Suppression of polypogenesis in a new mouse strain with a truncated Apc{Delta}474 by a novel COX-2 inhibitor, JTE-522

Hitoshi Sasai2, Michiko Masaki1 and Korekiyo Wakitani1

Pharmaceutical Frontier Research Laboratories, Japan Tobacco Inc., 1-13-2 Fukuura, Kanazawaku, Yokohama 236-4 and
1 Pharmaceutical/Biological Research Laboratories, Central Pharmaceutical Institute, Japan Tobacco Inc., 1-1 Murasaki-cho, Takatsuki, Osaka 569-11, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutations of the adenomatous polyposis coli gene (Apc) have been implicated in the occurrence of sporadic colon cancer. Various Apc knockout strains of mice have been created to better understand the function of this gene. In the present study, using gene targeting, we disrupted the mouse Apc gene at the end of exon 10 to compare its effect with the effects of other types of Apc gene disruption, all of which are on exon 15. The mice expressed a mutant form of mRNA that encoded 474 amino acids instead of 2845 amino acids due to exon duplication. In addition, these Apc{Delta}474 knockout mice developed intestinal and mammary tumors. Since the most severe cases of familial adenomatous polyposis are associated with mutations on exon 15, our mutation at exon 10 was expected to result in a mild phenotype. However, the number of polyps that our mice developed was similar to that of other Apc knockout mice such as ApcMin and Apc1309 mice. Cyclooxygenase-2 (COX-2) has been implicated in colorectal carcinoma. Apc{Delta}474 mice treated with JTE-522, a novel COX-2-selective inhibitor, showed a significantly reduced number of polyps. These results suggest that COX-2 plays an important role in polypogenesis and COX-2-selective inhibitors can be used as new preventive therapeutics against colorectal tumors.

Abbreviations: Apc, adenomatous polyposis coli; COX, cyclooxygenase; ES, embryonic stem; FAP, familial adenomatous polyposis; Min, multiple intestinal neoplasia; NSAIDs, non-steroidal anti-inflammatory drugs.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colon cancer is one of the common cancers in Western countries. Much effort is being devoted to the development of effective therapies for this disease, as well as to its prevention. Colon cancer is believed to develop partly from early cancer in adenoma and partly from de novo cancer. It is well known that the adenomatous polyposis coli gene (Apc) is mutated in patients with familial adenomatous polyposis (FAP) and sporadic colon cancer, and that these mutations initiate colon carcinogenesis (1). Indeed, mice with an artificially mutated Apc gene developed multiple intestinal neoplasia (Min) and have been used as a model of FAP in order to study colon carcinogenesis (2).

There is heterogeneity in the severity of FAP. Patients in some FAP families manifest more than 5000 polyps in the colon (profuse type) while patients in other families show less than 100 polyps (attenuated type). Although the cause of the difference in polyp number is not known, it is reported that the mutations found in attenuated FAP are segregated at the 5' end of the Apc gene while the ones in the profuse type are segregated mainly in exon 15 (3). So far, four strains of Apc knockout mouse have been created, and in each strain, the mutations were in exon 15 where the mutations of classical types of human FAP are clustered. To determine whether a mutation located near the 5' end of the Apc gene caused a less severe type of polyposis, we created mice with a mutation right after the end of exon 10, which resulted in a truncated Apc protein (Apc{Delta}474). All Apc{Delta}474 mice developed around 100 intestinal polyps and 18% of them manifested mammary tumors with the characteristics of adenoacanthoma.

We are also interested in the prevention of colon tumors by cyclooxygenase-2 (COX-2)-selective inhibitors, because aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) are reported to reduce the incidence or progression of colorectal cancer and polyps (4,5). Using Apc (+/–) Ptgs2 (–/–) mice which lack COX-2 or using a COX-2 inhibitor, MF tricyclic, Oshima et al. (6) clearly showed that COX-2 has an important role in colorectal polyposis and cancer. We have recently shown that JTE-522, 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide, selectively inhibits human COX-2. In addition, it has a potent anti-inflammatory activity and very little gastrointestinal ulcerogenicity in rats. We are now testing JTE-522 as an anti-inflammatory drug in Phase II clinical trials (79). Here we report that JTE-522 has a significant tumor-suppressive effect on polypogenesis in the Apc{Delta}474 mouse and that this compound can be the basis of a novel class of therapeutic drugs for polyposis and colorectal cancers.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Targeting vector constructs
The mouse Apc gene consists of 15 exons. We obtained a genomic clone, which contained exons 7–10 of the mouse Apc gene from a Balb/c genomic library. A 12 kb SalI fragment containing exons 7–10, and a XhoI/BamHI fragment of pMC-1/neo.polA (Stratagene) were inserted into the XhoI and SalI/BamHI sites of BlueScript KS (+) (Stratagene), respectively. For electroporation, the targeting vector was digested with SmaI, which is in the middle of exon 9 (Figure 1Go).



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Fig. 1. (A) The genomic structure around exon 9–10 of the normal mouse Apc gene. (B) The expected genomic structure of the targeted Apc locus in which the targeting vector (insertion type) is correctly integrated. Exon duplication (exon 7–8–9–10–7–8–9–10) is created by the insertion of the vector. The boxes filled with black are endogenous exons of Apc and the hatched boxes are exons from the targeting vector. The size of the homologous region in the targeting vector is ~12 kb. The probe used for the Southern blot analysis is indicated by a filled bar and the sizes of the BamHI/BstEII bands in the mutant and wild-type Apc alleles are also indicated. (C) Structure of the targeting vector digested with SmaI.

 
Embryonic stem (ES) cell line and cell culture
The ES cell line E14-1 was transfected with the linearized targeting vector and selection was carried out with G418 (300 µg/ml; Gibco BRL). After 6 days, drug-resistant clones were individually expanded and their genomic DNAs were prepared for Southern blot analysis using a 1.4 kb BstEII/SpeI fragment as a probe (Figure 1Go).

Production of germline chimeras
The targeted ES cells were injected into blastocysts of C57BL/6 mice, which were then transplanted into the uteri of pseudopregnant foster mothers. The resulting chimeric mice, which contain a significant contribution from the targeted ES cells, were crossed with C57BL/6 females to obtain the germ line transmission of the mutant Apc allele.

mRNA analysis in Apc{Delta}474 mice
mRNA was extracted from mouse intestine and cDNA was synthesized as described below. cDNA was synthesized from 4 µg mRNA using a First Strand cDNA Synthesis Kit (Pharmacia) with 10 pmol exon 8-rp primer (5'-TCGTGATCCACACGTGT-3'). PCRs were performed in 25 mM Tris–HCl pH 9.0, 50 mM KCl, 2 mM MgCl2, 200 mM of each dNTP, 50 pmol of exon 9-up primer (5'-CCTGATGACAAGAGAGGCA-3') and 2.5 U EX-Taq DNA polymerase (Takara) in a volume of 50 µl. After 3 min of denaturation at 94°C, 30 cycles were performed: a 1 min denaturation at 94°C, a 1 min annealing at 50°C and a 2 min extension at 72°C, followed by a final extension step at 72°C for 10 min. The PCR product was cloned into TA-cloning vector (Invitrogen) and its sequence was analyzed.

Feeding experiments with JTE-522 and polyp counting
The Apc{Delta}474 mice were prepared by artificial insemination using sperm derived from one male Apc{Delta}474 mouse. Three groups were established, each consisting of four females and four males. From an age of 4 weeks, mice were fed ad libitum with diets with either 0, 0.001 or 0.01% JTE-522 for 8 weeks until the end of week 11. Individual food intakes and body weights were monitored every week and the actual drug doses were calculated accordingly. The Apc{Delta}474 mice were killed by dislocation of cervical vertebrae when they reached an age of 12 weeks. The gut was ligated at the anterior end, filled with 10% formaldehyde in PBS from the anal end, and then opened longitudinally (6). The total number of gastrointestinal polyps >0.2 mm in diameter was counted three times under the microscope according to the double-blind method and the average number of polyps was calculated. The statistical difference between the total number of polyps of the groups was determined by Dunnett's two-tailed test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Constructions of Apc{Delta}474 mice
The mice were constructed according to the strategy shown in Figure 1Go. Five hundred G418-resistant ES cell clones were screened for homologous recombinant candidates by Southern blotting using a 1.4 kb BstEII/SpeI fragment as a probe. Nine homologous recombinant clones were identified. Two of these clones were injected into C57BL/6 blastocysts, and one of them was transmitted to the germline. Germline transmission of the mutant allele of Apc was confirmed by Southern blotting, in which the mutant allele showed a 14 kb band whereas the wild-type allele showed a 15 kb band when the genome was double digested with BamHI and BstEII (Figures 1 and 2GoGo). We used an insertional-targeting vector, which was expected to result in the duplication of exons 7–10 (7–8–9–10–7–8–9–10), which would cause a frameshift mutation. We analyzed mRNA derived from an Apc{Delta}474 mouse by RT–PCR using the above-described primer sets for exons 8 and 9, each of which went in opposite directions and amplified only the segment containing the exon duplication. We detected a distinct 357 bp band, which was specific to the mutant Apc (Figure 3Go). We cloned this fragment into a vector and determined its sequence, which showed the exact exon duplication. The exon duplication resulted in a frameshift at the junction between exon 10 and exon 7, which resulted in a stop codon at the eighth position from the junction (Figure 4Go).



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Fig. 2. Southern blot analysis of targeted Apc in ES cells and Agouti mice derived from chimeric mice created by the targeted ES cells. The genomic DNAs were digested with BamHI and BstEII and hybridized with a 1.4 kb ApaI/BstEII fragment immediately following exon 10.

 


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Fig. 3. The result of RT–PCR which amplified the mutant Apc allele that has exon duplication. The mRNA was purified from the intestines of Apc{Delta}474 and C57BL/6 mice and was amplified with the primers, exon 9-up and exon 8-rp.

 


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Fig. 4. Sequence of the RT–PCR product amplified from the mutant Apc allele. The border between exon10 and exon 7 is indicated by a dotted line.

 
Tumor development in Apc{Delta}474 mice
Although Apc{Delta}474 mice developed normally until 2–3 months of age, they became anemic later due to intestinal hemorrhage. No Apc{Delta}474 homozygous mouse was found in 33 pups which were born from Apc{Delta}474 heterozygous pairs (Table IGo). All of the Apc{Delta}474 mice started to develop tumors mainly in the small intestine and some of them developed them in the colon, stomach, duodenum and mammary gland at 8 weeks of age and died within 6 months due to severe anemia. The number of tumors in the small intestine was variable at an early generation, apparently due to heterogeneity in genetic background. However, after seventh generation backcrosses with C57BL/6 (N7), the number of polyps became stable (122.0 ± 9.1 at 12 weeks; Table IIGo). Macroscopically, the tumors in the small intestine appeared sessile shaped with a central depression (Figure 5AGo). Large tumors sometimes caused stenosis of the gut and hemorrhage. Microscopic examination showed hyperproliferation of intestinal glands and mild cellular and structural abnormalities (Figure 5BGo).


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Table I. Genotypes of offspring derived from heterozygous pairs
 

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Table II. Number of intestinal tumors in APC{Delta}474 mice
 


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Fig. 5. Macroscopic and microscopic findings concerning intestinal and mammary tumors. (A) Multiple sessile-type polyps. (B) Low-power field microscopic finding of an intestinal tumor showing hyperplasia with atypical morphology of epithelial cells. (C) Mammary tumor found in a 3-month-old Apc{Delta}474 mouse. An ulcer is visible at the center of the tumor. (D) Low-power field observation of a mammary tumor.

 
Some of the Apc{Delta}474 mice developed a characteristic mammary tumor between 3 and 5 months of age (Table IIIGo). The tumors were well demarcated from the surrounding normal tissue and developed to a size of ~2 cm in diameter. Cross-sections of the surface of the tumor were whitish and coarse. When they grew large, they had an ulcer in the central portion due to necrosis (Figure 5CGo). Microscopic observations of the tumors revealed adenoacanthomas. The tumor had a characteristic onion-skin-like structure and cancer-pearl-like keratinization, which are commonly observed in squamous cell carcinomas. No invasive cells were observed in the surrounding tissue (Figure 5DGo).


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Table III. Frequency of mammary tumors in APC{Delta}474 mice
 
Suppression of polyp formation by JTE-522
The tumor-suppressive effects of a novel COX-2-selective inhibitor, JTE-522 were tested as follows. Apc{Delta}474 mice were prepared by artificial insemination using one male N6 Apc{Delta}474 mouse. All of the mice used in the experiment were born on the same day. When the mice reached the age of 3 weeks (body weight 12–15 g), they were weaned and three groups (A–C) with eight mice in each were established. Due to the early weaning, we lost one mouse in group A and two mice in group C soon after the experiment was started. Because of the limited number of mutant mice obtained by artificial insemination, and because we found no significant difference in the number of polyps between females and males, we combined the data on males and females. All three groups were placed on a powdered diet for 1 week. Then JTE-522 was added to the diet of groups B and C (to give doses of 20 and 2 mg/kg body wt/day, respectively). Later, the actual doses were calculated to be 20.42 and 2.36 mg/kg body wt/day, respectively. Group A was a control. The 20 and 2 mg/kg/day doses of JTE-522 used in this experiment were based on the results of a prior study (7) in which rats with yeast-induced hyperalgesia was treated with JTE-522. In that study, JTE-522 showed a dose-dependent anti-nociceptive effect over a dose range of 3–30 mg/kg, p.o. The mice were kept on the three diets for an additional 8 weeks and then killed. Group A (control) had 123.3 ± 9.3 (mean ± SEM) intestinal polyps/mouse. Groups B and C had 83.8 ± 12.3 (P < 0.05) and 111.6 ± 6.7 polyps/mouse, respectively (Figure 6Go). The numbers of polyps in different size groups are shown in Figure 7Go. It can be seen that the number of polyps in each size group decreased in a dose-dependent manner. At these drug concentrations, JTE-522 had no significant effects on the body weight, food intake or general condition of the Apc{Delta}474 mice (Table IVGo).



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Fig. 6. Inhibitory effect of JTE-522 on the number of polyps in Apc{Delta}474mice. The mean numbers of total polyps are indicated with SEM. *P < 0.05 versus control, by Dunnett's two-tailed test. n = 6–8.

 


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Fig. 7. Size distribution of polyps found in Apc{Delta}474 mice that were treated or not treated with JTE-522. The mean number of total polyps are indicated with SEM. *P < 0.05 versus control, by Dunnett's two-tailed test. n = 6–8.

 

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Table IV. Food intake, body weight and the number of polyps of each group of mouse
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Based on epidemiological data, FAPs are classified into three types: profuse, sparse and attenuated, depending on the number of polyps (more than 5000, 1000–5000 and less than 1000, respectively). Accumulating data have suggested that the mutations found in attenuated FAP families are clustered (i) at the 5' end spanning exons 4 and 5, (ii) within exon 9 and (iii) at the 3' distal end of the Apc gene (10). On the other hand, the mutations found in the profuse type are segregated between codons 1250 and 1464 (11). It has been proposed that the variation in severity was due to variable dominant-negative activities of variable truncated Apc products, since Apc proteins are known to form homodimers (12,13). However, there are still arguments in support of the dominant-negative theory because a minigene of the mutant Apc in mouse did not cause the Apc phenotype (14). We still do not know the exact mechanism by which different types of truncated Apc proteins cause the variation in severity.

In the mouse model of FAP, there are five Apc knockout strains (including ours): Apc1638N (15), Apc1309 (16), ApcMin (2), Apc{Delta}716 (17) and Apc{Delta}474 (this report). These five strains have mutated Apc alleles encoding truncated Apc proteins of 1638, 1309, 850, 716 and 474 amino acids, respectively. The number of polyps in Apc{Delta}474 (approximately 100 at 16 weeks) was similar to the numbers in ApcMin (850 amino acids) and Apc1309 (1309 amino acids). Apc{Delta}716 (716 amino acids) had a much greater number of polyps (300–400), while Apc1638N (1638 amino acids) had much fewer polyps (approximately 10). The relationship between the severity and the position of the mutation in human FAP is not similar to the relationship found in the five knockout mouse strains. This may be due to a difference in protein structure among species or a difference in the stability of the different truncated Apc proteins, which interact with wild-type Apc protein in a dominant-negative way.

The incidence of mammary tumors in Apc{Delta}474 (18.5%) is a bit higher than that in Min mouse (<10%). This may be due to the longer life span of Apc{Delta}474 (~6 months) than Min mouse (~4 months). Moser et al. (18) reported that mammary tumors in Min mouse contained areas of adenocarcinoma and adenoacanthoma. We found a similar pathology in the mammary tumor of Apc{Delta}474 although the areas of adenoacanthoma were much greater than the areas of adenocarcinoma. Since adenoacanthoma is rare in human breast cancer and a germline mutation of Apc in a human does not necessarily cause breast cancer, the mammary carcinogenesis found in Apc mutant mice may reflect a mechanism that is different from that of human breast cancer.

There is evidence that colorectal carcinoma tissues from both human patients and rodent models contain elevated levels of COX-2, the inducible isozyme of COX (19,20). Oshima et al. (6) reported the induction of the COX-2 gene in interstitial cells in the intestine of Apc{Delta}716 mice at the early stage of polypogenesis, and obtained a dramatic reduction in the number of polyps by crossing these mice with COX-2 gene knockout mice or by administrating a COX-2 inhibitor, MF tricyclic. Other selective inhibitors of COX-2, such as nimesulide (21,22), celecoxib (23,24) and NS-398 (25,26) were reported to have significant chemopreventive effects in carcinogenesis. A comparison of the number of polyps in different size groups in the JTE-522-treated mice and the control mice (Figure 7Go) revealed a reduction in the number of polyps in all size groups in a dose-dependent manner. However, it is difficult to compare tumor suppressive effect of JTE-522, MF-tricyclic and nimesulide, because they were tested in different protocols and in different Apc mutant strains. JTE-522 has a strong, highly selective inhibitory activity against human COX-2, as compared with other COX-2 inhibitors (8,9). Therefore, we expect that JTE-522 would have a tumor-suppressing effect in humans (human trials have not yet been conducted).

The molecular mechanism by which COX-2 inhibition reduces intestinal neoplasm in Apc knockout mice is unclear. Tsujii and DuBois (27) expressed the COX-2 gene in rat intestinal epithelial cells and showed that these cells had an increased tumorigenic potential and an ability to escape from apoptosis. The signaling route from the COX-2 gene product to apoptosis is not known. Chan et al. (28) suggested that the apoptosis in colon cancer cell lines caused by NSAIDs was due to the accumulation of arachidonic acid, which is involved in the conversion of sphingomyelin to ceramide, a known mediator of apoptosis. This hypothesis is consistent with the fact that secretory type II phospholipase A2 (sPLA2: a modifier of Apc) reduced the number of polyps in Apc knockout mice (29), assuming that sPLA2 increased the arachidonic acid pool.

Giardiello et al. (30) reported that NSAIDs reduce the mortality from colorectal cancer and reduce the number and size of the colonic polyps in human. Although NSAIDs have COX-1 and -2 inhibitory activity, only COX-2 inhibition is likely to be critical for the suppression of neoplasma. Because COX-2 specific inhibitors, including JTE-522, avoid the side effects of COX-1 inhibition, such as gastrointestinal ulceration, renal toxicity and bleeding, these inhibitors show promise for the chemoprevention of colon carcinogenesis.


    Acknowledgments
 
We thank the following individuals for help, suggestions and discussions: M.Kumai, K.Ishii, S.Yokoyama, I.Suganuma, K.Iwata, H.Iwamura, Y.Nishi, A.Kushi and H.Iwatsuka.


    Notes
 
2 To whom correspondence should be addressed Email: hitoshi.sasai{at}ims.jti.co.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received August 2, 1999; revised August 2, 1999; accepted February 4, 2000.