Yoshihito Kurashige

High frequency of hypermethylation of p14, p15 and p16 in oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

BACKGROUND Oral squamous cell carcinoma and the most common oral pre-malignancies appear to be related to the habit of betel-quid chewing in Sri Lanka. Although hypermethylation of the tumour suppressor genes in oral cancer have been well documented, little information has been available concerning hypermethylation in oral pre-cancerous lesions. In the present study, we investigated the hypermethylation of p14, p15 and p16 in pre-cancerous lesions including epithelial dysplasia and submucous fibrosis. METHODS All samples were obtained from patients with a betel-quid chewing habit in Sri Lanka. Sixty-four patients were clinically diagnosed with leukoplakia, and histopathologically diagnosed with mild or severe dysplasia. Ten patients were diagnosed with submucous fibrosis without epithelial dysplasia. CpG island hypermethylation was assessed by a methylation-specific PCR method. Immunohistochemical staining was performed using anti-p53 antibodies. RESULTS A high frequency of hypermethylation of p14, p15 and p16 was detected in the pre-cancerous lesions, although no hypermethylation was found in normal epithelium. The frequency of hypermethylation was higher than that of positive staining for p53 mutation except in the case of p16 in mild dysplasia. No significant correlation was observed between p53-positive reactions and hypermethylation in any lesions. The hypermethylation was highly detectable even in p53-negative lesions, suggesting that hypermethylation of p14, p15 and p16 occur regardless of whether the lesions have p53 mutations or not. CONCLUSIONS The present study indicates that hypermethylation may be involved in the pathogenesis of oral pre-cancerous lesions associated with betel-quid chewing in Sri Lanka.

Nicotine induces upregulated expression of beta defensin-2 via the p38MAPK pathway in the HaCaT human keratinocyte cell line

Human beta-defensins (hBDs), a group of antimicrobial peptides, are involved in the protective barrier of the oral epithelium. Nicotine induces periodontal and oral epithelial diseases. The purpose of the present study was to investigate the effect of nicotine on the expression pattern of hBD-2 in keratinocytes. HaCaT cells, a keratinocyte cell line, were incubated with 8, 15, 30, or 80 μM nicotine for 24 h. Expression of hBD-2 was observed by RT-PCR, qRTPCR, and ELISA assay. The cells were treated with inhibitors for intracellular pathways (p38MAP kinase, NF-κB, JNK, MAPK-ERK) and with nicotinic acetylcholine receptor (nAChR) inhibitors in a series of experiments. Data were analyzed using Student’s t test. qRT-PCR revealed that the expression level of hBD-2 mRNA was significantly higher at 30 and 80 μM nicotine than the control without nicotine (P < 0.05). The 80 μM cell extraction contained significantly higher hBD-2 peptide levels than the control (P < 0.05). The p38MAP kinase inhibitor abolished the upregulated expression of hBD-2 by nicotine. Both nAChR inhibitors also abolished the upregulation of hBD-2 by nicotine. The present study demonstrated that nicotine causes upregulated expression of hBD-2 via the p38MAP kinase pathway in keratinocytes.

Involvement of toll‐like receptors in autoimmune sialoadenitis of the non‐obese diabetic mouse

The aim of this study was to characterize the expression of Toll-like receptors (TLRs) during the development of sialoadenitis in the non-obese diabetic mouse. Submandibular glands were dissected from non-obese diabetic mice at 4, 8, 10, 12, and 16 weeks of age. The mRNA expression levels of TLR1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, MyD88, and TRIF was quantified using real-time reverse transcription polymerase chain reaction. The mRNA expression levels in 4-week-old non-obese diabetic mice were used as controls. The expression levels of TLR1, 2, 4, and 9 were significantly higher at 8, 10, 12, and 16 weeks than the levels in the controls. The expression level of TLR3 was significantly higher at 16 weeks than in the controls. A group of mice were given drinking water containing 4.75% chloroquine starting at 4 weeks. Chloroquine caused a significant decrease in the expression of TLR1, 2, 3, 4, and 9 at 16 weeks compared with control mice who did not receive chloroquine. The areas of lymphocyte infiltration seen on serial sections of submandibular glands in the mice receiving chloroquine were significantly smaller than the areas of infiltration in control glands. Increased expression of Toll-like receptors may be involved in the development and/or progression of sialoadenitis in the non-obese diabetic mouse. Toll-like receptors may be a therapeutic target for autoimmune sialoadenitis.

Differentiation of mouse iPS cells into ameloblast-like cells in cultures using medium conditioned by epithelial cell rests of Malassez and gelatin-coated dishes

Induced pluripotent stem (iPS) cells are generated from adult cells and are potentially of great value in regenerative medicine. Recently, it was shown that iPS cells can differentiate into ameloblast-like cells in cultures using feeder cells. In the present study, we sought to induce differentiation of ameloblast-like cells from iPS cells under feeder-free conditions using medium conditioned by cultured epithelial cell rests of Malassez (ERM) cells and gelatin-coated dishes. Two culture conditions were compared: co-cultures of iPS cells and ERM cells; and, culture of iPS cells in ERM cell-conditioned medium. Differentiation of ameloblast-like cells in the cultures was assessed using real-time RT-PCR assays of expression of the marker genes keratin 14, amelogenin, and ameloblastin and by immunocytochemical staining for amelogenin. We found greater evidence of ameloblast-like cell differentiation in the cultures using the conditioned medium. In the latter, the level of amelogenin expression increased daily and was significantly higher than controls on the 7th, 10th, and 14th days. Expression of ameloblastin also increased daily and was significantly higher than controls on the 14th day. The present study demonstrates that mouse iPS cells can be induced to differentiate into ameloblast-like cells in feeder-free cell cultures using ERM cell-conditioned medium and gelatin-coated dishes.

Increased expression of b-defensin-2 and -3 during the development of autoimmune sialoadenitis in MRL/lpr mice

Abstractβ-Defensins (BD) are small cationic antimicrobial peptides that produced principally in the epithelial cells of a number of organs. The present study analyzed the expression patterns of BD-1, -2, and -3 during the development of sialoadenitis in MRL/lpr mice. Submandibular glands were dissected from MRL/lpr mice at 4, 8, 10, 12, 14, and 16 weeks of age. The expression of mouse (m) BD-1, -2, and -3 mRNAs was examined by RT-PCR and quantified using TaqMan real-time RT-PCR. No significant differences in the level of expression of mBD-1 were observed among mice of different ages. The level of expression of mBD-2 was significantly higher at 16 weeks than at 4 or 8 weeks. The expression level of mBD-3 was highest in 14-week-old mice and was significantly higher than in 4-week or 16-week-old animals. Immunohistochemical staining showed that BD-3 was clearly localized in the ductal cells with variable intensities. In the center of the foci of lymphatic infiltration, ductal staining was faint or often not present. The results indicate that BD-2 and -3 may be upregulated during the development of autoimmune sialoadenitis.

Profiling of differentially expressed genes in porcine epithelial cells derived from periodontal ligament and gingiva by DNA microarray

OBJECTIVE It is unknown which genes are differentially expressed in cultured epithelial cells derived from the epithelial rests of Malassez (ERM) in periodontal ligament and oral gingival epithelium (OGE). This study analysed the different gene expression of OGE and ERM cells using a DNA microarray technique. DESIGN Epithelial cells from ERM and OGE were isolated from porcine periodontal ligament and oral gingival epithelium. Each RNA sample extracted from the cells was reverse transcribed into cDNA and labelled with either cytidine 5-dUTP (Cy5) or cytidine 3-dUTP (Cy3). These labelled cDNA probes were then mixed and simultaneously hybridised to the Pig 13K microarray plate bearing 13,295 different genes (Operon, AL). Cellular enzyme-linked immunosorbent assay (CELISA) was performed to confirm the expression at protein level. RESULTS There were nine genes common to the triplicate microarrays in ERM cells and one in OGE cells. Four of the nine genes including tissue factor (TF), FAT cadherin (FAT) and two unknown genes were expressed at levels more than threefold higher in ERM cells than in OGE cells. The protein levels of both TF and FAT in ERM cells were significantly higher than those in OGE. CONCLUSION TF and FAT may act as markers to distinguish ERM cells from OGE cells in vitro.

hBD-2 is downregulated in oral carcinoma cells by DNA hypermethylation, and increased expression of hBD-2 by DNA demethylation and gene transfection inhibits cell proliferation and invasion

Human β-defensin-2 (hBD-2) is a type of epithelial antimicrobial peptide. The expression level of hBD-2 mRNA is lower in oral carcinoma cells (OCCs) than in healthy oral epithelium. Yet, it is still unknown how hBD-2 expression is downregulated in OCCs. The present study investigated DNA hypermethylation of hBD-2 in OCCs and the effect of the demethylation and increased expression of hBD-2 on cell proliferation and invasion. Six different types of oral carcinoma cell lines (OSC-19, BSC-OF, SAS, HSC-2, HSC-4 and HSY) and normal oral keratinocytes (NOKs) were used. The expression levels of hBD-2 in all OCCs were significantly lower than that in the NOKs. Treatment with DNA methyltransferase inhibitor, 5-aza-dC, at the concentration of 50 μM significantly induced upregulation of expression of hBD-2 in the OCCs. Using methylation-specific PCR, DNA hypermethylation was observed in all OCCs. These results suggest that DNA hypermethylation is, at least in part, involved in the decreased expression of hBD-2 in OCCs. We examined the effect of 5-aza-dC on the cell proliferation and invasive ability of OCCs. The cell invasion assays showed that the number of OCCs treated with 5-aza-dC on the filters was significantly lower than that of the controls. We examined whether increased expression of hBD-2 generated by gene transfection inhibited the proliferation and invasion of SAS cells. The number of SAS cells exhibiting increased expression of hBD-2 on the filters in the invasion assay were significantly lower on day 7 when compared with the control. hBD-2 may function as a tumor suppressor. Increased expression of hBD-2 induced by demethylation or increased expression generated by gene transfection may be useful therapeutic methods for oral carcinoma.

Effect of epithelial cells derived from periodontal ligament on osteoblast-like cells in a Transwell membrane coculture system.

BACKGROUND AND OBJECTIVE The epithelial rests of Malassez (ERM) are developmental residues of Hertwigs epithelial root sheath, and they are present throughout life in the periodontal ligament. Previous studies have shown that ERM induce cementogenesis or inhibit cement-osteogenesis. This study investigated how ERM cells are involved in osteoblast mineralization in an in vitro coculture system. MATERIALS AND METHODS ERM cells were isolated from porcine periodontal ligament by an outgrowth method. Osteoblast-like MC3T3-E1 cells were cocultured with ERM or gingival epithelium (GE) cells in six-well Transwell units. Mineralization of MC3T3-E1 cells was confirmed by Alizarin Red staining after 30 days of culture. Alkaline phosphatase (ALP) activity was measured in the culture media. To examine whether enamel matrix proteins are involved in the mineralization of MC3T3-E1 cells, an anti-amelogenin antibody was added to the culture media. RESULTS The staining by Alizarin Red of MC3T3-E1 cells cocultured with ERM cells was clearly weaker than that in the GE cell coculture. The ALP activity in the ERM cell coculture was significantly lower than that in the GE cell coculture (p < 0.05). Alizarin Red staining of MC3T3-E1 cells in the ERM cell coculture with anti-amelogenin antibody was clearly stronger than in those cocultures without the antibody at 30 days. The ALP activity in the ERM cell coculture with anti-amelogenin antibody was significantly higher than in those cultures without the antibody (p < 0.05). CONCLUSION This study demonstrated that ERM cells inhibit the calcification of osteoblast-like cells in a Transwell coculture system and that this inhibition is reversed with an anti-amelogenin antibody.

Expression profile of drosomycin-like defensin in oral epithelium and oral carcinoma cell lines.

OBJECTIVE Drosomycin-like defensin (DLD) is a recently discovered antimicrobial peptide mainly active against filamentous fungi. The present study investigated the expression profile of DLD in oral epithelium and oral squamous cell carcinoma (SCC) cell lines. METHODS Tissue sections of human oral mucosa, keratinocytes derived from oral mucosa (NOK) and eight kinds of SCC cell lines were used. In situ hybridization was performed on tissue sections of oral mucosa. Expression levels of DLD in the cells were observed by reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR assays. The cells were treated with IL-1β, IL-8 and TNF-α, and agonists for TLR2, TLR4 and β-glucan. DLD expression in cells was increased and decreased by the DLD gene and its siRNA transfection, respectively. The proliferation rates were assessed by cell counting. RESULTS By means of in situ hybridization, DLD mRNA positive staining was detected in the epithelial layer of the oral mucosa. An RT-PCR assay confirmed the expression of DLD mRNA in keratinocytes derived from oral epithelium. Expression of DLD in two out of eight cell lines was significantly lower than in NOK cells. The expression levels of DLD mRNA were not significantly changed in the cells stimulated with any cytokines or agonists. The cell proliferation rate where there was decreased expression of DLD was significantly lower than in the control. CONCLUSION DLD may be partially involved in the defence against filamentous fungal infection in the oral mucosa, and may also serve other functions, such as contribution to cell growth.