DNA methyltransferase immunohistochemical expression in odontogenic tumours.

BACKGROUND: Odontogenic tumours are a heterogeneous group of lesions formed from tissues that give rise to the tooth. DNA methylation, a covalent addition of a methyl group to the 5-carbon position of a cytosine nucleotide, is considered an important regulator of gene expression. The addition of the methyl radical is catalysed by DNA methyltransferases (DNMTs). Although some epigenetic studies have been conducted in odontogenic tumours, a study with the three types of DNMTs in several different members of this group is missing. This study analyses the expression of DNMTs in odontogenic tumours. METHODS: Formalin-fixed and paraffin-embedded tissue samples of 20 ameloblastomas, 10 calcifying cystic odontogenic tumours, 10 calcifying epithelial tumours, 10 adenomatoid odontogenic tumours, 10 keratocystic odontogenic tumours, five ameloblastic fibromas, two ameloblastic fibro-odontomas, four central odontogenic fibromas, seven peripheral odontogenic fibromas and 10 odontogenic myxomas were included. Immunohistochemical expression of DNMT1, 3A and 3B was assessed using a semi-quantitative analysis, and also a correlation with p21, p27 and E-cadherin immunoexpression was made. RESULTS: DNMT1, 3A and 3B were expressed in the nucleus and/or cytoplasm of all odontogenic tumours. DNMT1 expression was directly correlated with p27 expression in ameloblastomas. CONCLUSION: The high expression of DNMTs in odontogenic tumour cells suggests methylation as an important mechanism for this group of tumours.

Overexpression of Smad2 inhibits proliferation of gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Spatiotemporal inhibition of apical migration and proliferation of gingival epithelium are significant factors involved in periodontal regeneration. Transforming growth factor ß (TGF-ß) is important in multiple aspects of wound healing, and Smad2, a downstream transcription factor of TGF-ß, has an inhibitory effect on re-epithelialization during gingival wound healing. Therefore, we investigated the effects on migration and proliferation status, and intra/extracellular signaling regulated by Smad2 overexpression in gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells were isolated from the palatal gingival tissue of transgenic mice overexpressing Smad2 driven by the Keratin14 promoter. Smad2 expression was identified by western blotting and immunofluorescence analysis. Scratch assay and 5-bromo-2'-deoxyuridine staining were performed to assess cell migration and proliferation. To inactivate TGF-ß type I receptor, the cultures were supplemented with SB431542. Secreted TGF-ß was quantified by ELISA. Smad2 target gene expression was examined by real-time RT-PCR and in vivo immunofluorescence analysis of gingival junctional epithelium. RESULTS: Smad2-overexpressing cells were confirmed to have significant phosphorylated Smad2 in the nucleus. Scratch assay and 5-bromo-2'-deoxyuridine staining indicated that Smad2-overexpressing cells showed no significant differences in migration, but had reduced proliferation rates compared to wild-type controls. SB431542 significantly inhibited Smad2 phosphorylation, which coincided with restoration of the proliferation rate in Smad2-overexpressing cells. ELISA of TGF-ß release did not show any differences between genotypes. The cell cycle inhibitors, p15 and p21, showed significant upregulation in Smad2-overexpressing cells compared to wild-type controls. Moreover, junctional epithelium of the transgenic mice showed increased expression of P-Smad2, p15 and p21. CONCLUSION: The signaling activation triggered by overexpression of Smad2 was dependent on TGF-ß type I receptor, and the activated Smad2 increased p15 and p21 expression, responsible for inhibiting cell cycle entry, resulting in antiproliferative effects on gingival epithelial cells. Understanding of Smad2-induced signaling would be useful for possible clinical application to regulate gingival epithelial downgrowth.

Expression of p21 and p53 in rat gingival and human oral epithelial cells after cyclosporine A treatment.

BACKGROUND AND OBJECTIVE: Expression of p21 and p53 were examined, at gene and protein levels, in edentulous gingival epithelial cells from rats and from a human oral epidermoid carcinoma cell line, OECM1, after cyclosporine A therapy. MATERIAL AND METHODS: In vivo: 20 partially edentulous SD rats were assigned into cyclosporine A feeding and control groups. After the rats were killed, p21 and p53 in gingiva were evaluated by reverse transcription-polymerase chain reaction and immunohistochemistry. In vitro: after cyclosporine A treatment, p21 and p53 of OECM1 cells were evaluated by western blot and the luciferase assay. The distribution of OECM1 cells in each phase of the cell cycle was evaluated by flow cytometry. RESULTS: The mRNA expression of p21 was significantly higher in the cyclosporine A group than in the control group. A greater number of positive anti-p21-stained cells were observed in the gingival epithelium of the cyclosporine A group than in the control group. Significantly higher levels of p21 protein and activity were observed in OECM1 cells after cyclosporine A treatment than in cells without treatment. A relative increase of cells in G0/G1 phases, and a decrease of cells in G2/M phases, were observed in OECM1 cells after cyclosporine A treatment. CONCLUSION: In the present study, higher p21 mRNA and protein expressions were observed after cyclosporine A treatment. Thus, an up-regulation of p21 expression, via a p53-independent pathway, by cyclosporine A in gingival and oral epithelial cells was suggested.

Camphorquinone inhibits odontogenic differentiation of dental pulp cells and triggers release of inflammatory cytokines.

INTRODUCTION: Camphorquinone (CQ) is a photoinitiator that triggers polymerization of light-curing materials such as dental adhesives and composites. CQ does not become a part of the polymer network, suggesting that CQ can be leached out into surrounding environment including dental pulp and exert adversary effects on tissues. In order to understand the mechanisms of CQ-induced side effects, we investigated the effect of CQ on cell viability, cytokine secretion, and odontogenic differentiation of dental pulp stem cells in vitro. METHODS: Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after CQ exposure. Western blotting was performed for p16(INK4A), p21(WAF1), and p53. Secretory cytokines were evaluated using the membrane-enzyme-linked immunosorbent assay as well as conventional and quantitative reverse-transcription polymerase chain reaction. The effects of CQ on odontogenic differentiation were evaluated using alkaline phosphatase and alizarin red S staining methods. RESULTS: CQ treatment suppressed the proliferation of DPSCs and induced the expression of p16(INK4A), p21(WAF1), and p53. Levels of proinflammatory cytokines (eg, interleukin 6, interleukin 8, and matrix metalloproteinase-3 [MMP3]) were increased by CQ treatment. CQ also inhibited odontogenic differentiation and mineralization capacities of DPSC and MC3T3-E1 cells. CONCLUSIONS: Our study showed that CQ may trigger pulpal inflammation by inducing proinflammatory cytokine production from the pulpal cells and may impair odontogenic differentiation of dental pulp cells, resulting in pulpal irritation and inflammation.

Metastatic leiomyosarcoma in the oral cavity: case report with protein expression profiles.

Leiomyosarcoma (LMS) is a relatively uncommon malignant tumour derived from smooth muscle cells that rapidly metastasizes to distant regions. It rarely reaches oral tissues in which smooth muscle tissues are absent. We report the case of a 56-year-old woman who presented with LMS in the maxilla that had metastasized from a primary tumour in her uterus, received a total hysterectomy with bilateral salpingo-oophorectomy 9 months earlier. To reveal the poor prognosis of metastatic LMS, a total of 26 antibodies against different factors related to the proliferation, apoptosis, necrosis, and angiogenesis were simultaneously applied on the immunohistochemistry and immuno-blot detection in order to screen for expression n of different proteins in the metastatic LMS. Compared with the immunoreactions of primary uterine LMS, the different antibodies for cellular proliferation, i.e., proliferating cell nuclear antigen (PCNA), multiple primary neoplasm-2 (MPN-2), Max, p21, CDK4, p53, Rb-1, Bad, Bcl-2, epidermal growth factor receptor (EGF-R), hepatocyte growth factor (HGF), C-erbb2, Maspin, and DMBT-1, and those for angiogenesis, i.e., vWF, CD31, and Angiogenin, were more intensely expressed, while Bax, p16, Wnt-1, E-cadherin, and APC were relatively weakly expressed. In particular, beta-catenin was densely localized to the nuclei of tumour cells. These data suggest that rapid proliferation of the tumour cells is related to over-expression of different oncogenes, and that the infiltrative growth and early distant metastasis of these tumour cells are related to over-expression of angiogenesis factors. A total of seven cases of metastatic LMS to the oral cavity that had been published in the English literature were reviewed, and the reason for the poor prognosis in the metastatic LMS is suggested in this case report.

Potential of diallyl sulfide bearing pH-sensitive liposomes in chemoprevention against DMBA-induced skin papilloma.

Diallyl sulfide (DAS), an active component of garlic, possesses strong anti-neoplastic properties against various forms of cancer. In the present study, we have evaluated chemo-preventive effects of liposomized DAS (conventional egg PC and pH-sensitive liposomes) against DMBA-induced skin papilloma. Various liposome-based novel formulations of DAS (250 microg/mouse) were applied topically, after one hour of exposure to DMBA (52 microg/mouse/dose), to the animals. The animals were treated thrice weekly for the total period of 12 weeks. The efficacy of the various liposomal formulations of DAS was evaluated on the basis of parameters such as incidence of tumorogenesis and total numbers and sizes of induced tumor nodules. The liposomized DAS formulations also were assessed for their effect on the expression of p53wt, p53mut, and p21/Waf1. The results of the present study showed that liposomized DAS could effectively delay the onset of tumorogenesis and reduce the cumulative numbers and sizes of tumor papillomas in treated mice. Treatment of DMBA-exposed animals with the liposomal formulation of DAS ensued in upregulation of p53wt and p21/Waf1, while levels of p53mut expression reduced down. The promising chemo-preventive nature of liposomal DAS may form the basis for establishing effective means of controlling various forms of cancer, including skin papilloma.