Henriqueta Coimbra Silva

Polymorphisms of genes encoding P2X7R, IL-1B, OPG and RANK in orthodontic-induced apical root resorption

OBJECTIVE Orthodontic-induced external apical root resorption (EARR) is a complex phenotype determined by poorly defined mechanical and patient intrinsic factors. The aim of this work was to construct a multifactorial integrative model, including clinical and genetic susceptibility factors, to analyze the risk of developing this common orthodontic complication. MATERIALS AND METHODS This retrospective study included 195 orthodontic patients. Using a multiple-linear regression model, where the dependent variable was the maximum% of root resorption (%EARRmax) for each patient, we assessed the contribution of nine clinical variables and four polymorphisms of genes involved in bone and tooth root remodeling (rs1718119 from P2RX7, rs1143634 from IL1B, rs3102735 from TNFRSF11B, encoding OPG, and rs1805034 from TNFRSF11A, encoding RANK). RESULTS Clinical and genetic variables explained 30% of%EARRmax variability. The variables with the most significant unique contribution to the model were: gender (P < 0.05), treatment duration (P < 0.001), premolar extractions (P < 0.01), Hyrax appliance (P < 0.001) and GG genotype of rs1718119 from P2RX7 gene (P < 0.01). Age, overjet, tongue thrust, skeletal class II and the other polymorphisms made minor contributions. CONCLUSION This study highlights the P2RX7 gene as a possible factor of susceptibility to EARR. A more extensive genetic profile may improve this model.

MIA PaCa-2 and PANC-1 – pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors

Studies using cell lines should always characterize these cells to ensure that the results are not distorted by unexpected morphological or genetic changes possibly due to culture time or passage number. Thus, the aim of this study was to describe those MIA PaCa-2 and PANC-1 cell line phenotype and genotype characteristics that may play a crucial role in pancreatic cancer therapeutic assays, namely neuroendocrine chemotherapy and peptide receptor radionuclide therapy. Epithelial, mesenchymal, endocrine and stem cell marker characterization was performed by immunohistochemistry and flow cytometry, and genotyping by PCR, gene sequencing and capillary electrophoresis. MIA PaCa-2 (polymorphism) expresses CK5.6, AE1/AE3, E-cadherin, vimentin, chromogranin A, synaptophysin, SSTR2 and NTR1 but not CD56. PANC-1 (pleomorphism) expresses CK5.6, MNF-116, vimentin, chromogranin A, CD56 and SSTR2 but not E-cadherin, synaptophysin or NTR1. MIA PaCA-1 is CD24−, CD44+/++, CD326−/+ and CD133/1−, while PANC-1 is CD24−/+, CD44+, CD326−/+ and CD133/1−. Both cell lines have KRAS and TP53 mutations and homozygous deletions including the first 3 exons of CDKN2A/p16INK4A, but no SMAD4/DPC4 mutations or microsatellite instability. Both have neuroendocrine differentiation and SSTR2 receptors, precisely the features making them suitable for the therapies we propose to assay in future studies.

Antagonistic effect of IL1 variants in periodontitis and external apical root resorption: Evidence from a literature review

External apical root resorption (EARR) induced by orthodontic treatment and chronic periodontitis (CP) are complex phenotypes dependent on the interaction of multiple genetic and non-genetic risk factors. Apart from different environmental triggers, these phenotypes are caused by antagonistic biological mechanisms involving local immunoinflammatory reaction and alveolar bone metabolism, for which IL1 have a prominent role. Whereas EARR benefits from bone remodelling, CP is characterized by osteolytic damaged. Our aim was to verify if these two phenotypes have opposite genetic profiles, considering the most frequently analysed polymorphisms for both diseases. A review of the literature was performed searching for the association of rs1800587 from Interleukin-1 alpha (IL1A) gene and rs1143634 from interleukin-1 beta (IL1B) gene with EARR and CP. The electronic search included MEDLINE/PubMed, EBSCOhost, Cochrane and Web of Science databases. Twenty four articles met the inclusion and exclusion criteria. For IL1B polymorphism, two out of seven studies found a significant statistical association between EARR and CC genotype, whether for CP, there were eighth out of fifteen references describing a statistically significant associations with T allele. For IL1A variant, no significant association with EARR was described. In conclusion, literature review suggests that for IL1B SNP rs1143634, EARR and CP have an opposite genetic profile. For IL1A SNP, our hypothesis could not be confirmed.

Polymorphisms of estrogen receptors, ERα and GPR30: association with breast cancer susceptibility and prognosis

The purpose of this study was to clarify the roles of polymorphisms from the classical nuclear estrogen receptor ESR1 and from the recently described estrogen receptor coupled to G proteins GPR30 [1], in breast cancer susceptibility and prognosis. Three single nucleotide polymorphisms (SNPs), rs2234693 and rs9340799 from ESR1 and rs3808350 from GPR30 were genotyped in 260 breast cancer patients and 259 controls. SNPs were analyzed by PCR-RFLP and by real-time PCR with TaqMan probes. Genotypes were correlated with established breast cancer prognostic markers. For rs9340799, our results showed a significant association between A allele and breast cancer susceptibility, particularly for homozygous (OR-7.33, 95%CI, 4.3-12.6; p < 0.0005). The occurrence of polymorphisms rs2234693 and rs3808350 did not differ between breast cancer patients and controls. However, for rs2234693, CC genotype was significantly associated with higher (G2/G3) tumor grade (p < 0,05; OR-1.01, 95%CI, 1.01-4.98) and in postmenopausal women, the TT variant was associated with lower (G1) tumor grade (p = 0,02, OR-1.9, 95% CI, 1.09-3.45). No significant association was found with the presence of estrogen receptors or with HER2 overexpression in tumor samples. In conclusion, our work confirms the role of ESR1 polymorphisms in breast cancer: rs9340799 in breast cancer susceptibility and rs2234693 in breast cancer prognosis. For GPR30 SNP rs3808350, none association was found.

Evaluation of the role of mir-34b in modulation of radioresistance in non-small cell lung cancer

Radiotherapy is a major therapeutic weapon in lung cancer. However, the resistance to radiotherapy is frequent. The microRNAs of the miR-34 family, miR34a, miR-34b and miR-34c, described as effector molecules in the cellular response to activation of P53, have low expression levels in lung cancer [1]. The mRNA of the anti-apoptotic protein BCL-2 is among the targets of miR-34 family. The aims of our study were to clarify the involvement of miR-34b over-expression in the modulation of radiation response in NSCLC cell lines and the mechanisms involved. For these purposes we used two radioresistant NSCLC cell lines, A549, expressing P53, and H1299, not expressing P53. Cells transfected with pre-miR-34b or with a transfection control were exposed to different irradiation doses. The response to irradiation was assessed by cell survival curves obtained by clonogenic assay, and flow citometry allowed the characterization of cell death and the quantification of BCL-2, BAX and P53 protein expression levels. Our results showed that both cell lines had low expression levels of miR-34 family members, especially for miR-34b/c. Over-expression of miR-34b sensitized A549 cells to low doses of radiation and decreased BCL-2 expression, but without changing apoptosis levels. H1299 cells remained unchanged. These results suggest that in NSCLC expressing P53, response to radiotherapy is dependent on BCL-2 levels and may be modulated by over-expression of miRNA34b. Other cell death mechanisms than apoptosis, but also involving BCL-2, like autophagy, could to be involved. Author details Department of Molecular Biology, Faculty of Medicine, University of Coimbra (FMUC), Coimbra, Portugal. IBILI, FMUC, Coimbra, Portugal. Medical Genetics, FMUC, University of Coimbra, Coimbra, Portugal. CIMAGO, FMUC, Coimbra, Portugal. Center for Neuroscience and Cell Biology, University of Coimbra, Portugal.

Polymorphisms of GSTM1, GSTT1, GSTP1 and CYP1A1 genes and susceptibility to lung cancer

Biotransformation enzymes are related with lung cancer that arises as a consequence of exposure to mutagenic agents. CYP1A1 gene codifies the phase I enzyme, aryl hydrocarbon hydroxilase, belonging to the Cytochrome P450 system, that plays a major role in the bioactivation of tobacco procarcinogenes, while glutathione-S-transferases genes, GSTM1, GSTT1 and GSTP1, codify conjugation enzymes associated with detoxification processes of free radicals, xenobiotics and cytotoxic drugs [1]. Our main goal was to verify possible associations between polymorphisms of these genes and susceptibility to lung cancer. CYP1A1 polymorphisms, m1 (T6235C) and m2 (A4889G) were studied by RFLP assay, GSTM1 and GSTT1 (GSTM1*0 and GSTT1*0) by PCR multiplex and GSTP1 (rs1695) by real time PCR, in 197 patients and 237 controls. For CYP1A1 alleles and genotype distributions, no statistically significant differences were found between both populations. GSTT1 *0/*0 genotype was associated with a higher susceptibility to lung cancer (OR: 1.6; 95%CI: 1.02-2.44; p < 0.05). In the patient population, smoking burden of 21-100 pack-years were more frequently associated with GSTT1 *0/*0 genotype than in controls (p < 0.02). This difference was even more significant for ex-smokers (p < 0.001). Gene copy number assay exposed an association between GSTM1*1/*0 and lung cancer (p < 0.001). The results reveal a possible association between GSTT1 *0/*0 and susceptibility to lung cancer related with smoking habbits.