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,
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
Tumour and abnormality incidence and tumour spectrum
Histological tumour types
Incidence of tumours and/or abnormal lesions (% of animals)
Intestinal lesions (% of animals)
1 hepatocellular carcinoma
2 lymphomas (1 B-cell lymphoma)
1 periosis hepatis
1 lymphocyte infiltration
1 endometrial hyperplasia
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
2 hepatocellular carcinomas
4 small intestinal polyps
9 lymphomas (6 B-cell lymphomas)
2 endometrial hyperplasia
3 fused villi
2 lymphocyte infiltrations
1 swollen crypts
Number of animals.
P<0.05 compared to wild-type controls by chi-squared test.
One of these mice contained a small intestinal polyp and a fused villi. The other
mice contained only one lesion per mouse.
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
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)
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
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.