L. Mesquita

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.

New targeted therapies in myelodysplastic syndrome: the role of farnesyltransferase and proteasome inhibitors

One of the main mechanisms responsible for MDS molecular pathogenesis involves the activation of tyrosine-kinase receptors, such as FLT3, RAS proteins, and deregulation of apoptotic pathways. Regarding this, new drugs have been developed to target pathways involved in malignancy, such as Farnesyltransferase Inhibitors (IFTs) and proteasome inhibitors (PI). This work aims to clarify the role of IFTs and PI as potential therapeutic agents in Myelodysplastic Syndrome (MDS). For this, F-36P cells, were incubated with different concentrations of α-HFPA (IFT) and MG262 (PI), as single agents and in association with the conventional therapeutic drug, Cytosine Arabinoside (Ara-C). Cell growth and viability was evaluated by Trypan Blue test. Cell death was analyzed by optic microscopy and flow cytometry (FC). Expression of proteins involved in apoptosis and cell cycle regulation was evaluated by FC. The detection of RAS and FLT3 mutations was accessed by sequentiation and PCR, respectively. Our results show that α-HFPA and MG262, in monotherapy, induce a decrease in cell growth and viability in a time and dose-dependent manner (IC50, α-HFPA 125 μM; MG262 100 nM). The antiproliferative effect of α-HFPA could be related to RAS/MAPK pathway inhibition, as we observed a decrease in cyclin D1 levels, while the cytotoxicity induced by MG262 to an increase in BAX expression. Our results show that α-HFPA is effective independently of RAS mutations, once we didn’t identify mutations in none of the isoforms studied, but we observe ITD mutations in FLT3 gene. These results suggest that IFTs and PIs may constitute a potential therapeutic approach as single agents in MDS.

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.

Sondas de RNA ribossómico no diagnóstico da tuberculose humana

RESUMO Tendo em vista um mais rapido e especifico diagnostico laboratorial da tuberculose, os autores avaliaram esta tecnologia usando para tal 15 amostras desconhecidas, avaliaram ainda as condicoes ambientais do seu laboratorio, o que para este tipo de tecnologia e particularmente critica.