Carolina Cavaliéri Gomes

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.

REVIEW ARTICLE: Current concepts of ameloblastoma pathogenesis

Ameloblastoma is a locally destructive and invasive tumour that can recur despite adequate surgical removal. Molecular studies have offered interesting findings regarding ameloblastoma pathogenesis. In the present review, the following topics are discussed regarding its molecular nature: clonality, cell cycle proliferation, apoptosis, tumour suppressor genes, ameloblastin and other enamel matrix proteins, osteoclastic mechanism and matrix metalloproteinases and other signalling molecules. It is clear from the literature reviewed that translational studies are necessary to identify prognostic markers of ameloblastoma behaviour and to establish new diagnostic tools to the differential diagnosis of unicystic from multicystic ameloblastoma. Finally, molecular biology studies are also important to develop more effective alternative approaches to the treatment of this aggressive odontogenic tumour.

Review of the molecular pathogenesis of the odontogenic keratocyst

The odontogenic keratocyst (keratocystic odontogenic tumour) (OKC) is one of the most prevalent odontogenic tumours. Since its initial description, a number of studies have focused on different aspects of this lesion, attempting to explain its distinctive biological behaviour. In this review the authors address the main genetic and epigenetic alterations reported on this tumour. Although most of the knowledge on this field is not being used in the clinical practice, some perspectives of translational studies are discussed.

P21/ WAF1 and cyclin D1 variants and oral squamous cell carcinoma

BACKGROUND Genetic factors are known to be involved in oral squamous cell carcinoma (OSCC) development. METHOD We evaluated a possible association between CCND1 A870G and P21/WAF1 C98A polymorphisms and OSCC, as well as the impact of the genotypes on protein immunoexpression. The study group consisted of 80 individuals with histopathological diagnosis of OSCC and the control group consisted of 80 healthy individuals without oral lesions and matched by age, sex and tobacco usage. The genotypes were studied by the polymerase chain reaction and restriction fragment length polymorphic analysis. Paraffin-embedded sections were used for immunohistochemical analysis. RESULTS No statistical association between CCND1 and/or P21/WAF1 genotypes and OSCC was demonstrated, although we found that people harbouring the combined presence of at least one variant allele of both genes showed a 1.8 times more chance of developing OSCC compared to the referent genotype. OSCC tumours from individuals with P21 heterozygous genotype showed a significantly higher immunopositivity than tumours from wild-type individuals. CONCLUSION The present study did not demonstrate a significant association between CCND1 and / or P21 / WAF1 genotypes and OSCC. However, P21 protein expression in OSCC tumours is affected by P21 / WAF1 genotype.

Increased miRNA-146a and miRNA-155 expressions in oral lichen planus

Oral lichen planus (OLP) is a chronic inflammatory disease T helper 1 lymphocytes (Th1)-mediated. Interferon-gamma (IFN-γ) plays a central role in local immune response in this disease. MicroRNAs (miRNAs) are endogenously expressed non-coding RNAs that have important biological and pathological functions due to their potential mechanism regulating gene expression. Recently, some studies have demonstrated that miRNA-146a and miRNA-155 participate in immune response regulation, and are important in several chronic inflammatory and autoimmune diseases. The purpose of the present study was to investigate the expression of the miRNA-146a and miRNA-155 in 31 OLP lesions compared to normal oral mucosa and blood samples. Quantitative real-time polymerase chain reaction was used to analyze miRNA expressions. Our results showed increased expression of miRNA-146a and miRNA-155 in OLP lesions. In conclusion, these data highlight the possibility of miRNA-146a and miRNA-155 involvement in the regulation of the immune response in OLP.

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.

Clonal nature of odontogenic tumours.

BACKGROUND Although clonal origin is an essential step in the comprehension of neoplasias, there have been no studies to examine whether odontogenic tumours are derived from a single somatic progenitor cell. The purpose of this study was to investigate the clonal origin of odontogenic tumours. METHODS Fresh samples of seven ameloblastomas, two odontogenic mixomas, two adenomatoid odontogenic tumour, one calcifying odontogenic cyst, one calcifying epithelial odontogenic tumour (CEOT) and six odontogenic keratocyst (OKC) of female patients were included in this study. After DNA extraction, the HUMARA gene polymorphism assay was performed. RESULTS Most of the informative odontogenic lesions studied (12 out of 16) showed a monoclonal pattern. Among the polyclonal cases, two were OKC, one CEOT and one odontogenic mixoma. CONCLUSIONS Our results suggest that most odontogenic tumours are monoclonal.

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.

Assessment of TP53 Mutations in Benign and Malignant Salivary Gland Neoplasms

Despite advances in the understanding of the pathogenesis of salivary gland neoplasms (SGN), the molecular pathways associated with enhanced tumor growth and cell survival remain to be established. The aim of the present study was to investigate whether TP53 mutations are relevant to SGN pathogenesis and if they impact on p53 protein expression. The study included 18 benign and 18 malignant SGN samples. Two polymorphic microsatellite markers at the TP53 genetic locus were chosen to assess loss of heterozygosity (LOH) in the samples that had matched normal DNA. The TP53 exons 2–11 were amplified by PCR, and all of the products were sequenced. Reverse transcription-PCR of the TP53 open reading frame (ORF) was carried out in the samples that had fresh tissue available, and immunohistochemistry for the p53 protein was performed in all samples. TP53 LOH was only found in two pleomorphic adenomas. We found two missense mutations in exon 7 (one in a pleomorphic adenoma and the other in a polymorphous low grade adenocarcinoma), another in exon 8 (in a carcinoma ex pleomorphic adenoma) and a fourth missense mutation in exon 10 (in a mucoepidermoid carcinoma). In addition, a nonsense mutation was found in exon 8 of an adenoid cystic carcinoma. Several intronic and exonic SNPs were detected. Although almost all of the malignant samples were immunopositive for p53, approximately 37% of the benign samples were positive, including the sample harboring the missense mutation and one of the samples that showed LOH. The complete TP53 ORF could be amplified in all samples analyzed, including the IHC negative samples, the samples showing LOH and one sample displaying a missense mutation. In summary, our results show that TP53 mutations are not a frequent event in SGN and that p53 immunopositivity might not be associated with sequence mutations in SGN.