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Notes: To obtain monoclonal antibodies reactive with odontogenic but not other types of epithelium, mice were immunized wish honiogenates of fixed ameloblastoma tissues, and monoclonal antibodies Y4 and Mil were produced. Y4 reacted immunohistochemically with odontogenic epithelial components but not with those of squamous differentiation, while M11 reacted with odontogenic epithelial components and a part of keratotic epithelial tissues. Immunoglobulin isotypes of both antibodies were IgM as determined by Ouchterlonv immunodiffusion and enzyme-linked immunosorbent assays. Western blotting revealed that the antigen recognized by Y4 had a molecular mass of approximately 66 kDa; however, the antigen reactive with M11 was not identified by Western blotting in spile of various attempts in changing reaction conditions. These antibodies may be beneficial to histological analyses of odontogenic tissues and their related lesions.

Notes: Background:  To investigate the roles of the apoptosis signaling pathway mediated by mitochondria in oncogenesis and cytodifferentiation of odontogenic tumors, expression of pathway signaling molecules was analyzed in ameloblastomas as well as in tooth germs.Methods:  Tissue specimens of 12 tooth germs, 41 benign ameloblastomas, and five malignant ameloblastomas were examined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry to determine the expression of cytochrome c, apoptotic protease-activating factor-1 (APAF-1), caspase-9, and apoptosis-inducing factor (AIF).Results:  The mRNA expression of APAF-1, caspase-9, and AIF was detected in all samples of normal and neoplastic odontogenic tissues. Immunohistochemical reactivity for cytochrome c, APAF-1, caspase-9, and AIF was detected in both normal and neoplastic odontogenic tissues. Expression of cytochrome c and AIF was evident in odontogenic epithelial cells neighboring the basement membrane, and APAF-1 and caspase-9 were detected in most odontogenic epithelial cells. Immunoreactivity for cytochrome c in tooth germs was slightly weaker than that in benign and malignant ameloblastomas. Keratinizing cells in acanthomatous ameloblastomas and granular cells in granular cell ameloblastomas showed a decrease or loss of immunoreactivity for these mitochondria-mediated apoptosis signaling molecules. Expression of AIF was obviously low in ameloblastic carcinomas.Conclusion:  Expression of cytochrome c, APAF-1, caspase-9, and AIF in tooth germs and ameloblastomas suggests that the mitochondria-mediated apoptotic pathway has a role in apoptotic cell death of normal and neoplastic odontogenic epithelium. Expression of these mitochondrial apoptosis signaling molecules might be involved in oncogenesis, cytodifferentiation, and malignant transformation of odontogenic epithelium.

Notes: Background:  Alterations of human patched (ptc) homolog have been proven to be responsible for basal cell nevus syndrome (BCNS). Mandibular cysts in heterozygous ptc knockout mouse (ptc+/− mouse) were microradiologically, histologically, immunohistochemically, and genetically examined to investigate the possible role of the ptc gene and its associates in the jaw cysts.Methods:  The mandibular bones were prepared from 63 ptc+/− mice and 6 ptc+/+ mice. Soft X-ray radiographs and histological sections were examined for detection of the presence of mandibular cysts. The mandibular cysts were immunohistochemically investigated using anti-ptc, shh, and smo antibodies. PCR analysis of loss of heterozygosity (LOH) of ptc was performed in genomic DNA from the mandibular cysts.Results:  Six ptc+/+ mice showed no pathologic change in any examinations. Microradiologically, ptc+/− mice did not show any apparent lesion. Mandibular cysts were often multiple, and were histologically detected in the alveolar bones or periodontal ligaments of the molars in 16 (25.4%) ptc+/− mice. The mandibular cysts were lined by thin parakeratotic stratified squamous epithelium and contained keratinized materials. Immunohistochemical examination showed sonic hedgehog (shh) protein mainly in cyst lining epithelium, and ptc and smoothened (smo) proteins in cyst lining epithelium, and surrounding fibrous connective tissue. Expression of ptc protein in the cyst lining epithelium tended to be weak as compared with incisor enamel organs and gingival stratified squamous epithelium. LOH of the ptc gene couldn't be found in lining epithelium of mandibular cysts in any ptc+/− mice.Conclusions:  Ptc +/− mouse is a useful model of BCNS from the standpoint of occurrence of jaw cysts, and downregulation of ptc protein in cyst lining epithelium caused by gene targeting would be associated with formation of jaw cysts in ptc+/− mice.

Notes: Background:   Tumors derived from odontogenic epithelium exhibit considerable variation and are classified into several benign and malignant entities. To clarify the role of growth factors in oncogenesis, cytodifferentiation and progression of epithelial odontogenic tumors, expression of hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β) and their receptors were analyzed in these tumors as well as in tooth germs.Methods:   Specimens of five tooth germs, 34 ameloblastomas, three calcifying epithelial odontogenic tumors (CEOTs), two clear cell odontogenic tumors (CCOTs), five adenomatoid odontogenic tumors (AOTs), six calcifying odontogenic cysts (COCs) and six malignant ameloblastomas were examined immunohistochemically with the use of antibodies against HGF, TGF-β and their receptors.Results:   In tooth germs and epithelial odontogenic tumors, immunoreactivity for HGF and TGF-β was detected in both epithelial and mesenchymal cells, while expression of their receptors was found only in epithelial cells. In tooth germs and main types of ameloblastomas, HGF and TGF-β reactivity was marked in epithelial cells near the basement membrane, and their receptors were diffusely positive in most epithelial cells. In subtypes of ameloblastomas, reduced expression of HGF, c-Met and TGF-β and increased reactivity for TGF-β receptors were detected in keratinizing cells in acanthomatous ameloblastomas, and granular cells in granular cell ameloblastomas demonstrated little or no expression of HGF, TGF-β or their receptors. As compared with main types of ameloblastomas, basal cell ameloblastomas showed high HGF reactivity, and desmoplastic ameloblastomas exhibited elevated reactivity for TGF-β and its receptors. Neoplastic cells in CEOTs, AOTs and COCs showed reactivity for HGF, TGF-β and their receptors. Elevated HGF and TGF-β reactivity was found in pseudoglandular cells in AOTs, and high expression of their receptors was noted in ghost cells in COCs. Metastasizing ameloblastomas showed similar expression patterns of HGF, TGF-β and their receptors to those of benign ameloblastomas, while CCOTs and ameloblastic carcinomas had increased HGF expression and low reactivity for TGF-β and its receptors as compared with benign ameloblastomas.Conclusions:  Immunohistochemical localization of HGF, TGF-β and their receptors in tooth germs and epithelial odontogenic tumors supports the hypothesis that HGF and TGF-β act on epithelial cells via paracrine and autocrine mechanisms. Altered expression of the agents in these epithelial odontogenic tumors, especially subtypes of ameloblastomas, AOTs and COCs, suggests that HGF and TGF-β signaling might affect differentiation of neoplastic odontogenic epithelial cells. Activated HGF/c-Met pathway and reduced TGF-β signaling in CCOTs and ameloblastic carcinomas may be associated with the malignant potential of these epithelial odontogenic tumors.

Notes: Background:  The mechanisms responsible for activation and proliferation of lining epithelium involved in inflammatory processes in periapical inflammatory lesions remain unclear. In this study, the expression and distribution of inducible nitric oxide synthase (iNOS) and heat shock proteins (HSPs) were immunohistochemically investigated in periapical inflammatory lesions.Methods:  Control specimens of periodontal ligaments including Malassez epithelial rests from seven teeth and periapical inflammatory lesions (15 apical granulomas (AGs), 16 radicular cysts (RCs), and 10 residual radicular cysts (RRCs)) were prepared and examined by the standard streptavidin–biotin peroxidase complex method using anti-iNOS rabbit polyclonal antiserum, and anti-HSP27, -HSP60, -HSP70 mouse monoclonal antibodies.Results:  Immunoreactivity for iNOS was detected in macrophages, lymphocytes, and endothelial cells of granulation tissue and in lining epithelium of periapical inflammatory lesions. Malassez epithelial rests showed no or slight staining for iNOS. The epithelial staining intensity of iNOS in RCs was greater than that in Malassez epithelial rests and RRCs. Immunoreactivity for HSP27 was recognized in inflammatory cells, endothelial cells and lining epithelium of periapical inflammatory lesions and in Malassez epithelial rests. HSP60 was detected in some lymphocytes of granulation tissue and in lining epithelium of periapical inflammatory lesions, whereas Malassez epithelial rests showed no staining for HSP60. Epithelial HSP60 reactivity was more intense in RCs than in RRCs. HSP70 was expressed in lymphocytes, endothelial cells and lining epithelium of periapical inflammatory lesions and in Malassez epithelial rests. The staining intensity of HSP70 in Malassez epithelial rests was slightly lower than that in lining epithelium of RCs and RRCs.Conclusions:  These data demonstrate that the expressions of iNOS, HSP60, and HSP70 are involved in inflammatory processes and might play a role in the activation and proliferation of lining epithelium, leading to progression of periapical inflammatory lesions.

Notes: Background:  To clarify the possible role of nitric oxide (NO) and stress proteins in oncogenesis and cytodifferentiation of odontogenic epithelium. Inducible NO synthase (iNOS) and heat shock proteins (HSPs) were analyzed in ameloblastomas as well as in tooth germs.Methods:  Specimens of seven tooth germs, 36 benign ameloblastomas and five malignant ameloblastomas were examined by immunohistochemistry using antibodies against iNOS and 27-, 60- and 70-kDa HSPs (HSP27, HSP60 and HSP70).Results:  Immunoreactivity for iNOS was detected in normal and neoplastic odontogenic epithelial cells and was higher in malignant ameloblastomas than in tooth germs and benign ameloblastomas. HSP27 was expressed constitutively in all odontogenic epithelial cells in tooth germs and benign and malignant ameloblastomas. Expression of HSP60 and HSP70 was detected in normal and neoplastic odontogenic epithelial cells and was prominent in cells neighboring the basement membrane. HSP60 reactivity showed no apparent difference between normal and neoplastic odontogenic epithelium, whereas HSP70 expression was slightly higher in benign and malignant ameloblastomas than in tooth germs.Conclusions:  Activation of iNOS might be associated with malignant potential of epithelial odontogenic tumors. Elevated expression of HSP70 is considered to be involved in neoplastic transformation of odontogenic epithelial cells.

Notes: Background:  Expression of vascular endothelial growth factor (VEGF), a major angiogenic factor, and microvessel density (MVD), assessed by the use of anti-CD34 antibody, were immunohistochemically examined in benign and malignant ameloblastomas, as well as tooth germs, to clarify the possible role of angiogenesis in epithelial odontogenic tumors.Methods:  Specimens of 5 tooth germs, 35 benign ameloblastomas and 5 malignant ameloblastomas were examined by immunohistochemistry using anti-VEGF and CD34 monoclonal antibodies.Results:  Immunoreactivity for VEGF was detected in both normal and neoplastic odontogenic epithelial cells, and weakly in microvessels near odontogenic epithelial cells, suggesting that this angiogenic factor acts on endothelial cells via a paracrine mechanism in odontogenic tissues. Both benign and malignant ameloblastomas showed elevated VEGF expression as compared to tooth germs. VEGF expression was low in keratinizing cells in acanthomatous ameloblastomas and granular cells in granular cell ameloblastomas, and acanthomatous ameloblastomas showed the lowest VEGF reactivity among the subtypes of ameloblastomas. MVD in both benign and malignant ameloblastomas was higher than that in tooth germs, indicating increased demands for blood in the neoplastic tissues. CD34-positive microvessels in follicular ameloblastomas were numerous and small, whereas those in plexiform ameloblastomas were scattered and dilated. MVD tended to depend on VEGF expression levels in both benign and malignant ameloblastomas.Conclusions:  VEGF was considered to be an important mediator of angiogenesis in these epithelial odontogenic tumors, and up-regulation of VEGF might be associated with neoplastic or malignant changes of odontogenic epithelial cells.

Notes: Abstract: We examined the immunohistochemical expressions of cell-cycle- and apoptosis-related factors to investigate the possible role of these factors in odontogenic keratocyst (OKC). Expression of cyclin D1 and p16 protein was detected in the basal and parabasal cells in lining epithelium of OKCs and was found more frequently in basal cell nevus syndrome (BCNS)-associated OKCs than in primary or recurrent OKCs. Positivity for p21 protein was detected in basal to superficial cells, whereas that for p27 protein was detected in parabasal to superficial cells in lining epithelium of OKCs. DNA topoisomerase IIα reacted with nuclei in basal and parabasal cells of the lining epithelium of OKCs, and positive cells were observed in BCNS-associated OKCs significantly more frequently than in primary or recurrent OKCs. Expression of Fas in suprabasal to superficial cells and expression of Fas-L in basal and parabasal cells were detected in lining epithelium of OKCs. Immunoreactivity for caspase-3 was detected in basal to suprabasal or superficial cells in lining epithelium of OKCs. Single stranded (ss)DNA-positive nuclei were detected in superficial cells in lining epithelium of OKCs. Fas was more broadly distributed in BCNS-associated OKCs than in primary OKCs, and ssDNA-positive cells were observed in BCNS-associated OKCs significantly more frequently than in primary or recurrent OKCs. These results suggest that BCNS-associated OKCs might be a distinguishable entity from solitary OKCs.

Notes: Abstract: To determine whether cell cycle regulation or alteration plays a role in oncogenesis and cytodifferentiation of odontogenic epithelium, cell cycle-related factors, including cyclin D1, p16INK4a, p21WAF1/Cip1 and p27Kip1 proteins, DNA topoisomerase IIα and histone H3 mRNA, were examined in 8 tooth germs and 31 ameloblastomas. Cyclin D1 was expressed in epithelial cells near the basement membrane in tooth germs and ameloblastomas, suggesting that this protein participates in cell proliferation in odontogenic epithelium. Immunoreactivity for p16 protein was observed in most epithelial cells in tooth germs and ameloblastomas. Expression of p21 protein was detected in most epithelial cells in tooth germs and ameloblastomas, but not in keratinizing or granular cells in variants of ameloblastomas. Expression of p27 protein was chiefly found in central polyhedral cells and keratinizing cells in tooth germs and ameloblastomas. These cyclin-dependent kinase inhibitors were well preserved in ameloblastomas as compared with tooth germs, suggesting that the odontogenic epithelium is strictly regulated by these factors. The cell cycle phase/cellular proliferation markers, DNA topoisomerase IIα and histone H3 mRNA, were localized in scattered epithelial cells attached to the basement membrane in tooth germs and ameloblastomas.

Notes: Abstract: Telomerase activity is believed to be crucial for cell immortalization and cancerization, and is proven to be induced by c-myc protein. Telomerase reverse transcriptase (TERT) has been recently identified as a catalytic subunit of telomerase, whose expression is closely correlated with telomerase activity. We estimated telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and examined the immunohistochemical expression of TERT and c-myc protein in 21 ameloblastoma tissues. All ameloblastoma samples were positive for telomerase activity, and TERT expression was detected in the nuclei of neoplastic cells but not in those of stromal cells. Numerous peripheral columnar or cuboidal cells, sporadic central polyhedral cells and some granular cells in ameloblastomas reacted with anti-TERT antibody. These results suggest that telomerase activity is associated with the oncogenesis or proliferative potential of odontogenic epithelium. The expression of c-myc protein showed a similar distribution pattern to that of TERT, suggesting that c-myc protein might induce telomerase activity in ameloblastomas.