Triclosan inhibits sodium lauryl sulphate-induced changes in expression of cytokeratin genes in hamster cheek pouch epithelium

Triclosan is known to reduce the untoward side effects of sodium lauryl sulphate (SLS). The aim of the present study was to determine whether triclosan can inhibit SLS-induced changes in expression of cytokeratin (CK) genes in hamster cheek pouch epithelium. With a hybridohistochemical technique, using specific human cRNA probes, hamster CK mRNAs were identified by immunological detection of heterologous hybrids. In contrast to application of SLS-containing paste, application of paste containing SLS together with triclosan produced no marked changes in expression and distribution patterns of CK. mRNAs, compared to the normal cheek pouch epithelium. Therefore, we may accept that triclosan inhibits the effect of SLS on CK gene expression. However, the mechanism of this protection remains elusive. Conversely, the epithelial hyperplasia induced by application of SLS was histologically identical to that induced by application of SLS and triclosan. This suggests that the changes in CK. gene expression identified in the present study are not a simple consequence of epithelial hyperplasia. but rather are specific to the irritating agent. Establishment of the fact that SLS may influence gene expression, and that this may be prevented by triclosan, may be helpful in research on the cytological effects of SLS and the elucidation of protection mechanisms of triclosan against side effects of SLS.     

Differential expression of type I cytokeratins in hamster cheek pouch epithelium following treatment with dimethylbenzanthracene

Cytokeratin (CK) expression in untreated, paraffin-treated or dimethylbenzanthracene (DMBA)-treated hamster cheek pouch epithelium was investigated utilizing monoclonal antibodies AE1 or AE3, which react with type I or type II CKs, respectively, and by in situ hybridization utilizing type I CK-specific probes. The latter were isolated from a cDNA library of hamster cheek pouch mRNA and designated CK 13 and CK 10 based on their respective homologies (>95% amino acids) with murine CK 13 and human CK 10. Treatment of hamster cheek pouch epithelium with DMBA resulted in increased expression of type I CK, detected immunohistochemically with monoclonal AE1, but decreased expression of type II CKs detected with AE3. Despite an overall increase in type I CKs, in situ hybridization demonstrated differential expression of type I CKs with altered distribution of CK 13 mRNA and reduced expression of CK 10 mRNA, providing additional sensitive markers for DMBA-associated changes in CKs. These changes were constant at 2 to 22 weeks in the pre-neoplastic and neoplastic epithelium following the initial application of DMBA.     

In situ hybridization study of cytokeratin 4, 13, 16 and 19 mRNAs in human developing junctional epithelium

Cytokeratins (CKs) are now considered to be reliable markers for following the development and differentiation of epithelial tissue. We have investigated the pathway of differentiation in human developing junctional epithelium using monoclonal antibodies and two-dimensional gel electrophoresis of mcrodissected tissue to identify CK 19. CK 16. CK 14. CK 13. CK 6. CK 5. CK 4 in the junctional epithelium (JE) over partially erupted human teeth. The CK profile was similar to that of developing oral epithelia. suggesting that the junctional epithelium in teeth during eruption is of odontogenic origin. The present study used in situ hybridization to determine the distribution of the mRNAs of CKs 19. 16. 13 and 4 in human developing junctional epithelium and to examine the correlation between mRNAs and their encoded proteins. CK 19 mRNA was abundant in the basal cell layers of the primary junctional epithelium (PJE) but less concentrated in the suprabasal layers. CK16. 13 and 4 mRNAs were abundant in the basal cell layers of the PJE. Tlie parabasal cell layers reacted intensely to the cRNA probe complementary to CK16 mRNA. as were the reactions in the suprabasal cell layers of the PJE for the CK 13 and 4 probes. Our results demonstrate that the PJE express the genes encoding for CKs 16 and 4 that have been revealed previously only by electrophoresis. They therefore confirm that the PJE is a well-differentiated stratified epithelium with a complex unique phenotype that produces CKs specific for basal cells (CK 19), CKs associated with hyperproliferation (CK 16). and finally those associated with stratification (CKs 4 and 13). Only synthesis of CK 19 protein and mRNA are strictly parallel. CKs 4 and 13 mRNAs are present in basal and suprasal cells, while their encoded proteins were not. except for CK 13 in suprabasal cell layers of PJE. where the amount of its mRNAs was coincident with the expression of the protein.     

Immunolocalization and adenoviral vector-mediated manganese superoxide dismutase gene transfer to experimental oral tumors

The anti-oxidant enzyme system protects cellular macromolecules against damage from reactive oxygen species. One component of this system, manganese superoxide dismutase (MnSOD), has also been shown to display tumor suppressor gene-like activity. The purpose of this study was to examine changes in MnSOD expression during hamster cheek pouch carcinogenesis, and the effects of MnSOD overexpression using an adenoviral vector. Tumor induction was carried out using 7,12-dimethylbenz[alpha]anthracene. Animals were killed at periodic intervals, and cheek pouch tissues were excised and examined for MnSOD expression by immunohistochemistry and digital image analysis. We observed a reduction in MnSOD expression as early as 2 weeks after the start of carcinogen application. Low MnSOD expression persisted until the end of the 23-week experimental period. Solid hamster cheek pouch carcinoma xenografts were then established in nude mice. An adenoviral vector encoding the human MnSOD gene was delivered to the xenografts by direct injection. We observed high, immediate expression of MnSOD in the xenografts that persisted for 10 days following cessation of viral construct delivery. Delivery of the MnSOD construct resulted in a maximal 50% reduction in tumor growth compared with untreated controls. Our results suggest that MnSOD may be a tumor suppressor gene in the hamster cheek pouch model system.     

P73蛋白在金黄地鼠颊囊癌变过程中的表达及意义

目的 检测p73基因在口腔癌前病变和鳞状细胞癌发生发展中的表达，为明确p73在口腔癌前病变和鳞状细胞癌中的诊断和治疗价值提供理论依据。方法 采用免疫组化SABC法研究p73在60只地鼠颊囊黏膜癌前病变和鳞状细胞癌中的表达。结果 p73在口腔癌前病变和鳞状细胞癌中表达逐渐增加，证实p73参与了黏膜癌前病变和鳞状细胞癌的发生与发展，有可能用于肿瘤的基因治疗。结论 结果对口腔黏膜癌前病变和鳞状细胞癌的早期诊断、鉴别诊断、基因治疗提供了理论依据。     

Expression of bone sialoprotein in mineralized tissues of tooth and bone and in buccal-pouch carcinomas of Syrian golden hamsters

The expression of bone sialoprotein (BSP) is normally restricted to mineralized connective tissues of bones and teeth where it has been associated with mineral crystal formation. However, recent studies have revealed ectopic expression of BSP in various lesions, including oral and extraoral carcinomas, in which it has been associated with the formation of microcrystalline deposits and the metastasis of cancer cells to bone. To develop a model to study the induction of BSP in carcinoma development, BSP expression in squamous-cell carcinomas induced by chemical carcinogen in the hamster cheek-pouch epithelium was investigated. Hamster BSP cDNA was first isolated and characterized, then used to prepare probes for Northern and in situ hybridization. The protein sequence of hamster BSP displayed 86% amino acid identity with a consensus mammalian BSP sequence and retained polyglutamate sequences, the RGD sequence and sites of phosphorylation, glycosylation and sulphation. The tissue-specific expression of hamster BSP mRNA and protein was confirmed by in situ hybridization and immunolocalization in developing tissues. Squamous-cell carcinomas induced in the buccal pouches of 5-week-old male Syrian golden hamsters treated with chemical carcinogen had BSP mRNA and BSP in the proliferating neoplastic epithelium. In contrast, neither BSP mRNA nor the protein could be detected in the stroma within which islands of the transformed tissue had formed. Thus, the hamster cheek pouch is a well-characterized model that can be used to study the induced expression of BSP in association with the development of squamous-cell carcinomas.     

The effect of DMBA on hamster cheek pouch mucosa in vitro: constant exposure

Repeated topical application of DMBA to hamster cheek pouch mucosa in vivo has been shown to cause dysplastic changes and ultimately the development of squamous cell carcinomas in the epithelium. A technique has already been reported whereby neonatal hamster pouch mucosa can be maintained in vitro and dysplastic changes have been described in the epithelium after a single exposure to DMBA. By 35 days in organ culture, these changes disappeared and the epithelium regained its normal organotypic morphology. In this study, the tissue was maintained in vitro in medium containing various concentrations of DMBA for up to 49 days. Dysplastic changes were seen in the epithelium of the explants with some histological evidence of malignant behaviour. These changes were observed throughout the experimental period.     

P27、P16和pRb 蛋白在金黄地鼠颊囊癌变过程中的表达及意义

目的：检测pRb、p27和p16在地鼠颊囊黏膜癌前病变和鳞癌中的表达。方法：用60只金黄地鼠建立了颊囊粘膜癌前病变的动物模型，用免疫组化SABC法研究了p27、pRb和p16的表达。结果：p27、pRb和p16在口腔癌前病变和鳞癌中均有表达的异常。结论：p27、pRb和p16参与了黏膜癌前病变和鳞癌的发生与发展，它们的异常表达有助于口腔粘膜癌前病变和鳞癌的早期诊断，为研究癌前病变和鳞癌的基因治疗也提供了一定的理论依据。     

The effect of vitamin A on hamster cheek pouch mucosa in organ culture.

Vitamin A was added to the medium of neonatal hamster cheek pouch mucosa maintained in organ culture. The epithelium of the treated explants showed a reduction in keratinization and down-growth into the underlying connective tissue. The cytological and morphological changes in the epithelium were consistent with glandular metaplasia.     

Cytokeratins in hamster cheek pouch epithelium during DMBA-induced carcinogenesis

The pattern of keratin expression in hamster cheek pouch epithelium during 15-wk of DMBA-induced carcinogenesis was studied. The sequential changes in cytokeratins of premalignant and malignant tissues and comparative investigation of normal epithelial tissues were examined during a weekly sequential DMBA-induced chemical carcinogenesis. Keratin polypeptides of normal pouch epithelium appear in a molecular weight range of 43-67 kd and 5-6 proteins can be identified. The disappearance of high molecular weight keratin (61-67 kd) was observed from the 6-wk DMBA-treated premalignant group to the 15-wk DMBA-treated malignant group. An additional keratin polypeptide was noted initially on the 11th-wk-DMBA-treated group and remained to the 15th-wk-DMBA treated group.     

Influence of partial excision of carcinogen treated hamster cheek pouch on subsequent tumour development

Hamster cheek pouch was treated with carcinogen for 5 or 8 weeks. Part of the carcinogen treated area was excised and the lesion allowed to heal by secondary intention. At follow-up the biopsied pouches were compared with control pouches and potentiation of the development of carcinomas was seen. It is suggested that these results have relevance to the previously demonstrated tumour potentiating effect of cryosurgery in hamster cheek pouch, in that the important tumour potentiating influence may be regeneration involving dysplastic epithelium and not a function of cryosurgery per se.     

AgNOR mark epithelial foci in malignant transformation in hamster cheek pouch carcinogenesis

Hamster cheek pouch mucosa is an accepted model of oral carcinogenesis. We herein examined the value of morphometric evaluation of silver-stained nucleolar organizer regions (AgNOR) in the detection of epithelial foci in malignant transformation following dimethyl-1, 2-benzanthracene-induced carcinogenesis of hamster cheek pouch. AgNOR-related parameters were analyzed at different stages of the process of carcinogenesis (control epithelium, epithelium with no unusual microscopic features, "dysplastic" epithelium, exophytic and endophytic carcinomas). Morphometric evaluation of AgNOR revealed incipient cellular alterations which were not evident in routine preparations and contributed to the characterization of different stages of carcinogenesis in this model.     

Surface ultrastructural changes in chemically induced premalignant lesions in the hamster cheek pouch

The histopathological and topographical alterations occurring during the early stages of chemical carcinogenesis in the hamster cheek pouch using 7, 12-dimethylbenz (α) anthracene (DMBA) were studied by light and scanning electron microscopy. At the scanning electron microscopic level, cellular overlapping and mild disturbance of intercellular bridges were noticed as early as the second week of DMBA application; these changes progressed with time. Clear epithelial dysplastic changes at the light microscopic level were detected by the sixth week of the experiment. The results of this investigation demonstrate the usefulness of scanning electron microscopy as an adjunct tool to study early alterations in cell morphology which occur in the stratified squamous epithelium of the hamster cheek pounch in response to a chemical carcinogen.     

Surface ultrastructural changes in chemically induced premalignant lesions in the hamster cheek pouch

The histopathological and topographical alterations occurring during the early stages of chemical carcinogenesis in the hamster cheek pouch using 7,12-dimethylbenz (alpha) anthracene (DMBA) were studied by light and scanning electron microscopy. At the scanning electron microscopic level, cellular overlapping and mild disturbance of intercellular bridges were noticed as early as the second week of DMBA application; these changes progressed with time. Clear epithelial dysplastic changes at the light microscopic level were detected by the sixth week of the experiment. The results of this investigation demonstrate the usefulness of scanning electron microscopy as an adjunct tool to study early alterations in cell morphology which occur in the stratified squamous epithelium of the hamster cheek pounch in response to a chemical carcinogen.     

Enamel epithelium expresses bone sialoprotein (BSP)

Bone sialoprotein (BSP) is a major non-collagenous extracellular matrix protein in bone and other mineralized connective tissues. BSP is synthesized and secreted by bone-, dentin- and cementum-forming cells. In this study we hypothesized that BSP may be also involved in enamel formation through its postulated role in matrix mineralization. In situ hybridization with cRNA probes for rat and hamster BSP, respectively, showed strong mRNA signals in ameloblasts actively synthesizing enamel matrix in developing incisors. However, no hybridization signals were observed at an earlier developmental stage when bell-shaped molar tooth germs were being formed. Immunohistochemical analysis of tooth tissues from transgenic mice harboring a 2.7 kb rat BSP promoter ligated to a luciferase reporter gene revealed strong staining for luciferase in the enamel epithelium of the developing tooth germ. Interestingly, BSP expression was also observed in epithelial cells of an ameloblastoma. The neoplastic epithelial nests and cords demonstrated strong mRNA signals to the human BSP probe while the connective tissue stroma showed only a background level of silver grains. Immunostaining also showed deposition of BSP by the odontogenic cells of the tumor. These results demonstrate that BSP is expressed by the enamel-forming epithelium of developing teeth, suggesting a possible role for BSP in enamel formation and its subsequent mineralization. Expression of the BSP gene in ameloblastomas is consistent with the expression of BSP by the enamel epithelium and also with the expression of BSP by neoplastic tissues, suggesting a possible role in tumorigenesis.     

The effect of DMBA on hamster cheek pouch mucosa in vitro

Neonatal hamster cheek pouch mucosa was treated with DMBA for three h either before being explanted or after 7 days in vitro. The epithelium of the treated cultures exhibited dysplasic changes, most marked at 21 to 28 days in vitro. These changes were not seen in the explants maintained for 35 to 49 days in vitro indicating a return to an apparently normal morphology. Examination of the ultrastructure of DMBA treated and untreated explants showed changes consistent with those described in similar tissues in vivo. The epithelium of the carcinogen treated tissue exhibited widened intercellular spaces, tortuous cell membranes, an irregular and discontinuous basal lamina and pseudopodia of the basal cells extending into the underlying mesenchyme. Similar changes have been described in inflamed epithelium, in some other experimental preparations, and in premalignant lesions in vitro, both in animals and man.     

Mitochondrial volume densities in the smokeless tobacco-treated hamster cheek pouch epithelium

OBJECTIVES: To compare the morphological changes and quantitative distribution of mitochondria in the hamster cheek pouch (HCP) epithelium treated with smokeless tobacco (ST). MATERIALS AND METHODS: Archives of experimental material from previously published studies (Ashrafi et al., 1992) were utilized. Animals in experimental group received moist ST (snuff) in their right pouch, 5 days weekly for 24 months, while no snuff was given to control group. After 24 months, the epithelial tissues were processed for electron microscopy study. Volume densities of mitochondria were assessed by morphometry. MAIN OUTCOME MEASURES: Mitochondrial volume densities in the two groups, experimental vs control. RESULTS: In both control and experimental groups mitochondria were concentrated between the nucleus and basal cell plasma membrane. A decrease in the mean mitochondrial volume density (Vvmit) was observed from the basal layer to the more superficial layers in both groups. The experimental HCP displayed more mitochondria than control, and the granular epithelial cell layer in experimental group showed significantly a higher mean Vvmit than the control group (P = 0.03). It was concluded that greater numbers of mitochondria were retained in ST-treated granular cells of the hyperplastic epithelia than in the normal epithelium.     

诱导型一氧化氮合酶抑制剂对地鼠颊囊癌变的干预作用

目的研究一氧化氮(NO)在肿瘤发生发展过程中所起的作用,探讨一氧化氮合酶(NOS)抑制剂对地鼠颊囊粘膜癌变所起的干预作用。方法90只金黄地鼠分为实验1组(TI组)、实验2组(T2组)和空白对照组(C组),利用二甲基苯并葱(DMBA)诱导T1,T2组地鼠颊囊癌变,并在T2组给予一氧化氮合酶抑制剂I,.硝基精氨酸甲醋( L, NAME),观察T1和T2组颊囊豁膜癌变中病理改变,SABC免疫组化法检测iNOS,VEGF,珊因子动态表达,硝酸还原酶法测定NO量的变化。结果】)MBA诱导T1组和72组颊囊薪膜癌变率的差异有统计学意义,P < O.OS,iNOS, VEGF阳性表达增加,NO量和微血管密度不断增加。结论NO在地鼠颊囊癌的发生发展中起促进作用,而G NAME起干预作用。     

Early detection of alterations associated to oral cancer

The model of hamster cheek pouch carcinogenesis closely mimics the development of human oral cancer. The study of the interaction between chemical carcinogens and radiation in the process of oral carcinogenesis is of interest given that the oral cavity is frequently exposed to chemical carcinogens such as alcohol and tobacco and is the route of entry of therapeutic radiation. In this context, markers of incipient alterations associated to a process of malignant transformation would contribute to early diagnosis and follow-up. The aim of the present study was to assess the early changes produced by carcinogenic agents applied separately or combined in a two-stage carcinogenesis protocol in hamster cheek pouch. The cheek pouch of the hamsters was treated with a single dose of radiation (20 Gy) or 7,12-dimethylbenz(a)anthracene (DMBA) as initiating agents and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoting agent for 1 or 2 weeks. The end-points chosen to identify early alterations were hyperplastic foci and silver-stained nucleolar organizer regions (Ag NOR). The data show that both markers are useful in the detection of early alterations compatible with a process of malignant transformation.     

Sequential cytogenetic alterations in hamster oral keratinocytes during DMBA-induced oral carcinogenesis

Using the hamster cheek pouch oral cancer model, we have performed a comprehensive analysis of the cytogenetic changes in hamster oral keratinocytes during 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis. Tumour induction in the hamster cheek pouch required repeated application of the carcinogen for 14 weeks. We have found that this hamster oral cancer model to be suitable for cytogenetic studies. Unlike human oral cancers where chromosome breaks have been shown, this is only infrequently observed in DMBA-treated hamster oral keratinocytes. Of importance is the finding that at the beginning of the second week of DMBA treatment, there is a significant increase of karyotypes demonstrating tetraploid or near-tetraploidy. We propose that the significant increase in hamster oral keratinocytes exhibiting tetraploidy be further evaluated as a marker of premalignancy/malignancy.