Masato Saitoh

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.

Salivary Defensins and Their Importance in Oral Health and Disease

Saliva contributes significantly to the protective barrier of oral epithelium through its mechanical rinsing action and the unique peptides it contains. Saliva contains several types of antimicrobial peptides, including defensins, which may have an important role in innate host defense. Many types of human defensins have been discovered and characterized in the last decade. This review summarizes the recent literature on salivary defensins and discusses their importance in oral health and disease. Salivary defensins are possibly derived from salivary ductal cells, oral epithelial cells and some blood cells. The antimicrobial activity of defensins may be affected by the components of saliva. Salivary defensin levels can be altered in oral diseases, and therefore may be a useful marker for risk assessment, salivary diagnosis and therapeutic strategies.

Expression pattern of p63 in oral epithelial lesions and submucous fibrosis associated with betel-quid chewing in Sri Lanka

Betel-quid chewing, which is closely related to the high incidence of oral cancer, is prevalent in Sri Lanka. p63 has a remarkable structural similarity to p53, suggesting an aberrant expression in oral cancer. Using anti-p63 antibody and immunohistochemistry, the present study investigated the expression pattern of p63 in oral epithelial lesions, including different types of squamous cell carcinoma (SCC), different grades of epithelial dysplasia, and submucosal fibrosis associated with betel-quid chewing. Nuclear immunoreactivity for p63 was detected in all the cases, including normal oral epithelium and epithelial lesions. In normal oral epithelium, nuclear positivity for p63 was observed in some of the basal cell layers and focally in the parabasal layer. Nuclear positivity increased in the epithelial lesions. The percentage of positive nuclei in the epithelial lesions was significantly higher than in normal epithelium (P < 0.01) and was also significantly higher in oral submucosal fibrosis than in epithelial dysplasia (P < 0.05). The results indicate that the overexpression of p63 in oral precancerous lesions and SCC in betel-quid chewers in Sri Lanka may be a useful marker for oral precancerous lesions.

Localization of human β-defensin 3 mRNA in normal oral epithelium, leukoplakia, and lichen planus: an in situ hybridization study

 Human β-defensin 3 (hBD-3), an antimicrobial peptide, is produced by various epithelial and some nonepithelial tissues. hBD-3 mRNA is widely expressed in oral tissues, including oral epithelium and the salivary glands. Although the localization of hBD-1 and hBD-2 has been well demonstrated in tissue sections, the localization pattern of hBD-3 has not yet been shown. In the present study, we investigated the expression pattern of hBD-3 mRNA by in situ hybridization using specific RNA probes; the signal for hBD-3 was detected in upper spinous and granular layers in normal oral epithelium. In cases of leukoplakia, a strong signal of hBD-3 mRNA was observed in the granular layer. In lichen planus, the signal was strongly detected in the spinous and suprabasal layers. The signals were stronger than those of either normal oral epithelium or leukoplakia. The results indicate that the localization pattern of hBD-3 is very similar to that of hBD-2. hBD-2 and hBD-3 may function together or compensate each other for expressional loss.

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.

Apoptosis in the reduced enamel epithelium just after tooth emergence in rats

The reduced enamel epithelium transforms into a stratified squamous epithelium, i.e. a junctional epithelium, as the tooth erupts. In this study, we observed apoptosis in the reduced enamel epithelia of rats just after tooth eruption and before complete junctional epithelium formation, by the TUNEL method and electron microscopy. TUNEL-positive reactions were scattered in the reduced ameloblasts and in the external cells of the reduced enamel epithelium. Electron microscopic observation confirmed features of apoptosis, such as nuclei with chromatin condensation, cell shrinkage, and phagocytosis of apoptotic bodies by macro-phage-like cells and epithelial cells. These results suggest that apoptotic cell death is involved in the disappearance of reduced ameloblasts and the external cells of the reduced enamel epithelium during the formation of the junctional epithelium.

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.

Expression of claudin-4 and -7 in porcine gingival junctional epithelium

Junctional epithelium, a nonkeratinized stratified epithelium, extends apically in apposition to the surface of the enamel to form a seal between the epithelium and the tooth. Desmosomes and gap junctions adhere to the junctional epithelium through cell-cell contact, but no evidence of tight junctions has been found. Recently, tight junction hallmark proteins and tight junction-related structures have been identified in stratified squamous epithelium. The present study examined whether tight junction proteins were expressed in the junctional epithelium. We used immunohistochemical techniques to observe expression of claudin-1, -4, -5, -7, and occludin in porcine gingival junctional epithelium. Claudin-4 exhibited immunoreactivity in the intercellular spaces of all layers of the oral epithelium and the junctional epithelium. Stronger expression was observed in junctional epithelial cells adjacent to the inner and outer basal laminae than in the inner cell layers. Immunohistochemical positivity for claudin-7 was clearly observed in the junctional epithelium, but only a faint positivity was observed in the basal layer of the oral epithelium. No immunohistochemical positivity for claudin-1, -5, or occludin was observed in the junctional epithelium. RT-PCR assay confirmed expression of porcine claudin-4 and -7 mRNAs in the junctional epithelium. These findings indicate that claudin-4 and -7 may play a role in the junctional epithelium even in the absence of tight junctions.