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      Development of spontaneous tumours and intestinal lesions in Fhit gene knockout mice

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          Abstract

          The fragile histidine triad (FHIT) gene has been identified as a candidate tumour suppressor gene localized in FRA3B, the most sensitive common fragile site, at chromosome 3p14.2 (Ohta et al, 1996). Chromosomal deletion of the FHIT-containing locus or inactivation of FHIT is frequently observed in various types of cancers (for a review, see Huebner and Croce, 2003), consistent with a tumour suppressor function in a variety of organs. The Fhit protein carries a proapoptotic activity through a caspase-dependent pathway in human cancer cells, which may contribute to the tumour suppressor activity (Ji et al, 1999; Ishii et al, 2001; Roz et al, 2002). Previously, we have demonstrated that Fhit mutant mice develop tumours spontaneously in lymphatic tissues, sebaceous glands, liver, stomach, colonic submucosa, uterus, skin, salivary glands, and parathyroid glands (Zanesi et al, 2001). Moreover, Fhit knockout mice are susceptible to chemical carcinogen-induced tumour formation in the forestomach (Fong et al, 2000), which is reversed by adenoviral transduction of the human FHIT gene (Dumon et al, 2001). These results are consistent with the tumour suppressor function of FHIT. To further characterise tissue types affected by inactivation of Fhit, including nontumorous lesions, we have constructed a second mouse strain with a targeted Fhit gene, and conducted a thorough pathological analysis. These Fhit mutant mice develop tumours in a variety of tissues, including intestinal adenomatous polyps, and show abnormal tissue building of intestinal mucosa, suggesting avenues for further study of Fhit function in normal tissues. MATERIALS AND METHODS All in vivo experiments were carried out with ethical committee approval and met the standards required by the UKCCCR guidelines (Workman et al, 1998). The mouse Fhit gene-targeting vector was constructed to replace exon 5, containing the translation initiation codon, with a pGK-Neo-bpA cassette. Homologous recombination in ES cells (RW4) was confirmed by genomic Southern hybridisation (data not shown). Germline transmitted F1 (+/−) mice were intercrossed to obtain Fhit (+/+), (+/−) and (−/−) progeny. Both males and females of each genotype from 12 to 20 months of age were used for thorough pathological examinations. Tissue samples were first examined under a dissection microscope. Then, all tissues were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 16 h at 4°C, embedded in paraffin wax, sectioned at 4 μm thickness and stained with H&E. For immunostaining, sections were preincubated with 3% BSA/10% goat serum in PBS for 2 h and incubated with anti-β-catenin antibody (Sigma Chemical Co., St Louis, MO, USA) at 5000-fold dilution or with c-Kit antibody (Santa Cruz, CA, USA) at 400-fold. Signals were visualised using the Vectastain Elite Kit (Vector Laboratories, Burlingame, CA, USA). For mutation analysis and microsatellite instability (MSI) analysis, DNA samples were extracted from paraffin-embedded sections using DEXPAT (TaKaRa, Japan). Extracted DNA samples were subjected to PCR in 11 contiguous fragments spanning nucleotides 2750–4830 of exon 15 of the Apc gene, where germline and somatic mutations were frequently detected. Amplified DNA samples were sequenced directly with the respective primers using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems; Rotkreuz, Switzerland) and ABI Prism 377 DNA Sequencing System (Applied Biosystems). For MSI analysis, four primer sets, D1Mit4, D6Mit59, D9Mit67 and D10Mit2 (http://www.informatics.jax.org), were used. DNA samples extracted from polyps were amplified by PCR. After denaturation (100°C, 5 min), PCR products were loaded onto 5% Long Ranger Gel (TaKaRa) in 8 M urea, and electrophoresed at 150 V for 40 min. The dried gel was scanned in a BAS-1800 (Fujifilm, Japan). RESULTS AND DISCUSSION Fhit (+/−) and (−/−) mice were viable, fertile and clinically normal up to 12 months of age. However, upon necropsy, tumours and abnormal lesions were found in mice older than 12 months, consistent with observations of another Fhit mutant strain (Fong et al, 2000). Although several types of tumours developed spontaneously even in the age-matched wild-type littermates, overall incidences of tumours and abnormal lesions were significantly higher in both Fhit (+/−) and (−/−) mice than in the wild-type littermates (60, 77 and 30% in Fhit (+/−), (−/−) and (+/+) mice, respectively; Table 1 Table 1 Tumour and abnormality incidence and tumour spectrum           Histological tumour types Genotype n a Age (months) Incidence of tumours and/or abnormal lesions (% of animals) Intestinal lesions (% of animals) Forestomach papilloma Lymphoid malignancies Other neoplasms Fhit (+/+) 26 12–20 8 (30%) 0 3 (12%) 3 (12%) 1 hepatocellular carcinoma             2 lymphomas (1 B-cell lymphoma) 1 periosis hepatis             1 lymphocyte infiltration 1 endometrial hyperplasia                                 Fhit (+/–) 90 12–20 54 (60%)b 19 (21%) 22 (24%) 23 (26%) 1 hepatocellular carcinoma         6 small intestinal polyps   18 lymphomas (16 B-cell lymphomas) 1 hepatocellular adenoma         5 fused villi   5 lymphocyte infiltrations 1 liver hemangioma         8 swollen crypts     2 endometrial hyperplasia               1 uterine leiomyoma               1 lung adenoma               1 Leydig cell tumour               1 dermoid cyst                 Fhit (–/–) 26 12–20 20 (77%)b 7c (27%) 6 (23%) 11 (42%) 2 hepatocellular carcinomas         4 small intestinal polyps   9 lymphomas (6 B-cell lymphomas) 2 endometrial hyperplasia         3 fused villi   2 lymphocyte infiltrations 1 GISTd         1 swollen crypts       a Number of animals. b P<0.05 compared to wild-type controls by chi-squared test. c One of these mice contained a small intestinal polyp and a fused villi. The other mice contained only one lesion per mouse. d Gastrointestinal stromal tumour. ). In both Fhit (+/−) and (−/−) mice, lymphoid malignancies including B-cell lymphoma and lymphocyte infiltrations, hemangioma and adenoma of the liver, and uterine leiomyoma were found, which is consistent with the previous report (Zanesi et al, 2001). Gastrointestinal stromal tumour (GIST) was described in the other Fhit mutant strain. Here, we have confirmed the diagnosis by immunohistochemistry for c-Kit protein (Figure 1A Figure 1 Histology of tumours and abnormal intestinal mucosa in Fhit-deficient mice. (A) Gastrointestinal stromal tumour in a female Fhit (−/−) mouse at 20 months of age. Inset, immunostaining with an anti-c-Kit antibody. (B) Leydig cell tumour in a male Fhit (+/−) mouse at 15 months. (C) Dissection micrograph of a duodenal polyp in a Fhit (−/−) mouse at 15 months. (D) Schematic distribution of the small intestinal polyps. Asterisks indicate relative locations of the polyps. (E, F) Immunostaining for β-catenin in polyps of a Fhit (+/−) mouse at 12 months, and Fhit (−/−) at 15 months, respectively. Note the nuclear accumulation of β-catenin (white arrowheads) in (E) and basolateral staining (asterisks) in (F). (G, J) Dissection micrographs of swollen crypts (ileum) in a Fhit (+/−) mouse at 20 months, and fused villi (duodenum) in a Fhit (+/−) at 20 months, respectively. (H, K) Normal ileal mucosa adjacent to the lesions shown in (I, L), respectively. (I, L) Histological sections of ileal swollen crypts in a Fhit (+/−) mouse at 20 months, and fused villi in a Fhit (+/−) at 20 months, respectively. Bars in: (A) 500 μm; (B) 100 μm; (C) 250 μm; (E, F) 50 μm; (G, J) 250 μm; (H, I, K, L) 200 μm. inset). In the present study, several additional types of tumours were detected, such as Leydig cell tumour, endometrial hyperplasia and hepatocellular carcinoma (Table 1). However, the last two tumours were observed also in Fhit (+/+) mice, and no increase was found in incidence in Fhit mutant mice for either tumour type. One Leydig cell tumour was observed in one of 90 Fhit (+/−) mice (Figure 1B), although the number is too small to draw a statistical link with Fhit deficiency, and none was found in (−/−) animals. Interestingly, some adenomatous polyps were found in the small intestine of Fhit mutant mice (Figure 1C, D), whereas these polyps have never been observed in wild-type littermates (Table 1). Although the incidence of polyp development in Fhit (−/−) was higher (15.4%) than that in Fhit (+/−) mice (6.7%), the polyp multiplicity was not different in the two genotypes. These results suggest that tumours develop in Fhit (+/−) mice because of Fhit haploinsufficiency, either directly or through an indirect mechanism. Mutations in the gene encoding Apc or β-catenin result in intestinal adenomatous polyposis through Wnt signalling activation (Oshima et al, 1995; Harada et al, 1999). To examine whether the Wnt pathway was activated in the intestinal polyps of Fhit-deficient mice, we localised β-catenin in nine polyp tissues from both Fhit (+/−) and Fhit (−/−) mice by immunohistochemistry. β-Catenin had accumulated in the nucleus in two polyps of Fhit (+/−) mice, indicating Wnt activation in these adenoma cells (Figure 1E). β-Catenin was localised to the basolateral side of adenoma cells in the other seven polyps (Figure 1F). By sequence analysis of the Apc gene of the two nuclear β-catenin-positive polyps, we further found a nonsense mutation in the Apc gene at codon 1055 (wt: GAA (Glu) → mutant: TAA (STOP)) in one polyp. This mutation may explain the cause of at least a fraction of the polyps, although other genes may be mutated giving rise to other adenomas. As the intestines were not inspected in detail in the other Fhit knockout strain, it is conceivable that similar intestinal lesions existed at low frequencies. These results suggest that an insufficient Fhit level can induce Apc gene mutations, which is consistent with the enhanced survival and mutation frequency of Fhit-deficient cells after UVC or mitomycin C damage (Ottey et al, 2004). In contrast, MSI was not observed in genomic DNAs from Fhit mutant mouse tails or tumours (Fong et al, 2000). In the present study, we also examined MSI in cells from two intestinal polyps, using four different sets of markers, and confirmed no MSI in these samples (data not shown). Accordingly, it is possible that inactivation of Fhit results in tumorigenesis through induction of mutations in tumour suppressor genes. On the other hand, morphological abnormalities in the small intestine, such as swollen crypts (goblet cell hyperplasia, Figure 1G, H) and fused villi (aggregated villi, Figure 1I, J), were observed in both Fhit (+/−) and (−/−) mice at similar incidences. Such lesions were not found in the age-matched wild-type mice. Histologically, these lesions consisted of differentiated epithelial cells without any signs of dysplasia. Thus, it is conceivable that Fhit expression is necessary also for the maintenance of normal intestinal architecture, in addition to suppressing tumorigenesis.

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          Most cited references 10

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          The FHIT gene, spanning the chromosome 3p14.2 fragile site and renal carcinoma-associated t(3;8) breakpoint, is abnormal in digestive tract cancers.

          A 200-300 kb region of chromosome 3p14.2, including the fragile site locus FRA3B, is homozygously deleted in multiple tumor-derived cell lines. Exon amplification from cosmids covering this deleted region allowed identification of the human FHIT gene, a member of ther histidine triad gene family, which encodes a protein with 69% similarity to an S. pombe enzyme, diadenosine 5', 5''' P1, P4-tetraphosphate asymmetrical hydrolase. The FHIT locus is composed of ten exons distributed over at least 500 kb, with three 5' untranslated exons centromeric to the renal carcinoma-associated 3p14.2 breakpoint, the remaining exons telomeric to this translocation breakpoint, and exon 5 within the homozygously deleted fragile region. Aberrant transcripts of the FHIT locus were found in approximately 50% of esophageal, stomach, and colon carcinomas.
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            Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene.

            Mutations in the APC (adenomatous polyposis coli) gene appear to be responsible for not only familial adenomatous polyposis but also many sporadic cases of gastrointestinal cancers. Using homologous recombination in mouse embryonic stem cells, we constructed mice that contained a mutant gene encoding a product truncated at a 716 (Apc delta 716). Mendelian transmission of the gene caused most homozygous mice to die in utero before day 8 of gestation. The heterozygotes developed multiple polyps throughout the intestinal tract, mostly in the small intestine. The earliest polyps arose multifocally during the third week after birth, and new polyps continued to appear thereafter. Surprisingly, every nascent polyp consisted of a microadenoma covered with a layer of the normal villous epithelium. These microadenomas originated from single crypts by forming abnormal outpockets into the inner (lacteal) side of the neighboring villi. We carefully dissected such microadenomas from nascent polyps by peeling off the normal epithelium and determined their genotype by PCR: all microadenomas had already lost the wild-type Apc allele, whereas the mutant allele remained unchanged. These results indicate that loss of heterozygosity followed by formation of intravillous microadenomas is responsible for polyposis in Apc delta 716 intestinal mucosa. It is therefore unlikely that the truncated product interacts directly with the wild-type protein and causes the microadenomas by a dominant negative mechanism.
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              • Abstract: found
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              Muir-Torre-like syndrome in Fhit-deficient mice.

              To investigate the role of the Fhit gene in carcinogen induction of neoplasia, we have inactivated one Fhit allele in mouse embryonic stem cells and produced (129/SvJ x C57BL/6J) F(1) mice with a Fhit allele inactivated (+/-). Fhit +/+ and +/- mice were treated intragastrically with nitrosomethylbenzylamine and observed for 10 wk posttreatment. A total of 25% of the +/+ mice developed adenoma or papilloma of the forestomach, whereas 100% of the +/- mice developed multiple tumors that were a mixture of adenomas, squamous papillomas, invasive carcinomas of the forestomach, as well as tumors of sebaceous glands. The visceral and sebaceous tumors, which lacked Fhit protein, were similar to those characteristic of Muir-Torre familial cancer syndrome.
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                Author and article information

                Journal
                Br J Cancer
                British Journal of Cancer
                Nature Publishing Group
                0007-0920
                1532-1827
                05 October 2004
                12 October 2004
                18 October 2004
                : 91
                : 8
                : 1571-1574
                Affiliations
                [1 ] 1Department of Pharmacology, Graduate School of Medicine, Kyoto University, Yoshida-Konoé-cho, Sakyo-ku, Kyoto 606-8501, Japan
                [2 ] 2Laboratory of Biomedical Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan
                [3 ] 3Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Yoshida-Konoé-cho, Sakyo-ku, Kyoto 606-8501, Japan
                [4 ] 4Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, PA 19107, USA
                Author notes
                [* ]Author for correspondence: taketo@ 123456mfour.med.kyoto-u.ac.jp
                Article
                6602182
                10.1038/sj.bjc.6602182
                2410018
                15467769
                Copyright 2004, Cancer Research UK
                Categories
                Short Communication

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