also found in this group in comparison with male mice receiving 25 mg/kg PhIP (P < 0.001). Male Min/+ mice exposed via breast milk showed a 3-fold increase in the number of tumours compared with the controls, but this difference was not statistically significant. In the females, none of the PhIP treatments affected tumour numbers (Table II). No significant differences in gender were seen in the various PhIP-treated groups.
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LOH in the Apc gene
In male Min/+ mice, the frequency of LOH in the Apc gene was clearly lower in PhIP-induced small intestinal tumours than in tumours from spontaneous controls (Figure 2A
; Table I). This effect was most prominent in tumours from mice exposed via breast milk and in those exposed directly to 25 mg/kg PhIP, the frequencies being 65% (P = 0.011) and 68% (P = 0.017) in these groups, respectively, in comparison with 92% in tumours from the unexposed controls. The frequency of LOH (84%) in tumours from Min/+ mice exposed to 50 mg/kg PhIP was not significantly different from the controls (Table I).
In the females, PhIP apparently did not affect the LOH status in the small intestinal tumours since none of the PhIP treatments showed LOH frequencies significantly different from the controls (Table I).
Much fewer tumours were found in the colon than in the small intestine (Table II). Neither for males nor females were there any statistically significant differences in LOH frequencies between colonic tumours from the various PhIP-exposed groups and the controls (Table II). However, pooled data for males and females showed a significantly lower frequency of LOH in tumours from mice exposed to 50 mg/kg PhIP in comparison with the controls (P = 0.013) (Table II). However, this should be interpreted cautiously as it cannot be excluded that pooling might skew the results. The very low frequency of LOH (20%) in the colonic tumours from males exposed directly to 50 mg/kg PhIP, should, however, be noted since this was the only group that had a significantly higher number of colonic tumours following PhIP exposure (Table II). Only analysis of more PhIP-induced tumours would clarify whether PhIP induces colonic tumours primarily via loss of the wild-type Apc+ allele or by other mechanisms.
Truncated Apc proteins
The tumour samples apparently retaining the wild-type Apc+ allele according to the LOH analysis were examined for presence of truncated Apc proteins by the IVSP assay. Two overlapping segments of exon 15 in the murine Apc gene, codons 686–1217 (segment 2) and codons 1099–1693 (segment 3), were analysed.
Seventy-two small intestinal tumours with retained wild-type Apc+ allele were analysed by the IVSP assay (Table III). We were able to get PCR products from 52 and 54 of them for segments 2 and 3, respectively. Of these, we detected a truncated protein in eight samples, two in tumours from males and six in tumours from females, all in segment 2 (Figure 2B, Table III). All of these truncated proteins were found in tumours from Min/+ mice exposed to PhIP, with a frequency of 20% (8 of 40), and none were seen in the spontaneously occurring tumours from unexposed controls. Truncated proteins were found in all three PhIP-exposed groups. Two occurred in tumours from mice exposed via breast milk (one male and one female), two (one male and one female) and four (all in females) in tumours from mice exposed directly to 25 and 50 mg/kg PhIP, respectively. The length of the truncated proteins varied within ~10 kDa, and all truncations seemed to be in the region between codons 1080 and 1170.
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Fifteen colonic tumours with retained wild-type Apc+ allele were examined with the IVSP assay (Table IV
). PCR products were obtained in all analyses, both for segments 2 and 3. Two truncated Apc proteins among 12 colonic tumour samples (17%) were found in male mice exposed to the highest dose of PhIP, 50 mg/kg, both in segment 3 (Table IV). The location of these truncations was probably downstream of codon 1300. No truncated proteins were found in the other 13 samples (Table IV).
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Discussion |
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Previous studies have shown that the main mechanism for spontaneous intestinal tumour development in the Min/+ mice is loss of the wild-type Apc+ allele (Levy et al., 1994
; Luongo et al., 1994; Laird et al., 1995; Ritland and Gendler, 1999). Cross breeding studies have furthermore shown that the whole of chromosome 18 containing the wild-type allele is lost (Luongo et al., 1994). The great increase in the number of intestinal tumours, especially in the small intestine, in neonatal Min/+ mice after exposure to PhIP either directly or through breast milk, clearly demonstrates that neonatal Min/+ mice are highly susceptible to PhIP. We assume that the Min/+ mouse model would strongly select for inactivation of the wild-type Apc+ allele upon carcinogen-induced intestinal tumour development. There might be several mechanisms for carcinogen-induced inactivation of the wild-type Apc+ allele that may cause tumour development in Min/+ and similar animal models. For instance, tumours formed in Apc1638N mice exposed to ionizing radiation show LOH with partial intrachromosomal deletions of the wild-type Apc+ allele, but not loss of the whole of chromosome 18 (van der Houven van Oordt et al., 1997). Mutations in the Apc gene have been reported in intestinal tumours from Min/+ mice induced by N-ethyl-N-nitrosourea (ENU) (Shoemaker et al., 1997, 1998). In Min/+ mice cross-bred with homozygous MSH2 mismatch repair knockout mice, the full length Apc protein was shown to be lost by immunostaining in only five out of 34 adenomas (Reitmair et al., 1996).
In the present study, analyses of the allelic status clearly showed that one mechanism by which PhIP induces tumours is by causing LOH in the Apc gene, i.e. loss of the functional wild-type Apc+ allele. The mean frequency of LOH in tumours from the untreated controls, 84% in the small intestine and 70% in the colon, is in concordance with previous studies showing 78–100% LOH in spontaneous tumours from Min/+ mice (Levy et al., 1994; Luongo et al., 1994; Laird et al., 1995; Ritland and Gendler, 1999). The LOH frequencies in the small intestinal tumours from male mice exposed to the two lowest doses of PhIP, via breast milk, previously estimated to be ~7–12 mg/kg (Paulsen et al., 1999), or 25 mg/kg directly, were significantly lower than in tumours from unexposed controls. In addition, the LOH frequency in the 50 mg/kg PhIP group was also lower than in the controls, although it did not reach statistical significance. Therefore, in contrast to spontaneous tumours which are thought mainly to be caused by LOH in Apc, PhIP seems to induce tumours by other mechanisms as well. If some of the putative PhIP-induced tumours analysed in this study in reality were spontaneous tumours, this could cause a measured higher frequency of LOH, and therefore the real frequency of LOH induced by PhIP might be even lower. However, this has probably not affected the numbers significantly, since subtraction of the background tumours in the PhIP-exposed groups changed the frequencies of LOH by <10% (data not shown). In the female mice, the frequency of LOH in small intestinal tumours was not significantly affected by PhIP. However, we did find truncated proteins in some of the tumour samples with retained wild-type Apc+ allele in all three PhIP-exposed groups of females. In the few colonic tumours induced, no significant effects of PhIP treatment on frequency of LOH were observed in either males or females. For the colon, studies of more tumours are obviously needed to clarify the mechanisms of PhIP-induced tumourigenesis in the Min/+ mouse.
Our method for LOH analysis, which only determines the presence or absence of a minor fragment of the wild-type Apc+ allele, does not provide any information about whether the whole chromosome is lost or if only minor deletions have occurred. Because the Min/+ mice used were not on a hybrid background, i.e. the only genetic difference between the C57BL/6J-Min/+ and C57BL/6J-+/+ is the ApcMin mutation, we could not distinguish which one of the two chromosomes that had lost genetic material outside the Apc locus.
It is known that PhIP forms bulky DNA adducts mainly at the C8 position in guanine (Frandsen et al., 1992). One hypothesis is that the bulky PhIP–DNA adduct might block the DNA replication fork and thereby initiate events leading to recombination. Such a mechanism of LOH has been suggested for other carcinogens. Benzo[a]pyrene and 7,12-dimethyl-1,2-benz[a]anthracene induced LOH in the wild-type allele of the adenine phosphoribosyltransferase (Aprt) gene probably by mitotic recombination or chromosome loss in normal somatic cells from rodents (Mazur-Melnyk et al., 1996; Wijnhoven et al., 1998). This mechanism is in contrast to small alkyl adducts formed by carcinogens such as ENU, which mainly causes single-strand nicks upon activation of the base excision repair or nucleotide excision repair pathways (Wijnhoven et al., 1998). However, there are somewhat conflicting results from exposure studies with ENU. Wijnhoven et al. (1998) showed that ENU hardly induced LOH in the Aprt gene in T-lymphocytes from Aprt+/– mice, while Shoemaker et al. (1997) also found LOH in Apc in tumours from Min/+ mice. Interestingly, tumours from ENU-exposed Min/+ mice (Shoemaker et al., 1997) show a much lower frequency of LOH in Apc (39%) than we found with PhIP.
Truncation mutations have been reported to occur in the wild-type APC/Apc+ allele in tumours from FAP patients (Cottrell et al., 1992; Fodde et al., 1992; Miyoshi et al., 1992; Nagase et al., 1992; Groden et al., 1993; Olschwang et al., 1993; Powell et al., 1993; Varesco et al., 1993; Levy et al., 1994; Miyaki et al., 1995), ENU-exposed Min mice (Shoemaker et al., 1997, 1998) and Apc1638N mice crossed with Mlh1 mice (Edelmann et al., 1999). In our study, the wild-type Apc+ allele was apparently retained in a number of PhIP-induced tumours. These samples were analysed for the presence of proteins truncated in segment 2 or 3 in exon 15 of the Apc gene by IVSP assay. Truncated proteins were detected in eight small intestinal tumours and two colonic tumours, all tumours being from PhIP-exposed mice. However, this number might be an underestimation of truncation mutations caused by PhIP because we did not obtain a PCR product from ~25% of the tumours from the small intestine. The reason for the unsuccessful PCR amplifications may be the low amounts of DNA obtained or degradation caused by our crude method for DNA isolation (see Materials and methods).
In this study, only parts of the Apc gene have been analysed for truncated proteins. Nagao et al. (1997) previously showed that of four colonic tumours from PhIP-exposed rats harbouring five mutations in the Apc gene, three mutations occurred in exon 15 (in segments 2 and 3) and two in exon 14. However, in our study we were not able to examine exons 1–14 (segment 1, codons 1–804) by the IVSP assay because of lack of RNA. We were able to get PCR products both for segments 2 and 3 from all of the 15 colonic tumours. Since we found only two truncated proteins, both terminated in segment 3, among these samples, the other tumours could in theory have truncation mutations in other parts of the gene, e.g. in segment 1.
The putative truncation mutations detected in the small intestinal tumours in the present study all seemed to be in the region between codons 1080–1170 in segment 2, judged by migration of the proteins in the gels. This region is located between the ApcMin mutation and the human MCR, i.e. in the area for constitutive ß-catenin binding (Shoemaker et al., 1997). The two truncation mutations found in colonic tumours were both in segment 3, which is overlapping with segment 2. These two mutations must be in the region of segment 3 after the overlapping area with segment 2, i.e. downstream of codon 1217, since we did not find mutations in segment 2 in the same samples.
We only screened the tumour samples for truncated proteins, indicating presence of truncation mutations. Therefore, in the samples where we did not detect truncated Apc proteins there might be point mutations or minor deletions in the Apc gene that could not be detected by the IVSP assay.
In addition, it is possible that PhIP has affected genes other than Apc in some of these tumours, e.g. ß-catenin, genes in the DNA repair pathways or other genes in the Wnt signal pathway. ß-Catenin is an oncogene, and only one hit is needed to affect the function of its protein. Epigenetic events may also be involved. It has been shown that the wild-type APC+ allele may be inactivated by hypermethylation in human colorectal cancer (Hiltunen et al., 1997; Esteller et al., 2000).
In conclusion, this study shows that PhIP induces intestinal tumours in the Min/+ mouse both by causing LOH and truncation mutations in the wild-type Apc+ allele. However, since in a number of samples none of these two mechanisms were detected, PhIP may also induce intestinal tumours by other pathways. This is in contrast to spontaneous tumours in the Min/+ mice, where LOH seem to be the almost exclusive mechanism. Further studies of PhIP-induced intestinal tumourigenesis in Min/+ mice should be directed towards the study of mechanisms for LOH in the Apc gene, of the various types of mutations in the wild-type Apc+ allele in tumours not showing LOH, as well as towards genetic events in other relevant genes.
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Acknowledgments |
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We thank Hege Hjertholm and Marit Hindrum for excellent technical assistance. Å.A. is a post-doctoral fellow of the Norwegian Cancer Society, grant no. E98064/001, and I.-L.S. was a post-doctoral fellow of the Norwegian Research Council, grant no. 111931/431. This study has been carried out with financial support from the Commission of the European Communities specific RTD program Quality of Life and Management of Living Resources, QLK1-CT99-01197, Heterocyclic Amines in Cooked Foods—Role in Human Health. It does not necessarily reflect its views and in no way anticipates the Commission's future policy in this area.
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Notes |
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Å.Andreassen and R.Vikse contributed equally to this work
1 To whom correspondence should be addressed. Email: jan.alexander{at}folkehelsa.no 
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References |
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Received on November 13, 2000; accepted on February 16, 2001.
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