João Artur Ricieri Brito

Methylation of P16, P21, P27, RB1 and P53 genes in odontogenic keratocysts

BACKGROUND Odontogenic keratocyst (OKC) is a benign neoplasm with an aggressive clinical behavior and a high recurrence rate. Although epigenetic alterations have been reported in different tumors, these events were not investigated in OKC yet. The aim of this study was to investigate the presence of methylation in P16, P21, P27, P53 and RB1 genes in OKC tumors. METHODS Methylation-specific polymerase chain reaction (MSP) was used to evaluate the presence of methylation in 10 samples of OKCs, 10 samples of dental follicles and six samples of normal mucosa. RESULTS The methylation status of the P16 gene was similar among the three groups. In P21 gene, 30% of OKCs were methylated while no methylation could be detected in the other groups. High frequency of P27 methylation (90%) was observed in dental follicles, however, some OKC lesions (10%) and normal mucosa samples (33%) were also methylated. Concerning the RB1 gene, positive results were detected only in dental follicles (40%). No positive result was observed considering P53 gene. CONCLUSIONS Our data show methylation of the promoter of P21 gene in OKCs. In addition, methylation of the P27 and RB1 genes are commonly found in dental follicles. Further studies are necessary to determine the functional relevance of these alterations.

Expression, polymorphism and methylation pattern of interleukin-6 in periodontal tissues

Periodontitis is considered an inflammatory disorder of bacterial etiology that results in periodontal tissue destruction, as a result of complex interactions between periodontal pathogens, host and immune response. Genetic and epigenetic mechanisms may modulate the individual response since it is able to influence the gene expression. The aim of this study was to evaluate the impact of -174 G/C polymorphism and the methylation status of the promoter region of IL-6 gene on the expression of IL-6 in gingival samples from individuals with chronic periodontitis. Gingival biopsies were collected from 21 patients with chronic periodontitis and 21 controls. Histologic sections stained by hematoxylin-eosin were used for histopathological evaluation. The IL-6 gene expression was assessed by quantitative real-time PCR. The polymorphism IL-6 -174 C/G was studied by polymerase chain reaction (PCR) amplification and restriction endonuclease digestion (HspII). Methylation-specific polymerase chain reaction was used to verify the DNA methylation pattern. The number of inflammatory cells in tissue fragments from individuals with chronic periodontitis was higher than in the control group and the inflammatory infiltrate was predominantly mononuclear. The expression of IL-6 was higher in the group with periodontitis. In polymorphism assay, no statistical difference in the distribution of genotypes and alleles in both groups were observed. The most of samples were partially methylated. No difference was observed in methylation pattern from two different regions of the IL-6 gene among groups. The high expression of IL-6 is an important factor related to chronic periodontitis, but was not associated with methylation status or the -174 (G/C) genetic polymorphism, suggesting that other mechanisms are involved in this gene transcription regulation.

Methylation frequencies of cell-cycle associated genes in epithelial odontogenic tumours

OBJECTIVE The benign epithelial odontogenic tumours constitute a group of lesions derived from epithelial elements of the tooth-forming apparatus. This group includes lesions of different biological behaviour, such as ameloblastoma, calcifying cystic odontogenic tumour (CCOT) and adenomatoid odontogenic tumour (AOT). The pathogenesis of these neoplasms remains uncertain and the occurrence of methylation in cell-cycle related genes may be involved in their development. The aim of this study was to investigate the methylation status of P16, P21, P27, P53 and RB1 genes in epithelial odontogenic tumours. DESIGN Methylation-specific polymerase chain reaction (MSP) was used to evaluate the presence of methylation in 13 samples of ameloblastoma, six samples of CCOT, three samples of AOT and 14 samples of dental follicles, included as control. RESULTS Our results showed a distinct methylation profile in each group. In ameloblastoma, the highest methylated genes were P16 and P21, while in CCOT the P21 and RB1 genes were the most commonly methylated genes. Only the P16 and P21 genes were methylated in the AOT samples. In the dental follicle samples, P16, P27 and RB1 genes were commonly methylated. A high percentage of the odontogenic tumours analysed showed methylation of the P21 gene, in contrast to dental follicles. CONCLUSIONS Epithelial odontogenic tumours show a distinct methylation profile in cell-cycle associated genes. In addition to this, the current findings show that epigenetic alterations are common events in epithelial odontogenic tumours.

Methylation Pattern of the IFN-γ Gene in Human Dental Pulp

INTRODUCTION DNA methylation is characterized by the addition of methyl groups in cytosines within cytosine-phosphate-guanine (CpG) islands. Unmethylated islands are related with transcriptionally active structure, whereas methylated DNA recruits methyl-binding proteins that promotes chromatin compaction. Although epigenetic events can influence the expression of cytokines, such events have not been investigated in dental pulp yet. The purpose of the present study was to evaluate the methylation status of the interferon gamma (IFN-gamma) gene in human dental pulp affected by inflammation compared with pulp tissue of impacted molar teeth and to verify the impact of methylation status in the expression pattern of the gene. METHODS Methylation-specific polymerase chain reaction (MSP) was used to verify the DNA methylation status of the IFN-gamma gene in 16 human dental pulps affected by inflammation and in 16 pulp samples of impacted molar teeth. Histologic sections stained by hematoxylin-eosin were used for histopathological evaluation, and the expression of IFN-gamma was assessed by quantitative real-time PCR (qPCR). RESULTS Although total methylation was observed in 43.75% of the samples of normal dental pulp tissues, partial methylation or unmethylation was found in 93.75% of the samples of inflamed pulp tissues. All the samples with total methylation in MSP showed no transcription of IFN-gamma. The qPCR results showed expression of IFN-gamma in 5 of 10 samples with partial methylation. CONCLUSION The present study gives the first evidence of the possible participation of epigenetic events in the development of dental pulp inflammation.

Relationship between microRNA expression levels and histopathological features of dysplasia in oral leukoplakia

BACKGROUND Increased expression of microRNAs (miRNAs), miR-21, miR-345, and miR-181b has been demonstrated in oral leukoplakia (OL) that progresses to oral squamous cell carcinoma (OSCC), suggesting a miRNA signature with potential prognostic value. On the basis of these findings, this pilot study aimed to investigate the cytological and histopathological features that are used to grade oral dysplasia and determine associations with the expression of these 3 potentially cancer-related miRNAs. We also compared the expression levels of these miRNAs in OL with normal oral mucosa and OSCC. METHODS We evaluated miRNA expression by qPCR in 22 samples of OL demonstrating different grades of dysplasia, as well as 17 cases of OSCC, and 6 samples of normal oral mucosa. We associated the miRNAs expression profiles with cytological and histopathological features of OL. RESULTS OSCC cases showed increased expression of all 3 miRNAs when compared with OL and normal oral mucosa. Increased expression of miR-21 was also observed in OL when compared with normal oral mucosa. We found a higher expression of miR-21 and miR-181b in OL that presented with an increased number of mitotic figures, increased nuclear/cytoplasmic ratio, or hyperchromasia. Increased expression of miR-21 was also detected in OL with abnormally superficial mitosis. Higher expression of miR-345 was observed in OL with an increased number and size of nucleoli or increased nuclear/cytoplasmic ratio. CONCLUSIONS In conclusion, the present study shows that some cytological and histopathological parameters used to grade dysplasia are associated with altered miRNA expression.

PTCH1 isoforms in odontogenic keratocysts

Odontogenic keratocyst (OKC) is an aggressive benign odontogenic neoplasm associated with PTCH1 alteration. PTCH1 has several isoforms generated by use of different first exon (1b, 1d and 1e). These isoforms code for proteins with different functions, expression profiles and transcriptional regulation. The aim of the present study was to investigate the expression of PTCH1 first exons in OKC tumors to shed light on scenery whereby PTCH1 coordinates OKC tumorigenesis. Forty OKC, including 12 sporadic and 28 associated with Nevoid Basal Cell Carcinoma Syndrome (NBCCS), were included in the study. The variants 1b, 1d and 1e were investigated by RT-PCR. The exon 1b was detected in 90% of OKC and none of the dental follicle (control). Most of the OKC, sporadic and syndromic, and all of the samples of dental follicles demonstrated the expression of 1d mRNA. All primary OKC had 1b mRNA while 4 (24%) lesions marsupialized lost 1b expression. In addition, the pattern of exon 1 expression observed in oral mucosa adjacent to the OKC was similar to the OKC tumor. In conclusion, this report showed overactivity of Hedgehog (HH) pathway in OKC lesion and at adjacent oral mucosa. We also demonstrated that marsupialization could alter PTCH1 variants profiling in some OKC cases.

Alcohol Consumption and Periodontitis: Quantification of Periodontal Pathogens and Cytokines

BACKGROUND There are few studies on periodontal status related to microbiologic and immunologic profiles among individuals not or occasionally using alcohol and those with alcohol dependence. The aim of this study is to determine the effect of alcohol consumption on the levels of subgingival periodontal pathogens and proinflammatory cytokines (interleukin [IL]-1β and tumor necrosis factor [TNF]-α) in the gingival fluid among individuals with and without periodontitis. METHODS This observational analytic study includes 88 volunteers allocated in four groups (n = 22): individuals with alcohol dependence and periodontitis (ADP), individuals with alcohol dependence and without periodontitis (ADNP), individuals not or occasionally using alcohol with periodontitis (NAP), and individuals not or occasionally using alcohol without periodontitis (NANP). Levels of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Eikenella corrodens, and Fusobacterium nucleatum were determined by real-time polymerase chain reaction on the basis of the subgingival biofilm, and IL-1β and TNF-α were quantified by enzyme-linked immunosorbent assay in gingival fluid samples. RESULTS Individuals with alcohol dependence showed worse periodontal status and higher levels of P. intermedia, E. corrodens, F. nucleatum, and IL-1β than non-users. No significant correlations between TNF-α and bacterial levels were observed. However, in the ADP group, higher levels of E. corrodens were correlated with higher levels of IL-1β. CONCLUSION A negative influence of alcohol consumption was observed on clinical and microbiologic periodontal parameters, as well as a slight influence on immunologic parameters, signaling the need for additional studies.

Loss of heterozygosity of the PTCH gene in ameloblastoma.

Ameloblastoma is a locally aggressive benign neoplasm derived from odontogenic epithelium, with high recurrence rates. Alterations in the Sonic Hedgehog signaling pathway, including PTCH gene mutations, have been associated with the pathogenesis of some odontogenic tumors. The purpose of the present study was to assess loss of heterozygosity at the PTCH locus in ameloblastoma. Twelve ameloblastomas were included, and loss of heterozygosity was assessed by using 3 microsatellite markers D9S252, D9S127, and D9S287 and 3 single-nucleotide polymorphisms rs112794371, rs111446700, and rs357564, all located at the PTCH gene locus. Furthermore, we investigated GLI1 and GLI2 transcription levels by quantitative reverse transcription polymerase chain reaction in 8 ameloblastomas and, concomitantly, PTCH protein levels by immunohistochemical analysis. Loss of heterozygosity at 9q21.33-9q.31 was detected in 4 (40.0%) of 10 informative cases of ameloblastoma. All 8 analyzed samples expressed GLI1 messenger RNA and 7 cases GLI2 messenger RNA. Interestingly, loss of heterozygosity at the PTCH locus was not correlated with GLI1 or GLI2 transcription levels, nor was there any correlation with PTCH protein expression. In conclusion, our findings suggest that loss of heterozygosity in the PTCH region may be relevant to the pathogenesis of ameloblastoma but may target a different gene than PTCH.

NFATc1 and TNFα expression in giant cell lesions of the jaws

BACKGROUND Activation mutations of SH3BP2 gene have been demonstrated in cherubism and central giant cell lesion (CGCL). In the present study we first attempted to investigate the SH3BP2 gene in peripheral giant cell lesion (PGCL). The effect of SH3BP2 gene mutations on the transcription of the downstream genes nuclear factor of activated T cells (NFATc1) and the cytokine tumor necrosis factor-alpha (TNF-alpha) was also investigated together with the immunolocalization of NFATc1 protein in a set of cases of PGCL, CGCL and cherubism with and without SH3BP2 mutation. METHOD Fresh samples of five PGCL, five CGCL and one cherubism cases were included in this study. One of the samples of CGCL presented a somatic heterozygous mutation c.1442A>T in exon 11. The cherubism case showed a heterozygotic substitution c.320C>T in both blood and lesion. These mutations were previously published. All coding and flanking regions of the SH3BP2 gene were sequenced in the cases of PGCL. The real-time polymerase chain reaction (RT-PCR) was performed to analyze the transcription of NFATc1 and TNF-alpha genes. The immunohistochemical analysis of the NFATc1 protein was also performed. RESULTS No SH3BP2 gene mutation was found in PGCL. The RT-PCR showed increased expression of NFATc1 and decreased transcription of TNF-alpha in all the samples. The immunohistochemical analysis of the NFATc1 protein showed a predominant nuclear staining in the multinucleated giant cells. CONCLUSION The development of giant cells lesions of the jaws and cherubism are possibly mediated by overexpression of NFAT in the nucleus of the multinucleated cells.

Hypomethylation of tumor suppressor genes in odontogenic myxoma

Odontogenic myxoma (OM) is an ectomesenchymal benign odontogenic tumor characterized by spindle or stellate-shaped cells embedded in an abundant myxoid or mucoid extracellular matrix. DNA methylation is characterized by the addition of methyl groups in cytosines within CpG islands in the promoter gene. DNA methylation can decrease the expression of tumor suppressor genes and contribute to the development of neoplastic lesions. The aim of study was to evaluate the methylation pattern of the tumor suppressor genes P16 (CDKN2A), P21 (CDKN1A), P27 (CDKN1B), P53 (TP53) and RB1 in OM and dental pulp. Methylation was evaluated using methylation-specific polymerase chain reaction (PCR). The transcription was studied in some cases by using reverse transcription quantitative PCR. A higher frequency of unmethylated P27, P53, and RB1 samples was observed in the OM when compared with the dental pulp. OM expressed mRNA of all the genes evaluated. Considering all the samples together, the expression of Rb was higher in the unmethylated samples compared with the partially methylated samples. This investigation revealed hypomethylation of the genes P27, P53, and RB1 in OM. In addition, methylation of tumor suppressor genes was found to be an usual event in normal dental pulp.