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Background To evaluate roles of human epidermal growth factor receptor (HER) family molecules in ameloblastomas, protein expression and gene status were analyzed in odontogenic tissues. Methods Sixty five ameloblastomas, 10 dental follicles, and 11 dentigerous cysts were immunohistochemically examined with antibodies against epidermal growth factor receptor (EGFR) and HER2, HER3, and HER4. Amplification of EGFR and HER2 was evaluated by chromogenic in situ hybridization (CISH). In 18 ameloblastomas, EGFR exons 19 and 21 were analyzed by direct DNA sequencing. Results Immunohistochemical reactivity for EGFR and HER2, HER3, and HER4 was detected in odontogenic epithelium. Expression of EGFR and HER4 was remarkable in these odontogenic tissues, as compared with that of HER2 and HER3. The level of HER2 immunoreactivity was significantly lower in ameloblastomas than in dental follicles and dentigerous cysts. Follicular ameloblastomas showed significantly higher expression of HER2 and HER4 than plexiform ameloblastomas. Reactivity for EGFR and HER3 was slightly stronger in recurrent ameloblastomas than in primary ameloblastomas. CISH did not reveal obvious amplification of EGFR or HER2 in ameloblastomas; however, EGFR and HER2 gene signals were significantly higher in follicular ameloblastomas than in plexiform ameloblastomas. Direct DNA sequencing of EGFR did not show any gene alteration in ameloblastomas. Conclusion Expression of HER family molecules, especially EGFR and HER4, in odontogenic tissues suggests that growth signals mediated by these receptor molecules contribute to cell proliferation, survival, and differentiation in both normal and neoplastic odontogenic epithelial tissues. Some of these molecules might be useful for predicting outcomes in patients with ameloblastomas.

J Oral Pathol Med (2010) 39: 87–93 Background: To investigate the roles of stem cell-related molecules in oncogenesis and cytodifferentiation of odontogenic tumors, expression of CD133, Bmi-1, and ATP-binding cassette subfamily G member 2 (ABCG2) was analyzed in ameloblastic tumors as well as in tooth germs. Methods: Tissue specimens of 12 tooth germs, 47 ameloblastomas, and six malignant ameloblastic tumors were examined using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry to determine the expression of CD133, Bmi-1, and ABCG2. Results: mRNA expression of CD133, Bmi-1, and ABCG2 was detected in all samples of normal and neoplastic odontogenic tissues. Immunohistochemical reactivity for CD133 and Bmi-1 was evident in odontogenic epithelial cells neighboring the basement membrane in tooth germs, ameloblastomas, and metastasizing ameloblastomas, and ameloblastic carcinomas and clear cell odontogenic carcinomas showed reactivity for CD133 and Bmi-1 in most neoplastic cells. The level of CD133 immunoreactivity in malignant ameloblastic tumors was significantly higher than the levels in tooth germs and ameloblastomas. Immunoreactivity for ABCG2 in odontogenic epithelial components was detected in some ameloblastic tumors but not in tooth germ tissues. Some granular neoplastic cells in granular cell ameloblastomas showed ABCG2 expression. The level of ABCG2 immunoreactivity in malignant ameloblastic tumors was significantly higher than that in tooth germs. Conclusion: Expression of CD133, Bmi-1, and ABCG2 in tooth germs and ameloblastic tumors suggests that stem cell-related molecules might control the maintenance of odontogenic tissues. Expression of these molecules is possibly involved in oncogenesis, cell differentiation, and malignant potential of odontogenic epithelium.

Background: To evaluate the roles of Notch signaling in the oncogenesis and cytodifferentiation of odontogenic tumors, expression of Notch receptors and ligands was analyzed in ameloblastomas as well as in tooth germs. Methods: Tissue specimens of nine tooth germs and 32 ameloblastomas were examined by reverse transcriptase polymerase chain reaction and by in situ hybridization to determine the expression of Notch1, Notch2, Notch3, Delta1, and Jagged1. Results: mRNA expression of Notch1, Notch2, Notch3, Delta1, and Jagged1 was detected in all samples of normal and neoplastic odontogenic tissues. In tooth germs, Notch receptors were expressed in odontogenic epithelium (except for inner enamel epithelium), and expression of Notch ligands was lower in inner enamel epithelium than in other epithelial components. Odontogenic mesenchymal components were weakly reactive with these Notch signaling molecules. Ameloblastomas showed expression of Notch receptors and ligands in central polyhedral neoplastic cells. Notch2, Delta1, and Jagged1 were expressed in some neoplastic cells neighboring the basement membrane. Expression of Notch receptors and ligands was not found in keratinizing cells or granular cells in ameloblastoma variants. Stromal cells were weakly reactive with these Notch signaling molecules. Conclusion: Expression of Notch receptors and ligands in tooth germs and ameloblastomas suggests that Notch signaling might control cell differentiation and proliferation of normal and neoplastic odontogenic epithelium.

BACKGROUND: To evaluate the roles of extracellular matrix (ECM)-degrading serine proteinase in progression of odontogenic tumors, expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and maspin was analyzed in ameloblastic tumors as well as in tooth germs. METHODS: Tissue specimens of 10 tooth germs, 45 ameloblastomas, and 5 malignant ameloblastic tumors were examined immunohistochemically with the use of antibodies against uPA, uPAR, PAI-1, and maspin. RESULTS: Immunohistochemical reactivity for uPA, uPAR, PAI-1, and maspin was detected in normal and neoplastic odontogenic tissues: uPA was recognized predominantly in mesenchymal cells, uPAR was evident in epithelial cells, PAI-1 was found in both epithelial and mesenchymal cells, and maspin was expressed only in epithelial cells. The levels of uPA and uPAR immunoreactivity in ameloblastic tumors were slightly higher than the levels in tooth germs, while PAI-1 reactivity in ameloblastomas tended to be lower than that in tooth germs. The level of maspin immunoreactivity in ameloblastomas was significantly higher than that in tooth germs, and ameloblastic carcinoma showed decreased maspin reactivity. CONCLUSION: Expression of uPA, uPAR, PAI-1, and maspin in tooth germs and ameloblastic tumors suggests that interactions among these molecules contribute to ECM degradation and cell migration during tooth development and tumor progression. Altered expression of the serine proteinase and its associated molecules in ameloblastic tumors may be involved in oncogenesis of odontogenic epithelium.

BACKGROUND: To evaluate roles of mitogen-activated protein kinases (MAPKs) in oncogenesis and cytodifferentiation of odontogenic tumors, expression of phosphorylated JNK (p-JNK), p38 MAPK (p-p38 MAPK), and ERK5 (p-ERK5) was analyzed in ameloblastic tumors as well as in tooth germs. METHODS: Ten tooth germs, 47 ameloblastomas, and 5 malignant ameloblastic tumors were examined immunohistochemically with the antibodies against p-JNK, p-p38 MAPK, and p-ERK5. RESULTS: Immunoreactivity for p-JNK was detected in epithelial or neoplastic cells detached from the basement membrane in 7 tooth germs and 7 ameloblastomas, and the expression levels of p-JNK in ameloblastic tumors were significantly lower than that in tooth germs. Expression of p-p38 MAPK was found in epithelial or neoplastic cells in tooth germs and ameloblastic tumors except for two ameloblastomas, and increased expression was found in keratinizing cells of acanthomatous ameloblastomas. The expression level of p-p38 MAPK in ameloblastomas was significantly higher than the levels in tooth germs and malignant ameloblastic tumors. Immunoreactivity for p-ERK5 was found predominantly in epithelial or neoplastic cells near the basement membrane in tooth germs and ameloblastic tumors. The expression levels of p-ERK5 in ameloblastic tumors were slightly higher than that in tooth germs, and plexiform ameloblastomas showed significantly higher p-ERK5 expression than follicular ameloblastomas. CONCLUSION: Expression of p-JNK, p-p38 MAPK, and p-ERK5 in tooth germs and ameloblastic tumors suggests that these MAPK signaling pathways contribute to cell proliferation, differentiation, or apoptosis in both normal and neoplastic odontogenic tissues. Altered expression of these phosphorylated MAPKs in ameloblastic tumors may be involved in oncogenesis and tumor cell differentiation.

Background: To evaluate the roles of growth factors in oncogenesis and cytodifferentiation of odontogenic tumors, expression of insulin-like growth factors (IGFs), platelet-derived growth factor (PDGF), and their receptors was analyzed in ameloblastic tumors as well as in tooth germs. Methods: Tissue specimens of 10 tooth germs, 47 ameloblastomas, and five malignant ameloblastic tumors were examined immunohistochemically with the use of antibodies against IGF-I, IGF-II, IGF-I receptor (IGF-IR), PDGF A-chain, PDGF B-chain, PDGF α-receptor, and PDGF β-receptor. Results: Immunohistochemical reactivity for IGFs, PDGF chains, and their receptors was detected predominantly in odontogenic epithelial cells near the basement membrane in tooth germs and in benign and malignant ameloblastic tumors. The expression levels of IGF-II and PDGF chains were significantly higher in ameloblastic tumors than in tooth germs. Malignant ameloblastic tumors showed higher reactivity for PDGF chains than benign ameloblastomas and higher reactivity for platelet-derived growth factor receptors than tooth germs. The expression levels of PDGF chains were significantly higher in follicular ameloblastomas than in plexiform ameloblastomas. Desmoplastic ameloblastomas showed higher expression of IGFs and IGF-IR when compared with other ameloblastoma subtypes. Conclusion: Expression of IGFs, PDGF, and their receptors in tooth germs and ameloblastic tumors suggests that these growth factor signals contribute to cell proliferation or survival in both normal and neoplastic odontogenic tissues. Expression of these molecules in odontogenic tissues possibly affects interactions with the bone microenvironment during tooth development and intraosseous progression of ameloblastic tumors. Altered expression of the ligands and receptors in ameloblastic tumors may be involved in oncogenesis, malignant potential, and tumor cell differentiation.

Background: To clarify the roles of rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK) signaling pathway in oncogenesis and cytodifferentiation of odontogenic tumors, K-Ras gene status and expression of Ras, Raf1, MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)1, and ERK1/2 proteins were analyzed in ameloblastomas as well as in tooth germs. Methods: Paraffin sections of 10 tooth germs and 46 benign and 6 malignant ameloblastomas were examined immunohistochemically for the expression of K-Ras, Raf1, MEK1, and ERK1/2. Frozen tissue samples of 22 benign ameloblastomas and 1 malignant (metastasizing) ameloblastoma were analyzed by direct DNA sequencing to detect K-Ras gene alteration. Results: Immunohistochemical reactivity for K-Ras, Raf1, MEK1, and ERK1/2 was detected in both normal and neoplastic odontogenic epithelium, and these molecules were reactive chiefly with odontogenic epithelial cells neighboring the basement membrane. Plexiform ameloblastomas showed slightly stronger expression of these Ras/MAPK signaling molecules than follicular ameloblastomas. Keratinizing cells and granular cells showed decreased reactivity for the signaling molecules. Basal cell ameloblastomas showed slightly stronger reactivity for the signaling molecules than did the other subtypes. K-Ras immunoreactivity in malignant ameloblastomas was lower than that in dental lamina of tooth germs. Direct DNA sequencing showed a GGT to GCT point mutation at codon 12 of K-Ras gene in one ameloblastoma. Conclusion: Expression of K-Ras, Raf1, MEK1, and ERK1/2 in tooth germs and ameloblastomas suggests that Ras/MAPK signaling pathway functions to regulate cell proliferation and differentiation in both normal and neoplastic odontogenic epithelium. K-Ras gene status implied that K-Ras mutations might play a minor role in oncogenesis of odontogenic epithelium.

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.

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-beta (TGF-beta) 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-beta and their receptors. Results In tooth germs and epithelial odontogenic tumors, immunoreactivity for HGF and TGF-beta 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-beta 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-beta and increased reactivity for TGF-beta receptors were detected in keratinizing cells in acanthomatous ameloblastomas, and granular cells in granular cell ameloblastomas demonstrated little or no expression of HGF, TGF-beta 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-beta and its receptors. Neoplastic cells in CEOTs, AOTs and COCs showed reactivity for HGF, TGF-beta and their receptors. Elevated HGF and TGF-beta 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-beta and their receptors to those of benign ameloblastomas, while CCOTs and ameloblastic carcinomas had increased HGF expression and low reactivity for TGF-beta and its receptors as compared with benign ameloblastomas. Conclusions Immunohistochemical localization of HGF, TGF-beta and their receptors in tooth germs and epithelial odontogenic tumors supports the hypothesis that HGF and TGF-beta 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-beta signaling might affect differentiation of neoplastic odontogenic epithelial cells. Activated HGF/c-Met pathway and reduced TGF-beta signaling in CCOTs and ameloblastic carcinomas may be associated with the malignant potential of these epithelial odontogenic tumors.

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.

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.

Method and Results: Calcifying odontogenic cysts (COCs) were examined histopathologically and immunohistochemically to characterize the histological and cytological properties of these lesions. Histopathologically, COCs showed thin or thick lining epithelium with ghost cells. COCs were classified according to proliferative type or nonproliferative type lining epithelium, the presence or absence of ameloblastomatous appearance, and the presence or absence of odontoma in the cyst walls. Immunohistochemically, amelogenin protein was expressed chiefly in ghost cells, whereas cytokeratin 19 (CK19) and bcl-2 proteins were expressed chiefly in lining epithelial cells. The proportion of cases positive for bcl-2 protein was slightly higher in COCs with odontoma than in those without odontoma. Lining epithelial cells sporadically showed positive reactions for Ki-67 antigen. Mean Ki-67 labeling index was slightly greater in COCs with proliferative type lining epithelium, COCs with ameloblastomatous appearance of the cyst walls, and COCs with odontoma of the cyst walls than in COCs without these histological features. Our results suggest that ghost cells or lining epithelial cells show ameloblastic cytodifferentiation or odontogenic epithelial characteristics, that bcl-2 protein is associated with survival of lining epithelial cells in COCs, and that high proliferation potential is associated with ameloblastomatous proliferation or combined odontoma. COCs exhibited various histological features with several transitional forms, and immunohistochemical examinations revealed little or no difference in cytodifferentiation and cellular activity among COCs. Conclusion: We conclude that COCs with various histological features have neoplastic potential and may not be separate entities within the same histological spectrum.

Background: To clarify the possible role of apoptotic cell death in oncogenesis and cytodifferentiation of odontogenic epithelium, apoptosis-related factors – Fas, Fas ligand (FasL), caspase-3 and single-stranded DNA (ssDNA) – were analyzed in ameloblastomas as well as in tooth germs. Methods: Specimens of 5 tooth germs, 29 benign ameloblastomas and 5 malignant ameloblastomas were examined by immunohistochemistry using anti-Fas, FasL, caspase-3 and ssDNA polyclonal antibodies. Results: Immunoreactivity for Fas and FasL was detected in normal and neoplastic odontogenic epithelial cells. Fas expression in ameloblastomas was slightly lower than that in tooth germs, whereas FasL expression was similar in tooth germs and ameloblastomas. Malignant ameloblastomas showed downregulation of Fas expression and upregulation of FasL expression, as compared with benign ameloblastomas, indicating escape from cell death attack by immune cells. Immunoreactivity for caspase-3 was detected chiefly in cells neighboring the basement membrane in tooth germs and ameloblastomas. Expression of caspase-3 and Fas tended to be low in basal cell ameloblastomas and high in desmoplastic ameloblastomas, as compared with other variants of ameloblastomas. Caspase-3 expression was more intense in malignant ameloblastomas than in tooth germs and benign ameloblastomas. Apoptotic bodies reactive with anti-ssDNA antibody were detected in normal and neoplastic odontogenic epithelial cells detached from the basement membrane. Keratinizing cells in acanthomatous ameloblastomas and granular cells in granular cell ameloblastomas showed increased numbers of apoptotic bodies and increased expression of Fas and caspase-3, as compared with other neoplastic cells. Apoptotic reactions in malignant ameloblastomas were less frequent than in benign ameloblastomas, indicating abnormal regulation of cell turnover in odontogenic epithelial cells. Conclusion: These apoptosis-related factors were detected in various patterns in normal and neoplastic odontogenic epithelium, suggesting that these factors might be associated with oncogenesis and cytodifferentiation of epithelial odontogenic tumors.

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 IIalpha 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.

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, p21(WAF1/Cip1) and p27Kip1 proteins, DNA topoisomerase IIalpha 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 IIalpha and histone H3 mRNA, were localized in scattered epithelial cells attached to the basement membrane in tooth germs and ameloblastomas.

Apoptotic cell death in granular cell ameloblastomas was examined by immunohistochemistry using anti-single-stranded DNA (ssDNA) antibody and transmission electron microscopy. Routinely prepared sections of granular cell ameloblastomas showed various quantities of granular cells with some apoptotic nuclear fragments. Immunoreactivity for ssDNA was higher in granular cells than in other neoplastic cells. Ultrastructural examination revealed abundant lysosomes in the cytoplasm of granular cells. Numerous apoptotic cell fragments with condensed nuclei in granular cell clusters were phagocytosed by adjacent granular cells. On immunohistochemical characterization of cellular differentiation, granular cells were positive for cytokeratin, CD68, lysozyme and alpha-1-antichymotrypsin, but negative for vimentin, desmin, S-100 protein, neuron-specific enolase and CD15, indicating epithelial origin and lysosomal aggregation. These features suggest that the cytoplasmic granularity in granular cell ameloblastomas might be caused by increased apoptotic cell death of neoplastic cells and associated phagocytosis by neighboring neoplastic cells.

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.

A case of clear-cell odontogenic tumor with unusual histological features is presented. A 61-year-old Japanese man was admitted because of swelling of the left premolar-molar region of the mandible. Radiological examination revealed a multilocular radiolucency with irregular margins. Histological examination of the resected specimen showed infiltrative proliferation of both clear and eosinophilic cells into the adjacent soft tissue without encapsulation, suggesting the malignant potential of the tumor. The tumor cells sporadically formed cystic lesions. In addition, several tumor cell nests showed duct-like characteristics, and many eosinophilic dentin-like structures were attached to the tumor cell nests, suggesting the potential for epithelial-mesenchymal induction. Histochemically, the clear tumor cells possessed cytoplasmic glycogen granules. Both clear and eosinophilic tumor cells showed positive immunoreactivities for cytokeratin 19, epithelial membrane antigen and filaggrin, indicating an odontogenic epithelial origin.