Mucin Depleted Foci (MDF) Have B-catenin Gene Mutations, Altered Expression of its Protein and Are Dose- and Time–Dependent in the Colon of 1,2-Dimethylhydrazine (DMH)–Treated Rats.

Department of Pharmacology, University of Florence, Firenze, Italy.
International Journal of Cancer (Impact Factor: 5.09). 08/2005; 116(1):9-15. DOI: 10.1002/ijc.20981
Source: PubMed


Mucin-depleted foci (MDF) are purported preneoplastic lesions that can be easily visualized in the unsectioned colon of carcinogen-treated rats stained with high-iron diamine alcian blue (HID-AB). In F344 rats treated twice with 150 mg/kg of 1,2-dimethylhydrazine (DMH) and sacrificed after 5, 9, 13 and 28 weeks, MDF increased over time from 5 to 13 weeks, whereas they decreased at 28 weeks, when tumors appear. MDF multiplicity (crypts/MDF) linearly increased with time. Increasing doses of DMH (100, 150 and 200 mg/kg x 2 times) caused a dose-related increase in MDF. Mutations in Ctnnb1 gene codifying for beta-catenin were identified with PCR amplification and direct sequencing in 6/15 tumors (40%), 7/28 MDF (25%) and 2/27 (7%) aberrant crypt foci (ACF) identified in HID-AB-stained colon. All mutations in tumors and MDF caused amino acid substitution, while one mutation in ACF was silent. Beta-catenin detected at membrane level by immunohistochemistry was markedly reduced in MDF and tumors and, to a lesser extent, in ACF identified with HID-AB. By contrast, nuclear localization of beta-catenin was significantly increased in MDF and tumors, while no variation was observed in ACF. Beta-catenin cytoplasmic expression was also significantly increased in MDF and tumors but to a lesser extent in ACF. In conclusion, MDF are induced dose-dependently by DMH, increase in size with time, have mutations in the beta-catenin gene and marked alterations in beta-catenin cellular localization. Since all these phenomena are considered specific steps for colon tumorigenesis, these results further support the hypothesis that MDF are cancer precursors and can be proposed as endpoints in short-term carcinogenesis experiments.

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Available from: Benedetta Bendinelli, Nov 13, 2014
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    • "The rats developed approximately 4 and 8 MDF/colon at 7 and 15 weeks, respectively, after the start of the experiment, while 271–289 ACF/colon occurred during the same period. Mutations in β-catenin, Apc, and K-ras genes and cytoplasmic β-catenin expression were found in MDF induced by DMH46,47,48. Among these, β-catenin gene mutations included 32G→A (Asp→Asn), 37C→T (Ser→Phe), 33C→T (Ser→Phe), and 41C→T (Thr→Ile). "
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    ABSTRACT: The animal model is a powerful and fundamental tool in the field of biochemical research including toxicology, carcinogenesis, cancer therapeutics and prevention. In the carcinogenesis animal model system, numerous examples of preneoplastic lesions have been isolated and investigated from various perspectives. This may indicate that several options of endpoints to evaluate carcinogenesis effect or therapeutic outcome are presently available; however, classification of preneoplastic lesions has become complicated. For instance, these lesions include aberrant crypt foci (ACF), dysplastic ACF, flat ACF, β-catenin accumulated crypts, and mucin-depleted foci. These lesions have been induced by commonly used chemical carcinogens such as azoxymethane (AOM), 1,2-dimethylhydrazine (DMH), methylnitrosourea (MUN), or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Investigators can choose any procedures or methods to examine colonic preneoplastic lesions according to their interests and the objectives of their experiments. Based on topographical, histopathological, and biological features of colon cancer preneoplastic lesions in the animal model, we summarize and discuss the character and implications of these lesions.
    Full-text · Article · Dec 2013 · Journal of Toxicologic Pathology
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    • "A group of animals (n=4) was sacrificed after 15 weeks from DMH to harvest the pre-neoplastic lesions MDF as described [8]. Another group of animals (n=9) continued the same dietary regimen until 24 weeks after DMH administration, a time at which macroscopic tumours (adenomas) were already developed [7,8]. At this time point, animals were divided into two experimental groups as follows: 1) rats (n=4) continued on the same experimental diet (controls); 2) rats (n=5) were administered a diet supplemented with celecoxib (Celebrex, Pfizer) at 250 ppm. "
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    ABSTRACT: Background Colon cancer stem cells may drive carcinogenesis and account for chemotherapeutic failure. Although many markers for these cells have been proposed, there is no complete agreement regarding them, nor has their presence in the early phases of carcinogenesis been characterized in depth. Methods The expression of the putative markers LGR-5 (leucine-rich-repeat-containing G-protein-coupled receptor 5), MSI-1 (Musashi-1) and DCAMKL-1 (doublecortin and calcium/calmodulin-dependent protein kinase-like-1) was studied in normal colon mucosa (NM), in the precancerous lesions Mucin Depleted Foci (MDF) and in macroscopic tumours (adenomas) of 1,2-dimethylhydrazine-treated rats. Co-localization between these markers and nuclear β-catenin (NBC), an attributed feature of cancer stem cells, was also determined. Moreover, since PGE2 could increase NBC, we tested whether short-term treatment with celecoxib, a COX-2 inhibitor (2 weeks, 250 ppm in the diet) could reduce the expression of these markers. Results LGR-5 expression in NM was low (Labelling Index (LI): 0.22±0.03 (means±SE)) with positive cells located mainly at the base of the crypts. Compared to NM, LGR-5 was overexpressed in MDF and tumours (LI: 4.7±2.0 and 2.9±1.0 in MDF and tumours, respectively, P<0.01 compared to NM). DCAMKL-1 positive cells, distributed along the length of normal crypts, were reduced in MDF and tumours. Nuclear expression of MSI-1, located mainly at the base of normal crypts, was not observed in MDF or tumours. In both MDF and tumours, few cells co-expressed LGR-5 and NBC (LI: 1.0±0.3 and 0.4±0.2 in MDF and tumours, respectively). Notwithstanding the lower expression of DCAMKL-1 in tumours, the percentage of cells co-expressing DCAMKL-1 and NBC was higher than in NM (LI: 0.5±0.1 and 0.04±0.02 in tumours and NM, respectively). MSI-1 and NBC co-localization was not observed. Celecoxib did not reduce cells co-expressing LGR-5 and NBC. Conclusions Based on its prevalent localization at the base of normal crypts, as expected for stem cells, and on the overexpression in precancerous lesions and tumours, we support LGR-5, but not MSI-1 or DCAMKL-1, as putative neoplastic stem cell marker. In both MDF and tumours, we identified LGR-5-positive cells co-expressing NBC which could be a subpopulation with the highest stem cell features.
    Full-text · Article · Feb 2013 · BMC Cancer
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    • "MDF were first discovered in the colon of carcinogen treated rats [21] and they have also been observed in patients with FAP and with sporadic CRC [22], [23]. MDF, devoid of mucin which is secreted by goblet cells, also exhibit preneoplastic features e.g., dysplasia [21], [23]–[25], mutations in β-catenin [26] and Apc [24] gene, over-expression of survivin [27], cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (i-NOS) and macrophages [28] and reduced expression of MUC2 (a mucin abundantly expressed in the normal colon) and intestinal trefoil factor, a marker of goblet cell lineage [25]. While the expression of p21 and p16 (inbibitors of cyclin-dependent kinases) have been found to be reduced in ACF as well as MDF [29]. "
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    ABSTRACT: Colon carcinogenesis is a multistep process and it emanates from a series of molecular and histopathological alterations. Glycyrrhizic acid (GA) is a natural and major pentacyclic triterpenoid glycoside of licorice roots extracts. It has several pharmacological and biological properties such as anti-inflammatory, anti-viral, and anti-cancer. In the present study, we investigated the chemopreventive potential of GA against 1,2-dimethyhydrazine (DMH)-induced precancerous lesions i.e., aberrant crypt foci (ACF) and mucin depleted foci (MDF), and its role in regulating the hyperproliferation, inflammation, angiogenesis and apoptosis in the colon of Wistar rats. Animals were divided into 5 groups. In group III, IV and V, GA was administered at the dose of 15 mg/kg b. wt. orally while in group II, III and IV, DMH was administered subcutaneously in the groin at the dose of 20 mg/kg b.wt once a week for first 5 weeks and animals were euthanized after 9 weeks. GA supplementation suppressed the development of precancerous lesions and it also reduced the infiltration of mast cells, suppressed the immunostaining of Ki-67, NF-kB-p65, COX-2, iNOS and VEGF while enhanced the immunostaining of p53, connexin-43, caspase-9 and cleaved caspase-3. GA treatment significantly attenuated the level of TNF-α and it also reduced the depletion of the mucous layer as well as attenuated the shifting of sialomucin to sulphomucin. Our findings suggest that GA has strong chemopreventive potential against DMH-induced colon carcinogenesis but further studies are warranted to elucidate the precise mechanism of action of GA.
    Full-text · Article · Jan 2013 · PLoS ONE
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