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.

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.

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

beta-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.

Ameloblastic carcinoma ex ameloblastoma: report of a case-possible involvement of CpG island hypermethylation of the p16 gene in malignant transformation.

Ameloblastic carcinoma is described in the updated World Health Organization (WHO) classification as a rare malignant lesion. Ameloblastic carcinoma meeting the WHO criteria may arise either as a result of malignant change in a pre-existing benign ameloblastoma (carcinoma ex ameloblastoma) or as a primary malignant ameloblastoma not preceded by an ordinary ameloblastoma (de novo carcinoma). We report a case of ameloblastic carcinoma ex ameloblastoma and examine how this case underwent malignant transformation. The DNA was extracted separately from benign and malignant areas in paraffin sections of the tumor. Direct sequencing showed no genetic mutation of exons 5-8 of the p53 gene. Hypermethylation of CpG islands of the p16 gene was detected in the malignant parts of the tumor. The results indicate that hypermethylation of p16 may have been involved in the malignant transformation of the ameloblastoma in the present case.

Effect of defensin peptides on eukaryotic cells: primary epithelial cells, fibroblasts and squamous cell carcinoma cell lines.

BACKGROUND: Although the usefulness of the antimicrobial peptides known as defensins has been suggested against oral and skin infections, possible adverse effects of the defensins on the host should be understood before clinical applications can be contemplated. OBJECTIVE: In the present study, we investigated how alpha-defensin (HNP-1) and beta-defensins (hBD-1, -2, -3) affect cells including primary epithelial cells, fibroblasts and squamous cell carcinoma cell lines, SCC-9 and KB. METHOD: Cell proliferation was assessed by the direct cell counting and XTT assay. RESULTS: We found that alpha-defensin promotes proliferation of the epithelial cells at low concentration but has a cytotoxic effect at high concentration. In contrast, beta-defensins have little effect on these cells at any concentration, suggesting that beta-defensins may have no adverse effects on the host. CONCLUSION: Therefore, in terms of host response beta-defensins may be more suitable antimicrobial agents for clinical applications than alpha-defensins.