Browsing by Issue Date, starting with "2014-11-25"
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- Rac1b expression reverts B-Raf-V600E-induced senescence in colorectal cellsPublication . Henriques, Andreia; Barros, Patrícia; Matos, Paulo; Jordan, PeterMutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that overexpression of splice variant Rac1b occurs in around 80% of colorectal tumors carrying a mutation in BRAF. Using both BRaf-V600E-directed RNAi and overexpression we demonstrate that this mutation does not directly lead to Rac1b overexpression, indicating the latter as an independent event during tumor progression. Nonetheless, we observed that expression of oncogenic BRaf-V600E in non-transformed colonocytes (NCM460 cell line) increased both the transcript and protein levels of p14 ARF, p15 INK4b and p21 CIP1 and led to increased expression of β-galactosidase, all indicators of OIS induction. Importantly, the co-expression of Rac1b with B-Raf-V600E reverted the OIS phenotype, reducing the protein expression levels of the cell-cycle inhibitors and β-galactosidase activity to those of control cells. These data identify increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.
- Validation of the Portuguese self-administered computerised 24-hour dietary recall among second-, third- and fourth-grade childrenPublication . Carvalho, M.A.; Baranowski, T.; Foster, E.; Santos, O.; Cardoso, B.; Rito, A.; Pereira Miguel, J.Background: Current methods for assessing children's dietary intake, such as interviewer-administered 24-h dietary recall (24-h DR), are time consuming and resource intensive. Self-administered instruments offer a low-cost diet assessment method for use with children. The present study assessed the validity of the Portuguese self-administered, computerised, 24-h DR (PAC24) against the observation of school lunch. Methods: Forty-one, 7–10-year-old children from two elementary schools, in Lisbon, were observed during school lunch followed by completion of the PAC24 the next day. Accuracy for reporting items was measured in terms of matches, intrusions and omissions; accuracy for reporting amounts was measured in terms of arithmetic and absolute differences for matches and amounts for omissions and intrusions; and accuracy for reporting items and amounts combined was measured in terms of total inaccuracy. The ratio of the estimated weight of food consumed with the actual weight consumed was calculated along with the limits of agreement using the method of Bland and Altman. Results: Comparison of PAC24 against observations at the food level resulted in values of 67.0% for matches, 11.5% for intrusions and 21.5% for omissions. The mean for total inaccuracy was 3.44 servings. For amounts, accuracy was high for matches (−0.17 and 0.23 servings for arithmetic and absolute differences, respectively) and lower for omissions (0.61 servings) and intrusions (0.55 servings). PAC24 was found to under-estimate the weight of food on average by 32% of actual intake. Conclusions: PAC24 is a lower-burden procedure for both respondents and researchers and, with slight modification, comprises a promising method for assessing diet among children.
- Alternative splicing of tumour-related Rac1b in colorectal cells is regulated by protein phosphorylation of splicing factor SRSF1Publication . Gonçalves, Vânia; Henriques, Andreia; Pereira, Joana; Neves-Costa, Ana; Moyer, Pat; Ferreira Moita, Luís; Gama-Carvalho, Margarida; Matos, Paulo; Jordan, PeterThe pre-messenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications such as protein phosphorylation, which are determined by signal transduction pathways. Here we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells we found that depletion of AKT2, AKT3, GSK3β and SRPK1 significantly decreased endogenous Rac1b levels. Whereas knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insights into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.
- Human UPF1: a cap-independent translation initiation mechanism and a cryptic promoter regulate its gene expression in cancer cellsPublication . Lacerda, Rafaela; Marques-Ramos, Ana; Teixeira, Alexandre; Romão, LuísaCervical (CC) and colorectal (CRC) cancers are among the leading causes of death worldwide and their development and progression is dependent on regulation of gene expression. Eukaryotic cells possess a variety of post-transcriptional mechanisms by which they regulate gene expression, namely at translation initiation level. Although in most cases it occurs via the cap-dependent mechanism, several oncogenes, growth factors or proteins involved in the regulation of programmed cell death, among others, have a different translation initiation mechanism. It involves the direct recruitment of the ribosome to the vicinity of the initiation codon without the involvement of the cap structure. This allows the maintenance of protein synthesis under several cellular stresses and promotes tumourigenesis. Apart from its role in nonsense-mediated decay, the human up-frameshift 1 (UPF1) DNA and RNA helicase protein plays a crucial role in telomere replication and homeostasis, and in cell cycle progression. In addition, its expression levels are maintained in every phase of the cell cycle, thus indicating that its translation may occur via a cap-independent mechanism. To test this hypothesis, we cloned the human UPF1 5’UTR in a dicistronic vector and transfected CC and CRC cell lines with either this construct or the control counterparts. We observed a 15- to 25-fold increase in relative luciferase activity of the UPF1 5’UTR-containing construct compared to the levels obtained from the empty counterpart in all tested cell lines, suggesting a cap-independent translation initiation. To control whether these levels of luciferase activity could be due to the presence of a cryptic promoter, we transfected cells with promoterless plasmids and observed the same result, demonstrating that UPF1 5’UTR contains a cryptic promoter. To check the cap-independent translation activity alone, we transfected cells with in vitro transcribed, capped and polyadenylated mRNAs and observed a 2-fold increase in protein levels, which shows that translation can occur in a cap-independent way. This is maintained under conditions of global protein synthesis inhibition. Deletional analysis of UPF1 5’UTR revealed that first 50 nucleotides are essential for both cryptic promoter and cap-independent activities. These results provide new insights on the regulation of UPF1 expression in human cancer cells.
- Estudo do papel da imunidade inata nas infeções por micobactériasPublication . Sousa, Sara; Jordão, Luísa; Telhada, Maria Margarida Blasques[PT] Desde os primórdios da humanidade que existem relatos de infecções respiratórias causadas por micobactérias. A incidência de infecções por Micobactérias Não-Tuberculosas (MNT), consideradas oportunistas, tem aumentado gradualmente atingindo sobretudo a população imunodeprimida. As MNTs são maioritariamente ambientais e ubíquas. Sendo o difícil diagnóstico, a resistência aos antibióticos e o escasso conhecimento da patogenicidade destas micobactérias, os principais impulsores do seu estudo. Os macrófagos alveolares, em infecções do sistema respiratório, são as primeiras células do sistema imunitário que contactam com as MNTs. Estes irão desencadear de imediato uma resposta imunitária inata. Este trabalho teve como principal objectivo avaliar a resposta imunitária durante uma infecção com MNTs utilizando células THP-1 como modelo de macrófagos alveolares humanos. Para tal, foi avaliada a sobrevivência de algumas MNTs no interior dos macrófagos, seguida do estudo de vários componentes da resposta imunitária: maturação do fagossoma; produção de molécula pró- inflamatórias; e indução da morte celular. Foi possível observar a existência de 3 perfis de persistência intracelular: M. smegmatis é eliminado; M. fortuitum ATCC6841 apresenta um perfil de latência; o M. fortuitum 747/08, M. abscessus 549/08, M. avium ATCC25291 e M.avium 60/08 apresentam um perfil de crescimento. A maturação dos fagossomas de MNT é bloqueada duma forma menos eficiente do que a dos fagossomas de M. tuberculosis. Apenas o M. avium 60/08 foi capaz de induzir a produção de NO e IL-10. E ainda que existe a indução da apoptose diferenciada entre as estirpes estudadas, sendo o M. avium 60/08 o melhor indutor.