Neoplastic Odontogenic Tissues Research Articles

Comparison of calretinin expression in dentigerous cysts and ameloblastoma: An immunohistochemical study.

Background:Calretinin is a calcium-binding protein of 29-kilodalton (kDa), which is widely expressed in normal human tissues and tumorigenic tissues. Its expression in the odontogenic epithelium during odontogenesis and in neoplastic odontogenic tissues has been demonstrated. Unicystic ameloblastoma poses a diagnostic challenge, as its histologic presentation can be sometimes mistaken for cystic odontogenic lesions. In the present study, an attempt is made to overcome the confusion encountered in the diagnosis of dentigerous cyst and unicystic ameloblastoma, using the expression of calretinin in both lesions and to compare this expression with conventional ameloblastoma to accurately diagnose and differentiate these lesions.Materials and Methods:A total of eighty cases, in which twenty cases each of ameloblastoma, unicystic ameloblastoma, dentigerous cyst, and odontogenic keratocyst (OKC) were included in the study. Slides were made from the archival blocks of each case and were stained immunohistochemically with calretinin.Results:Correlation between calretinin staining and histopathological diagnosis was done, and it was found that all twenty cases of ameloblastoma showed positivity for calretinin, whereas 17 of twenty cases of unicystic ameloblastoma showed positivity for calretinin staining. All the cases of OKC and dentigerous cyst were negative for calretinin.Conclusion:Calretinin may serve as an important diagnostic adjunct in the differential diagnosis of ameloblastoma and cystic odontogenic lesions.

COMPARATIVE STUDY OF CALRETININ EXPRESSION IN ODONTOGENIC CYSTS AND TUMORS

Calretinin is a calcium-binding protein with molecular weight of 29 kDa (KiloDalton). It is expressed in a variety of normal and tumorigenic tissues. Its expression in odontogenic epithelium during odontogenesis and in neoplastic odontogenic tissues has been demonstrated. This study was undertaken to evaluate the expression of Calretinin in a group of odontogenic cysts and tumors. The expression of Calretinin was evaluated immunohistochemically in 42 samples including 8 cases of periapical cyst, 7 cases of dentigerous cyst, 7 cases of keratocystic odontogenic tumor, 8 cases of solid (multicystic) ameloblastoma, 6 cases of unicystic ameloblastoma and 6 cases of adenomatoid odontogenic tumor. Positive immunohistochemical reaction was found in all cases of solid ameloblastoma (100%), in 4 cases of unicystic ameloblastoma (67%) and 4 cases of keratocystic odontogenic tumors (57%) whereas none of the other odontogenic cysts and adenomatoid odontogenic tumors included in this study showed reactivity. Intensity was higher in the ameloblastomas compared with the keratocystic odontogenic tumors. On the basis of these results, it is suggested that calretinin might be used as a specific immunohistochemical marker for the ameloblastomas and could play an important role in the differentiation of aggressiveness of different odontogenic tumors.

Role of podoplanin expression in deciding the invasive potential of ameloblastoma – A retrospective IHC study

BackgroundThe aim of this study was to investigate the podoplanin expression in epithelial odontogenic tumors both non-aggressive and aggressive, tumors with and without ectomesenchyme and remnants of the odontogenic epithelium from dental follicles (DF) of unerupted teeth and to examine its role in progression and invasion of tumors. MethodThirty paraffin embedded specimens AM (15 were non-aggressive ameloblastomas and 15 ameloblastoma showing aggressive behavior), 15 paraffin embedded specimens of AOT, 15 paraffin embedded specimens of CEOT, 15 paraffin embedded specimens of CCOT were obtained and were analyzed by immunohistochemistry using anti-human podoplanin. Podoplanin expression in odontogenic epithelial cells was evaluated using a scoring method, along with calculation of the percentage of positive odontogenic cells. ResultsPodoplanin was expressed strongly at the invasive front (in the peripheral odontogenic epithelial cells) of most tumors and dental follicles. Membranous expression of podoplanin in ameloblastomas was stronger in cases of ameloblastomas showing aggressive behavior than (NA) non-aggressive ameloblastomas. ConclusionExpression of podoplanin at the invasive front (in peripheral cells) of odontogenic tumors considered to be associated with neoplastic odontogenic tissues. This molecule might play a role in progression and local invasion of odontogenic tumors. The migration and invasion mediated by podoplanin in odontogenic tumors could be related to cytoskeletal reorganization.

Calretinin as a diagnostic adjunct for ameloblastoma.

Background. Calretinin is a 29 kDa calcium-binding protein of the EF-hand family which is expressed in a variety of normal and tumorigenic tissues. Its expression in odontogenic epithelium during odontogenesis and in neoplastic odontogenic tissues has been demonstrated. Unicystic ameloblastoma poses a diagnostic challenge, as its histologic presentation can be sometimes mistaken for keratocystic odontogenic tumor (KCOT). This study was performed to assess the usefulness of calretinin as a confirmatory marker for ameloblastic tissue. Methodology. Total of 40 cases: 16 unicystic ameloblastoma, 4 multicystic ameloblastoma, and 20 KCOT, were evaluated immunohistochemically for the presence, localization, distribution, and intensity of calretinin expression. Statistical analysis was done using Chi-square test to intercompare the expression between ameloblastoma and KCOT. Results. Sixteen cases of ameloblastoma (12 unicystic, 4 multicystic) showed positive calretinin staining of ameloblastic epithelium and only one case of KCOT was positive for calretinin, with the positivity restricted to the stellate reticulum like epithelium. Intercomparison between two groups revealed statistically significant difference (P = 0.000). Conclusion. Calretinin appears to be a specific immunohistochemical marker for neoplastic ameloblastic epithelium and may be an important diagnostic adjunct in the differential diagnosis of ameloblastoma and KCOT.

Detection of Beclin1 and ATG5 in ameloblastic tumors

To investigate the roles of autophagy-related molecules in odontogenic tumors, we analyzed the expression of Beclin1 and ATG5 in tissue specimens of 11 tooth germs, 41 ameloblastomas, and 6 malignant ameloblastic tumors by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. RT-PCR and Western blot analyses confirmed expression of Beclin1 and ATG5 in all samples of normal and neoplastic odontogenic tissues. Immunohistochemical reactivity for Beclin1 was evident in odontogenic epithelial cells neighboring the basement membrane in tooth germs, ameloblastomas, and metastasizing ameloblastomas. ATG5 reactivity was found in most epithelial components of tooth germs, while ameloblastomas and metastasizing ameloblastomas showed ATG5 expression in many peripheral and some central neoplastic cells. Immunoreactivity for Beclin1 and ATG5 was evident in granular neoplastic cells in granular cell ameloblastomas, and ameloblastic carcinomas and clear cell odontogenic carcinomas showed Beclin1 and ATG5 expression in most neoplastic cells. These findings suggest that autophagic cell death might contribute to cell kinetics in odontogenic tissues. The molecular machinery for autophagy is possibly involved in oncogenesis and cell differentiation of odontogenic epithelium.

Calretinin Expression in Unicystic Ameloblastoma: An Aid in Differential Diagnosis

Calretinin is a 29 kDa calcium-binding protein, which is widely expressed in the central and peripheral neural tissue. It has also been demonstrated in odontogenic epithelium during odontogenesis and in neoplastic odontogenic tissues. The lining epithelium of eight cases of unicystic ameloblastoma, six cases of dentigerous cyst, six cases of odontogenic keratocyst, reclassified as keratocystic odontogenic tumor (KCOT), and four cases of solid/multicystic ameloblastoma was examined for the expression of calretinin. No positive staining was observed in any of the dentigerous cysts and keratocystic odontogenic tumor linings. In comparison, coarse dark brown staining was seen in the stellate reticulum of solid multicystic ameloblastoma and more superficial epithelial layers of unicystic ameloblastoma. In conclusion, we have highlighted calretinin to be a specific immunohistochemical marker for neoplastic ameloblastic tissue that can be used as an important diagnostic aid in the differential diagnosis of unicystic ameloblastoma and cystic odontogenic lesions.

Enhanced expression of podoplanin in ameloblastomas

Podoplanin, a mucin-type transmembrane glycoprotein, is specifically expressed by lymphatic but not blood vascular endothelial cells, and is also widely expressed in various specialized cell types throughout the body. Recent studies have demonstrated that it mediates a pathway leading to collective cell migration and invasion in vivo and in vitro. In the present study, we carried out an immunohistochemical investigation of podoplanin to clarify whether it is expressed in human ameloblastomas (AMs), which are characterized by locally aggressive behavior with a high rate of recurrence. In addition, we examined the localization of the epithelial marker E-cadherin and the mesenchymal marker vimentin to clarify whether AMs show epithelial-mesenchymal transition (EMT). Paraffin-embedded tissue specimens of 38 AMs were examined immunohistochemically using antibodies against podoplanin, E-cadherin, and vimentin. Immunohistochemical reactivity for podoplanin was detected in the cell membrane and cytoplasm of most odontogenic tumor epithelial cells in AMs. Podoplanin was expressed strongly in peripheral columnar cells and slightly in central stellate reticulum-like cells. E-cadherin was expressed in central stellate reticulum-like cells and showed decreased expression in peripheral columnar cells. Immunoreactivity for E-cadherin was weak or negative in keratinizing cells of acanthomatous AMs, suggesting terminal differentiation of the tumor cells. Immunohistochemical reactivity for vimentin was found in stromal cells, but partial or no reaction was observed in neoplastic cells. Expression of podoplanin in AMs is considered to be associated with neoplastic odontogenic tissues; this molecule might play a role in the collective cell migration of tumor nests in AMs. The pattern of expression of E-cadherin and vimentin suggests that invasion in AMs occurs in the absence of EMT. The migration and invasion mediated by podoplanin in AMs could be related to cytoskeletal reorganization.

Detection of CD133, Bmi-1, and ABCG2 in ameloblastic tumors

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. 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. 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. 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.

Immunohistochemical detection of BH3‐only proteins in ameloblastic tumors

To evaluate expression of BH3-only proteins in odontogenic tumors, expression of Bid, Bim, Bad, Noxa, and Puma was analyzed in ameloblastic tumors as well as in tooth germs. Nine tooth germs, 37 ameloblastomas, and five malignant ameloblastic tumors were examined immunohistochemically with antibodies against Bid, Bim, Bad, Noxa, and Puma. Immunohistochemical reactivity for Bid, Bim, Bad, Noxa, and Puma was detected in the cytoplasm of cellular components in normal and neoplastic odontogenic tissues. Expression of these BH3-only proteins was evident in odontogenic epithelial cells near the basement membrane in tooth germs and ameloblastic tumors. Acanthomatous ameloblastomas showed no reactivity for Bid, Bim, Bad, Noxa, or Puma in keratinizing cells, whereas granular cells in granular cell ameloblastomas reacted with these BH3-only proteins. Basal and desmoplastic ameloblastomas and ameloblastic carcinomas showed immunoreactivity for the BH3-only proteins in most neoplastic cells. Expression of Bid, Bim, Bad, Noxa, and Puma in tooth germs and ameloblastic tumors suggests that the BH3-only proteins have a role in apoptotic cell death of normal and neoplastic odontogenic epithelium. Distinctive expression patterns of these BH3-only proteins in ameloblastoma variants suggest that the BH3-only proteins might be involved in tumor cell differentiation of ameloblastomas.

Detection of Notch signaling molecules in ameloblastomas

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. 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. 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. 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.

Immunohistochemical detection of uPA, uPAR, PAI‐1, and maspin in ameloblastic tumors

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. 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. 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. 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.

Immunohistochemical detection of phosphorylated JNK, p38 MAPK, and ERK5 in ameloblastic tumors

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. 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. 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. 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.

Immunohistochemical detection of phosphorylated Akt, PI3K, and PTEN in ameloblastic tumors

To evaluate roles of the Akt signaling pathway in oncogenesis and cytodifferentiation of odontogenic tumors, expression of phosphorylated Akt (pAkt), PI3K, and PTEN was analyzed in ameloblastic tumors as well as in tooth germs. 11 tooth germs, 40 ameloblastomas, and 5 malignant ameloblastic tumors were examined immunohistochemically with antibodies against pAkt, PI3K, and PTEN. Immunoreactivity for pAkt, PI3K, and PTEN was detected predominantly in odontogenic epithelial cells near the basement membrane in tooth germs and ameloblastic tumors. The levels of immunoreactivity for pAkt and PI3K were slightly higher in ameloblastic tumors than in tooth germs. Plexiform ameloblastomas showed significantly higher expression of PI3K than follicular ameloblastomas, and PI3K immunoreactivity in ameloblastomas without cellular variation was significantly higher than that in acanthomatous ameloblastomas. The level of PTEN immunoreactivity was significantly lower in ameloblastomas than in tooth germs. Expression of pAkt, PI3K, and PTEN in tooth germs and ameloblastic tumors suggests that these signaling molecules regulate cell survival and growth in normal and neoplastic odontogenic tissues by mediating growth factor signals. Increased expression of pAkt and PI3K and decreased expression of PTEN in ameloblastic tumors may participate in oncogenesis of odontogenic epithelium by activating the Akt signaling pathway.

Immunohistochemical detection of insulin‐like growth factors, platelet‐derived growth factor, and their receptors in ameloblastic tumors

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. 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 alpha-receptor, and PDGF beta-receptor. 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. 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.

Expression of bone morphogenetic proteins and their associated molecules in ameloblastomas and adenomatoid odontogenic tumors

To further clarify the roles of regulators of embryonic development, bone morphogenetic protein (BMPs) and their associated molecules, in oncogenesis and cytodifferentiation of odontogenic tumors, the expression of these regulator molecules were analyzed in epithelial odontogenic tumors as well as in tooth germs. Tooth germs, ameloblastomas, adenomatoid odontogenic tumors, and malignant ameloblastomas were examined by RT-PCR and immunohistochemistry for detection of BMP-2, -4, -7, BMP receptors I and II (BMPR-I, BMPR-II), core-binding factor alpha1 (CBFA1), and osterix. mRNA expression of BMPs, BMPRs, CBFA1, and osterix was detected in all odontogenic tissues. Immunohistochemical reactivity for BMPs, BMPRs, and CBFA1 was detected in both epithelial and mesenchymal cells of tooth germs and epithelial odontogenic tumors. BMPs and BMPRs were evidently expressed in odontogenic epithelial cells in tooth germs and epithelial odontogenic tumors. Acanthomatous ameloblastomas showed increased BMP-7 reactivity in keratinizing cells. Nuclear CBFA1 expression was detected scatteredly in odontogenic epithelial cells in normal and neoplastic odontogenic tissues, as well as in some mesenchymal cells in tooth germs and in some stromal cells in epithelial odontogenic tumors. Ameloblastic carcinomas showed low reactivity for BMPs, BMPRs, and CBFA1. BMPs and their associated molecules might play a role in cytodifferentiation of normal and neoplastic odontogenic epithelium via epithelial-mesenchymal interactions.

Immunohistochemical detection of retinoblastoma protein and E2 promoter‐binding factor‐1 in ameloblastomas

To clarify the roles of cell cycle regulation in oncogenesis and cytodifferentiation of odontogenic tumors, expression of retinoblastoma protein (RB) and E2 promoter-binding factor-1 (E2F-1) was analyzed in ameloblastomas as well as in tooth germs. Tissue specimens of 10 tooth germs, 40 benign ameloblastomas, and five malignant ameloblastomas were examined immunohistochemically with the use of antibodies against RB, E2F-1, and phosphorylated RB. Ki-67 antigen immunostaining was made as a marker of cell proliferation. Immunohistochemical reactivity for RB, E2F-1, phosphorylated RB, and Ki-67 was detected in the nuclei of odontogenic epithelial cells near the basement membrane in tooth germs and benign and malignant ameloblastomas. The number of cells positive for phosphorylated RB was nearly equal to or slightly less than the number of cells positive for RB or E2F-1. The number of Ki-67-positive cells was slightly more than the numbers of cell positive for RB, E2F-1, or phosphorylated RB. The levels of immunoreactivity for RB, E2F-1, phosphorylated RB, and Ki-67 were slightly higher in benign and malignant ameloblastomas than in tooth germs. Plexiform ameloblastomas showed significantly higher expression of RB than follicular ameloblastomas. Ki-67 immunoreactivity was significantly higher in ameloblastic carcinomas than in metastasizing ameloblastomas. Similar immunoreactivity for RB, E2F-1, phosphorylated RB, and Ki-67 in tooth germs and ameloblastomas indicated cellular expression of phosphorylated RB and active-free E2F-1 in both normal and neoplastic odontogenic tissues. Expression of RB, E2F-1, and phosphorylated RB was considered to be involved in cell proliferation and differentiation of odontogenic epithelium via control of the cell cycle.

Transforming Growth Factor Beta (TGF-β) Gene Family Members in Developing and Neoplastic Odontogenic Tissues

During the last decade, several molecules regulating tooth development have been identified (http://bite-it.helsinki.fi/). Especially members of the transforming growth factor beta (TGF-β) supergene family, structurally relates cytokines, are essential for teeth to develop. These include TGF-βs, activin A, bone morphogenetic proteins (BMPs), and their cell surface receptors. In addition, follistatin, an extracellular protein that interacts with activin and BMP signalling, appears to be an important mediator of associated inductive signals. Enhanced or impaired TGF-β/BMP signalling may play a role in odontogenic tumorigenesis. This review focuses on findings of our studies on the roles played by of TGF-β receptors, their ligands and extracellular modifiers during tooth development and in odontogenic tumours.

Detection of mitochondria‐mediated apoptosis signaling molecules in ameloblastomas

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. 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). 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. 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.

Expression of tumor necrosis factor α, TNF‐related apoptosis‐inducing ligand, and their associated molecules in ameloblastomas

To clarify the roles of the apoptosis signaling pathway mediated by death receptors in oncogenesis and cytodifferentiation of odontogenic tumors, expression of tumor necrosis factor alpha (TNFalpha), TNF-related apoptosis-inducing ligand (TRAIL), and their associated molecules was analyzed in ameloblastomas as well as in tooth germs. Tissue specimens of 10 tooth germs, 40 benign ameloblastomas, and five malignant ameloblastomas were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry to determine the expression of TNFalpha, TNF receptor I (TNFRI), TRAIL, TRAIL receptor 1 (TRAIL-R1), TRAIL-R2, caspase-8, and nuclear factor-kappaB (NF-kappaB). Expression of TNFalpha, TNFRI, TRAIL, TRAIL-R1, TRAIL-R2, and NF-kappaB mRNA was detected in most samples of normal and neoplastic odontogenic tissues. Expression of caspase-8 mRNA was identified in six of 33 ameloblastomas, but not in 10 tooth germs or one malignant ameloblastoma. Immunohistochemical reactivity for TNFalpha, TRAIL, their receptors, and NF-kappaB was detected in both normal and neoplastic odontogenic tissues. Epithelial expression of TNFalpha was focal in about 50% of tooth germs and ameloblastomas, and TNFalpha expression in neoplastic cells was significantly higher in follicular ameloblastomas than in plexiform ameloblastomas. TRAIL reactivity was evident in epithelial cells neighboring the basement membrane. Receptors for TNFalpha and TRAIL were diffusely expressed in both normal and neoplastic odontogenic epithelium. Expression of caspase-8 was found in some neoplastic cells in three of 37 ameloblastomas, but not in 10 tooth germs or five malignant ameloblastomas. Nuclear NF-kappaB expression was much lower than cytoplasmic expression in both normal and neoplastic odontogenic epithelium. Expression of TNFalpha, TRAIL, and their receptors in tooth germs and ameloblastomas suggests that these death factors might be involved in cytodifferentiation of odontogenic epithelium and tissue structuring of ameloblastomas. Expression of caspase-8 and NF-kappaB suggests that signaling of TNFalpha and TRAIL minimally affects the biological properties of odontogenic epithelial components.

Expression of p63 and p73 in ameloblastomas

To clarify the role of p53 homologs in oncogenesis and cytodifferentiation of odontogenic tumors, expression of p63 and p73 was analyzed in ameloblastomas as well as tooth germs. Tissue specimens of nine tooth germs and 48 benign and five malignant ameloblastomas were examined by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) for the expression of p63 and p73. Immunoreactivity for p63 and p73 was evident in epithelial cells neighboring the basement membrane in developing and neoplastic odontogenic tissues. p63 expression in desmoplastic ameloblastomas was significantly higher than in acanthomatous and granular cell ameloblastomas, and ameloblastic carcinomas showed higher p63 expression than metastasizing ameloblastomas. p73 expression was significantly higher in plexiform ameloblastomas than in follicular ameloblastomas, and basal cell ameloblastomas showed higher p73 expression than granular cell ameloblastomas. mRNA transcripts for Delta Np63 and TAp73 were detected in all developing and neoplastic odontogenic tissues. TAp63 mRNA was expressed in five of eight tooth germs, 16 of 34 ameloblastomas, and one of one malignant ameloblastoma, whereas Delta Np73 mRNA was recognized in one of eight tooth germs, nine of 34 ameloblastomas, and one of one malignant ameloblastoma. The expression of p63 and p73 suggests that these p53 homologs play a role in differentiation and proliferation of odontogenic epithelial cells. Variations of predominantly expressed isoforms suggest that p63 and p73 might differentially function in odontogenic tissues.